Molecular Characterization and Developmental Expression Patterns of the Zebrafish twist Gene Family Yeo Gare Hoon (B.Sci, University of Melbourne) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE YOOG LOO LIN SCHOOL OF MEDICINE (DEPARTMENT OF PAEDIATRICS) NATIONAL UNIVERSITY OF SINGAPORE 2009 Table of Contents _____________________________________________________________ Acknowledgements v List of Tables vi List of Figures vii Abbreviation ix Summary x Abstract xiii ________________________________________________________________________ Chapter 1: Introduction 1 1.1 TWIST gene family 1 1.1.1 TWIST1 gene 1 1.1.2 TWIST2 gene 5 1.1.3 Twist3 gene 6 1.2 Why zebrafish is used as an animal model in this study? 7 1.3 Phylogenetics 8 1.3.1 DNA or protein sequences 9 1.4 Gene duplication 10 1.4.1 Evolutionary fates of duplicate genes 12 Chapter 2: Materials and Methods 16 2.1 Animal stocks and maintenance 16 2.2 Isolation of genomic DNA and total RNA 16 2.2.1 Isolation of genomic DNA 16 2.2.2 Isolation of total RNA 17 2.3 Full-length cDNA sequence 17 2.3.1 Rapid amplification of complementary DNA ends 2.3.2 (RACE) of zebrafish twist1b 17 Assembly of zebrafish twist1b full-length cDNA 20 ii 2.4 Genomic sequence of zebrafish twist1b 21 2.5 RT-PCR 22 2.6 Synthesis of RNA probes for in situ hybridization analysis 2.6.1 Identification of unique 3’UTR sequences of the zebrafish twist gene family 2.6.2 23 23 Isolation of unique 3’UTR sequences of zebrafish twist gene family 23 2.6.3 Linearization of plasmid DNA 28 2.6.4 RNA labeling with Digoxenin / Fluorescein RNA labeling kits (SP6/T7/T3)(Roche) 29 2.6.5 Purification of RNA probe 29 2.7 Whole mount in situ hybridization 30 2.8 Cryosection 34 2.9 Image processing 35 2.10 Phylogenetic analysis 35 2.10.1 Alignment and phylogenetic tree 35 2.10.2 Calculation of genetic distances 36 2.10.3 Comparative synteny analysis 37 Chapter 3: Results 38 3.1 Characterization of zebrafish full-length cDNA of twist1b 38 3.2 Genomic organization of zebrafish twist1b 39 3.3 Alignment of TWIST family peptides 41 3.4 Comparison and alignment of zebrafish twist gene family 3.5 41 Identification and confirmation of the true orthologs of zebrafish twist genes 47 3.5.1 Comparison of zebrafish twist gene family with other species 47 3.5.2 Phylogenetic analysis 48 iii 3.5.3 Calculation of genetic distances 50 3.5.4 Comparative synteny analysis 51 3.6 Embryonic expression patterns of the zebrafish twist gene family 58 3.6.1 RTPCR analysis 58 3.6.2 In situ hybridization analysis 60 Chapter 4: Discussion 76 4.1 The zebrafish twist gene family 76 4.2 Phylogenetic relationships of the twist genes in fish 77 4.3 Genetic distance analysis of twist1a and twist1b among the fishes 78 4.4 Comparative synteny analyses 79 4.5 Comparison of zebrafish twist family expression pattern with other species. 83 4.5.1 Zebrafish twist1a and twist1b genes 83 4.5.2 Zebrafish twist2 85 4.5.3 Zebrafish twist3 86 4.6 Shared and unique expression sites of the zebrafish twist genes 86 4.6.1 Importance of using unique 3’UTR sequences as riboprobes 86 4.6.2 Comparison of zebrafish twist genes expression sites with other publications 86 4.7 Evolutionary fates of the zebrafish twist gene family 91 Chapter 5: Conclusions 94 Chapter 6: References 95 iv Acknowledgement ________________________________________________________________________ Firstly, I thank God for the strength and perseverance that have sustained me through this research project. Thank God for His wisdom and for directing my path. My utmost gratitude goes to my supervisor, Assoc Prof Samuel Chong for giving me the opportunity to further my studies in his lab. Thank you for your kind understanding, encouragement and patient supervision to me. Special thanks to Assoc Prof Christoph Winkler for your constructive advice, for sharing with me your invaluable knowledge. Your help is very much appreciated. Thank you Prof Byrappa Venkatesh for your enormous help in phylogenetic analyses, for enlightening me on the topics of evolution and phylogeny, an area which I am very green. To Assoc Prof Vladimir Korzh, thank you for your precious recommendation and time. Your insightful advice has been most helpful. Thank you too, for the gifts of pax2.1 and wt1 plasmids. To Dr Karuna Sampath, I am very grateful for both your helpful technical advice and your patient guidance. Special thanks to Felicia and Ben Jin, for your words of encouragement and support and for sharing with me your laboratory expertise and personal experiences on time management as a part-time student. Big thanks to Haibo, Shanta and Xiaoyu for helping with the care and maintenance of the fish system. Without your meticulous care, I wouldn’t have healthy embryos and fish for my project. To Arnold, Wang Wen, Weijun, Chia Yee, Pooi Eng, Clara, Yvonne, Jack, Siew Hoon and Victor thanks for the words of encouragement, your friendship and moral support along the way. Thanks to Monte Westerfield and Andrew D. Sharrocks for their gifts of dlx2a and fli1a (pAS160) plasmids respectively. And last but not least, thanks to my parents, my sisters and Patrick for giving me the love and support to press on. v List of Tables ________________________________________________________________________ Table 1 : The synthesis of RNA probes and in situ hybridization conditions Table 2: Nucleotide identity of the coding region, bHLH domain, and WR domain of the zebrafish twist genes Table 3: Comparison of zebrafish twist gene sequences with TWIST sequences from other species Table 4: Comparative synteny analysis of chromosomal regions around zebrafish twist1a and twist1b and human TWIST1 Table 5: Comparative synteny analysis of chromosomal regions around zebrafish twist1a and medaka twist1a and twist1b Table 6: Comparative synteny analysis of chromosomal regions around zebrafish twist1b and medaka twist1a and twist1b Table 7: Comparative synteny analysis of chromosomal regions around twist2 from zebrafish, human and medaka Table 8: Comparative synteny analysis of chromosomal regions around zebrafish twist3 and medaka twist3a and twist3b Table 9: Expression domains of the four zebrafish twist genes Table 10 : Twist expression sites in selected species vi List of Figures ________________________________________________________________________ Figure 1.1: Terminologies used to classify homologs Figure 1.2: The model of synfunctionalization: a mechanism for gene loss or function shuffling. Figure 2.1: The incomplete cDNA sequence of zebrafish twist1 gene Figure 2.2: Agarose gel electrophoresis of 5’ and 3’ RACE experiment Figure 2.3: 5’ UTR sequences of the zebrafish twist1 (twist1b) gene obtained from 5’RACE experiment. Figure 2.4: 3’ UTR sequences of the zebrafish twist1 (twist1b) gene obtained from 3’RACE experiment. Figure 2.5: The complete full-length cDNA sequence of the zebrafish twist1 gene. Figure 2.6: cDNA sequence of zebrafish twist1a Figure 2.7: cDNA sequence of zebrafish twist1b Figure 2.8: cDNA sequence of zebrafish twist2 Figure 2.9: cDNA sequence of zebrafish twist3 Figure 3.1: Full-length cDNA sequence of zebrafish twist1 (twist1b) and its deduced amino acid sequence. Figure 3.2: Genomic DNA sequence of zebrafish twist1 (twist1b). Figure 3.3: Alignment of predicted Twist proteins Figure 3.4: Alignment of zebrafish full-length cDNAs Figure 3.5: Cladogram and unrooted radial tree of Twist proteins generated by the neighbor-joining method Figure 3.6: Gene structure of twist1a, twist1b, twist2 and twist3 Figure 3.7: RT-PCR of zebrafish twist genes Figure 3.8: Expression of zebrafish twist genesexpression during the cleavage period vii Figure 3.9: Expression of zebrafish twist genesexpression during the gastrula period Figure 3.10: Expression of zebrafish twist genesexpression during the early segmentation period Figure 3.11: Expression of zebrafish twist genes during mid-somitogenesis Figure 3.12: Zebrafish twist expression along the trunk Figure 3.13: Zebrafish twist1b and twist3 expression during somitogenesis Figure 3.14: Zebrafish twist1b expression in the somites Figure 3.15: Expression of zebrafish twist genesexpression during the prim-5 stage Figure 3.16: Expression of zebrafish twist genesexpression during the long-pec stage Figure 3.17: Expression of zebrafish twist genesexpression during the hatching period Figure 4.1: A model for the evolutionary history of twist genes. viii Abbreviations ________________________________________________________________________ cDNA complementary DNA dNTP Deoxyribonucleotide triphosphate UTR Untranslated Region PCR Polymerase Chain Reaction hpf hours post fertilization dpf days post fertilization DEPC diethylpyrocarbonate SDS sodium dodecyl sulfate RACE Rapid amplification of complementary DNA ends bHLH basic Helix-Loop-Helix WR tryptophan-arginine RT Reverse Transcription SCS Saethre-Chotzen Syndrome ORF Open reading frame LG Linkage Group ix Summary ________________________________________________________________________ The Twist gene is essential for development and survival, and is present in animals ranging from Drosophila to humans, either in single copy or as a gene family of two to five members. In 2007, a paralog of twist1 was identified by Gitelman, who renamed the genes according to their relationships with those of other species (Gitelman, 2007). This study aims to characterize the zebrafish twist family of genes, their phylogenetic and evolutionary relationships, and their developmental expression profiles. I performed a comprehensive alignment, phylogenetic and comparative synteny analysis to determine the relationship of these genes to each other and to those of other species. Phylogenetic analysis showed that the Twist peptides were clustered into three clades, with Twist1, Twist2 and Twist3 peptides in each clade. Interestingly, the Twist1b peptides of the Acanthopterygii (medaka, fugu, spotted green pufferfish and stickleback) were clustered together with the Twist3 peptides instead of Twist1 peptides whereas zebrafish twist1a and twist1b peptides were clustered with the Twist1 peptides. Comparative nucleotide substitution analyses revealed a faster nucleotide mutation/substitution in the acanthopterygian twist1b compared to the zebrafish twist1b, thus explaining the anomalous clustering of the former group of Twist1b peptides. x Comparative synteny analysis of the chromosomal regions flanking the zebrafish, medaka, and human twist genes showed that the zebrafish twist1a and twist1b are coparalogs and co-orthologs of human TWIST1. Furthermore, zebrafish twist1a and twist1b are orthologous to medaka twist1a and twist1b, respectively, despite the different phylogenetic clusterings of zebrafish and medaka twist1b. The orthology of zebrafish twist2 to human and medaka TWIST2/twist2, was also confirmed. Finally, zebrafish twist3 showed greater chromosomal synteny to medaka twist3b than to the medaka twist3a. Based on these results, a model for the evolutionary history of the twist genes has been reconstructed. I also performed a comprehensive developmental expression analysis of all four twist genes. All four genes were expressed in the pharyngeal arches. Zebrafish twist1a and twist1b were expressed in the sclerotome and twist3 in the somite during the segmentation period. Zebrafish twist1b and twist3 were found to be present as maternal transcript. Many expression sites were unique. Transcripts of twist1a were detected specifically in the premigratory neural crest cells during early somitogenesis and in the heart valve at the hatching period. Zebrafish twist1b was expressed in the intermediate mesoderm during segmentation period and in the olfactory placode at the hatching period. Zebrafish twist2 expression was observed in the organizer at the shield stage, presumptive vasculature during the segmentation period, and in the hypochord and dorsal aorta during the prim-5 stage. Zebrafish twist1a and twist3 were expressed in the fin bud, with twist3 expression concentrated in the endochondral disc and twist1a expression xi strongest in the actinotrichs. Minimal expression overlap was observed among the four twist genes using unique 3’UTR sequences for riboprobes. The contents of this thesis have been published in two paper, “Zebrafish twist1 is expressed in craniofacial, vertebral, and renal precursors (Yeo et al., 2007) and “Phylogenetic and evolutionary relationships and developmental expression patterns of the zebrafish twist gene family” (reference in press). xii Abstract _____________________________________________________________ Four members of the twist gene family (twist1a, 1b, 2 and 3) are found in the zebrafish, and they are thought to have arisen through three rounds of gene duplication, two of which occurred prior to the tetrapod-fish split. Phylogenetic analysis groups most of the vertebrate Twist1 peptides into clade I, except for the Twist1b proteins of the acanthopterygian fish (medaka, pufferfish, stickleback), which clustered within clade III. Paralogies and orthologies among the zebrafish, medaka, and human twist genes were determined using comparative synteny analysis of the chromosomal regions flanking these genes. Comparative nucleotide substitution analyses also revealed a faster rate of nucleotide mutation/substitution in the acanthopterygian twist1b compared to the zebrafish twist1b, thus accounting for their anomalous phylogenetic clustering. Based on these analyses, a model for the evolutionary history of the twist genes has been reconstructed. I observed minimal expression overlap among the four twist genes using unique 3’UTR sequences for riboprobes, suggesting that despite their significant peptide similarity, their regulatory controls have diverged considerably, with minimal functional redundancy between them. xiii Chapter 1: Introduction _____________________________________________________________ 1.1 The TWIST gene family The TWIST genes are a group of transcription factor genes whose peptides contain two highly conserved domains, the basic helix-loop-helix (bHLH) domain and the tryptophan-arginine (WR) domain (Atchley and Fitch, 1997; Spring et al., 2000). The bHLH domain can be found in a number of other proteins and is involved in growth regulation, myogenesis and neurogenesis (Jan and Jan, 1993). The function of the WR motif is unclear although it has been suggested to be required either for TWIST activity, for the stability of its mRNA or for normal protein folding (Gripp et al., 2000; Castanon and Baylies, 2002). Twist was first isolated in Drosophila as a zygotic gene involved in the establishment of dorso-ventral patterning, mesoderm specification and myogenesis (Thisse et al., 1987; Thisse et al., 1988; Baylies and Bate, 1996). At gastrulation, homozygous twist mutant embryos were abnormal and failed to differentiate their mesoderm (Simpson, 1983; Thisse et al., 1987). Since this initial discovery, Twist orthologs and paralogs have been identified in many other animal species. 1.1.1 TWIST1 The TWIST1 gene is located on human chromosome 7p21.2 and has been reported to be the causative gene for Saethre-Chotzen Syndrome. Twist1 has been the most intensively studied gene among the TWIST gene family and its expression profile has 1 been reported in many species including the mouse (Wolf et al., 1991; Fuchtbauer, 1995; Stoetzel et al., 1995), rat (Bloch-Zupan et al., 2001), Xenopus (Hopwood et al., 1989; Stoetzel et al., 1998), chick (Tavares et al., 2001), medaka (Yasutake et al., 2004) and zebrafish (Rauch, 2003; Germanguz et al., 2007; Yeo et al., 2007). In the mouse, maternal transcript of Twist1 was first detected in the extraembryonic tissue and embryonic ectodermal cells of the primitive streak (Stoetzel et al., 1995). As the embryo develops, Twist1 is expressed in the head region, trunk and limbs. In the head region, transcripts of Twist1 is found in the vicinity of the neural structures, including the forebrain and area of the nasal placodes, the diencephalon and the optical vesicles, the rhombencephalon and around the otic vesicles. Furthermore, a high level of expression was observed in the branchial arches. In the trunk, Twist1 expression is detected in the sclerotome and somatopleura. In addition, Twist1 expression is also found in the posterior limb buds and tail, the mesenchyme cells forming the internal ear, face, lingua and the skin (Wolf et al., 1991; Fuchtbauer, 1995; Stoetzel et al., 1995). In Xenopus, twist1 is also present as a maternal transcript (Stoetzel et al., 1998). Expression of twist1 is also detected in head, body and tail region. In the head, twist1 transcript accumulates in the internal mesoderm. In the trunk region, expression of twist1 is detected in the notochord, neural crest, lateral mesoderm and somites (Hopwood et al., 1989). 2 Expression of Twist1 has also been described in other species including rat (Bloch-Zupan et al., 2001), chick (Tavares et al., 2001), medaka (Yasutake et al., 2004) and zebrafish (Tavares et al., 2001; Yasutake et al., 2004; Germanguz et al., 2007; Yeo et al., 2007). The functions of Twist1 have been reported in many species. In mouse, Twist1 protein is known to be involved in myogenesis. Mouse Twist1 proteins can interfere with the activity of myogenic transcription factor MyoD (myogenic determination) and MEF2 (myocyte-enhancing factor 2) by preventing the formation of functional MyoD-E proteins heterodimers and inhibiting MEF2-mediated transactivation process (Spicer et al., 1996). In addition, a study done in a metastatic breast cancer mouse model showed that Twist1 is necessary for the onset of metastasis (Yang et al., 2004). Twist1 is also known to participate in transcription regulation. It has been reported that TWIST1 functions as a prometastic oncogene. TWIST1 protein can interact directly with two independent HAT (histone acetyltransferases) domains of p300 and PCAF (p300/CBP-associated factor) acetyltransferases via its N-terminus. The binding of Twist inhibits the acetyltransferase activities of p300 and PCAF, thereby preventing subsequent histone acetylation process that is essential for unwinding the densely packed chromatin to allow the access of transcriptional machinery during transcription process (Hamamori et al., 1999; Massari and Murre, 2000). 3 TWIST1 also plays a role in human osteoblast metabolism. The level of TWIST1 protein can influence osteogenic gene expression and it may act as a master switch in initiating bone cell differentiation by regulating the osteogenic cell lineages (Lee et al., 1999). Twist1 has also been reported to induce epithelial to mesenchymal transition (EMT) by repression of E-cadherin and induction and regulation of N-cadherin (Yang et al., 2004; Alexander et al., 2006). Additionally, overexpression of Twist1 has been described to induce angiogenesis and chromosomal instability (Mironchik et al., 2005). In knockout mice, the Twist1-/- null mice died at embryonic day 11.5, exhibiting a failure of neural tube closure specifically in the cranial region. They also had defects in head mesenchyme, branchial arches, somites, and limb buds, suggesting that Twist1 is involved in regulating the cellular phenotype and behavior of head mesenchymal cells that are essential for the morphogenesis of cranial neural tube (Chen and Behringer, 1995). Further studies show that absence of Twist activity in the cranial mesenchyme region causes improper closure of the cephalic neural tube and this subsequently leads to a malfunction of the branchial arches in Twist1-/- null mice. The authors later found that Twist1 activity is required in both the cranial mesenchyme for directing neural crest cells migration as well as the neural crest cells within the first branchial arch to ensure correct localization of the progenitor cells. Furthermore, Twist1 is also required for the proper differentiation of the first branchial arch tissues into bone, muscle, and teeth (Soo et al., 2002). 4 In medaka twist knockdown morphants, the neural arches were absent. Subsequent experiments performed suggest that twist is involved in the differentiation process of sclerotomal cells into neural arch-forming osteoblasts (Yasutake et al., 2004). 1.1.2 TWIST2 Twist2 (previously known as Dermo1) is another family member that is found in human (Lee et al., 2000), mouse (Li et al., 1995), rat (Maestro et al., 1999), chick (Scaal et al., 2001), medaka (Gitelman, 2007), Fugu (Gitelman, 2007) and zebrafish (NM_001005956). Its expression profile has been described in mouse (Li et al., 1995), chick (Scaal et al., 2001) and zebrafish (Thisse and Thisse, 2004; Germanguz et al., 2007). In mouse, Twist2 is expressed in both the sclerotome and dermatome of the somite, the cranial mesenchymal cells around the nose, pharyngeal arches and tongue, whiskers, somites, limb and branchial arches (Li et al., 1995). In chick, Twist2 is expressed in the somites, head mesenchyme, limbs, branchial arches and mesenchyme of the feather buds (Scaal et al., 2001). Twist 2 is involved in transcriptional regulation and is a transcriptional repressor of p65 (an NF-kB subunit) and myocyte enhancer factor 2 (MEF2) (Gong and Li, 2002; Sosic et al., 2003). A study showed that Twist2 protein bound the E-box consensus sequence in the presence of E12. Furthermore, Twist2 act as a repressor in Myo-D mediated transactivation via its C-terminal and HLH domains and has been suggested to 5 regulate gene expression in a subset of mesenchymal cell lineages including developing dermis (Li et al., 1995; Gong and Li, 2002). Furthermore, Twist2 interacted directly with MEF2 and selectively repressed MEF2 transactivation domain (Gong and Li, 2002; Sosic et al., 2003). Additionally, Twist2 has been identified to be an interacting protein with adipocyte determination and differentiation dependent factor 1 (ADD1)/sterol regulatory element binding protein isoform (SREBP1c). ADD1/SREBP1c is a transcription factor in fatty acid metabolism and insulin dependent gene expression. Overexpression of Twist2 specifically suppresses the transcriptional activity of ADD1/SREBP1c by interfering with ADD1/SREBP1c binding to its target DNA and histone deacetylation (Lee et al., 2003). Twist2 is also suggested to function as an oncoprotein, antagonizing the activation of p53-dependent apoptosis in response to DNA damage (Maestro et al., 1999). It is found that Twist2 is expressed in osteoblastic cells and it possibly act as a negative regulator of the differentiation of osteoblast (Tamura and Noda, 1999). 1.1.3 Twist 3 A third family member Twist3 is absent in mammals but found in Xenopus, chick, medaka, stickleback and zebrafish (Gitelman, 2007). In contrast to Twist1 and Twist2, little is known about the role of Twist3. 6 1.2 Why zebrafish is used as an animal model in this study? The laboratory mouse Mus musculus has become the predominant model organism used to study human development, however, the zebrafish Danio rerio has emerged as a promising complement for embryological, genetic/genomic, cellular/biochemical and other functional studies. The zebrafish was first introduced by George Streisinger as a system for genetic analysis of vertebrate development (Streisinger et al., 1981; Walker and Streisinger, 1983). Its increased use in research is attributed to the many advantages of the zebrafish. Firstly, the zebrafish is small in size (up to 6 cm) and thus can be economically maintained with relative ease in the laboratory compared to mouse and Xenopus. Secondly, it has a short generation time of about 3 months. Thirdly, zebrafish eggs are fertilized externally and each mating can generate approximately 100 eggs. In addition, zebrafish embryos are transparent and develop rapidly. Rudimentary organs such as eyes, ears, brain and heart can be observed one day after fertilization. Moreover, zebrafish form essentially all of the same skeletal and muscle tissue types as their higher vertebrate counterparts, but in much more simple spatial patterns composed of smaller numbers of cells and this is achieved within a short period of time (Schilling, 2002). Furthermore, many of the features that govern craniofacial development in higher vertebrates are conserved and zebrafish contain craniofacial elements similar to those of higher vertebrates (Schilling, 2002; Yelick and Schilling, 2002). 7 1.3 Phylogenetics Phylogenetics is the study of evolutionary history in which the nucleotide characters in DNA or protein sequences are compared among different species. This is based on the assumption that closely related organisms have sequences that are similar and more distantly related organisms have sequences that differs greatly. These sequences are known as homologs and they are believed to be inherited from a common ancestor. Other terminologies are used to classify homologs. Homologs that are produced by speciation are known as orthologs. They represent genes that were derived from a common ancestor that diverged because of divergence of the organism. Orthologs may or may not have the same functions. Homologs that are produced by gene duplication are known as paralogs. They represent genes that were derived from a common ancestral gene that duplicated within an organism and diverged. Paralogs are believed to have different functions (Figure 1.1). Phylogenetics reconstructs the evolutionary relationship between species and allows the estimation of the time of divergence between two organisms since they last shared a common ancestor. 8 Early globin gene GENE DUPLICATION α chain gene Frog α Chick α β chain gene Mouse β Mouse α PARALOG Chick β Frog β PARALOGS ORTHOLOG ORTHOLOG HOMOLOG Figure 1.1: Terminologies used to classify homologs. An example of the globin gene. (Adapted from: http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/Orthology.html) There are generally two classes of phylogenetic trees, namely, the rooted and unrooted tree. A rooted tree has a particular node (root), representing a common ancestor from which a unique path leads to any other nodes. An unrooted tree only specifies the relationship among species, without identifying a common ancestor or evolutionary path. 1.3.1 DNA or protein sequences? Both nucleotide characters in DNA and protein sequences are used for constructing phylogenetic trees, in estimating phylogenetic relationships and times of divergence among taxa. In general, DNA sequences are used for relatively recent events, for example, in closely related species such as human and chimpanzee. This is because the protein sequences between human and chimpanzee are too conserved to be useful 9 (Hedges, 2002). Both the coding and non-coding regions of the DNA sequence can be used. The rate of mutation is assumed to be the same in both coding and non-coding regions; however, there is a difference in the substitution rate. It is important to note that non-coding DNA regions have more substitutions than coding regions. Proteins are much more conserved since they “need” to conserve their function. Hence, protein sequences are more useful for more ancient events – for example, in human and fish – when DNA sequences are usually too divergent to make accurate estimates on the basis of nucleotide substitutions of DNA (Hedges, 2002). However, there is a limitation of using either nucleotide or protein sequences because unequal base or amino acid composition among the genomes of different species is common. In addition, sequence length is a limiting factor, in that the average gene (coding) or protein sequence (~1,000 nucleotides, ~350 amino acids) is usually not long enough to yield a robust phylogeny or time estimate, and therefore many genes and proteins must be used (Hedges, 2002). 1.4 Gene Duplication In 1936, Bridges observed gene duplication in a mutant of the fruit fly Drosophila melanogaster, where the doubling of a chromosomal band results in extreme reduction in eye size (Bridges, 1936; Zhang, 2003). Gene duplication is a key mechanism in evolution. Duplicated genes contribute genetic raw material for the emergence of new functions through natural selection. Lynch and Conery (2000) reported that there are 10 around 15% of genes in the human genome there are found to be duplicated. The average rate of duplication of a eukaryotic gene is estimated to be on the order of 0.01/gene/million years, which is of the same order of magnitude as the mutation rate per nucleotide site (Lynch and Conery, 2003). A number of mechanisms have been described to attribute gene duplication. These are unequal crossing over, retroposition, gene conversion and chromosomal (or genome) duplication (Ohta, 2000; Zhang, 2003; Hurles, 2004). However, depending on the mode of duplication different outcomes are generated. Unequal crossing over usually results in tandem gene duplication in which the duplicated genes are linked in a chromosome. The duplicated region can contain a portion of a gene, the entire gene or several genes, depending on the exact position of crossing over (Zhang, 2003). Retroposition is the integration of reverse transcribed mature RNAs at random sites in a genome. The resultant duplicated genes, known as retrogenes, usually display several molecular features such as lack of introns and regulatory sequences, the presence of poly-A tails and presence of flanking short direct repeats (Zhang, 2003; Hurles, 2004). In addition, a duplicated gene generated by retroposition is usually unlinked to the original gene as the insertion of cDNA process is random (Zhang, 2003; Hurles, 2004). 11 Chromosomal (or genome) duplication occurs probably by a lack of disjunction among daughter chromosomes after DNA replication (Zhang, 2003). Many of these duplicated segments are located in regions that are hot spots of chromosomal and/or evolutionary instability (Samonte and Eichler, 2002). In the TWIST gene family, gene duplication is observed. TWIST1 and TWIST3 genes are found to be duplicated in some species. Duplication of this Twist1 gene (twist1a and twist1b) has been observed specifically in Actinopterygii (ray-fined fishes) (Gitelman, 2007). In stickleback and medaka, there are also two copies of the twist3 gene (Gitelman, 2007). 1.4.1 Evolutionary fates of duplicate genes The consequences of gene duplication play a key mechanism of evolution as it is the survival and fitness of the organism harboring the newly duplicated gene/genome that determine whether either copy of the gene persists or not. Different mechanisms/models have been described to contribute to different evolutionary fates of duplicate genes. The nonfunctionalization model explains how one copy of the duplicate genes is assumed to be redundant and acquires degenerative mutations that eventually eliminate its function (silenced). The non-functional copy is referred to as pseudogene (Woollard, 2005; Sjodin et al., 2007). Pseudogenes can be classified into processed and unprocessed pseudogenes. Processed pseudogenes are generated by the integration of reverse transcription products of processed mRNA transcript into the genome whereas 12 unprocessed pseudogene are generated by the integration of non-processed RNA transcript and thus has retained the original exon-intron structure of the functional gene (Zhang et al., 2008). The neofunctionalization model specifies that one member of a duplicate gene acquires mutation that results in a novel, beneficial function (Conant and Wolfe, 2008). This requires varying numbers of amino acid substitutions. Here, directional selection or by genetic drift can contribute to the duplicate fixation (Conant and Wolfe, 2008). The subfunctionalization model showed that both members of the duplicate gene are stably maintained or persevered by purifying selection and they differ in some aspects of their functions (Conant and Wolfe, 2008). Each daughter gene adopts part of the function of their parental gene. The division of gene expression occurs after duplication. The synfunctionalization model provides a mechanism for gene loss or function shuffle (Figure 1.2). After gene duplication and prior to synfunctionalization, each copy of the duplicate gene is retain in the genome as they possess unique function. In the process of synfunctionalization, one copy of the duplicate gene acquires a unique expression domain of the other and hence, all unique functions of this gene can be found on one copy of the duplicates. Therefore, the other copy of the duplicate gene becomes redundant and leads to gene loss (Figure 1.2) (Gitelman, 2007). If both copy of the duplicate gene are retained due to their remaining unique functions, function shuffling 13 could result whereby a function found in both copies of the duplicate gene may now be redundant in one copy and lost (Figure 1.2) (Gitelman, 2007). Gene X Gene Duplication Gene X1 α Gene X2 β β ε δ synfunctionalization α β α gene loss α β α Gene X1 Figure 1.2: β ε δ Gene X2 β ε δ function shuffling α β α Gene X1 ε δ Gene X2 The model of synfunctionalization: a mechanism for gene loss or function shuffling. Boxes indicate expression domains. Adapted from (Gitelman, 2007). Phylogenetic and gene expression studies are invaluable tools that aid in our understanding of the regulation and function of conserved genes and gene families such as the TWIST gene family. Together, they provide important clues to the evolutionary events and functional changes that have occurred in these genes in different species. 14 1.5 Aims In this study, I aimed to: 1. Determine the complete cDNA sequence, genomic structure, and map location of the zebrafish twist genes 2. Sort out the confusion in evolutionary orthology of the zebrafish twist genes with their mammalian counterparts through comparative gene structure and linkage synteny analyses 3. Characterize and compare the developmental and tissue-specific expression profiles of the zebrafish twist genes during embryogenesis and in adult zebrafish 4. Compare expression patterns in zebrafish with other species. 15 Chapter 2: Materials and Methods _____________________________________________________________ 2.1 Animal stocks and maintenance Singapore wild-type zebrafish were maintained in an aquarium system at 28oC. Embryos were obtained by natural spawning and kept at 28oC and staged according to Kimmel et al. (1995) using standard morphological criteria. Older embryos were treated with 0.003% (2nM) 1-phenyl-2-thiourea to inhibit pigment formation and facilitate whole mount examination. 2.2 Isolation of genomic DNA and total RNA 2.2.1 Isolation of genomic DNA A healthy adult male fish was transferred into a 2-ml tube and frozen in liquid nitrogen. The frozen fish was immediately grinded into powder and homogenized in 10 ml genomic DNA extraction buffer (10 mM Tris pH8.0, 100 mM EDTA pH8, 0.5% sodium dodecyl sulfate (SDS) and 200 μg/ml proteinase K (Sigma)). The mixture was mixed well and incubated at 50oC for 4 hours with gently swirling. After incubation, the mixture was cooled to room temperature and extracted twice with one volume of equilibrated phenol and once with phenol:chloroform:isoamyl alcohol (25:24:1). The mixture was mixed gently until an emulsion has formed and centrifuged at 3000-5000 x g for 10 minutes. The aqueous phase was transferred into a fresh tube and precipitated with 16 200mM sodium chloride (NaCl2) and 2 volumes of ethanol. The DNA was washed with 70% ethanol, air-dried and dissolved in 5 ml of TE buffer (10 mM Tris pH8, 5 mM EDTA) and treated with 100 μg/ml RNAse A (Sigma) at 37oC for 3 hours. DNA solution was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) to remove RNAse A. The DNA was precipitated with 0.1 volume of 7.5 M ammonium chloride and two volumes of ethanol. The DNA was washed twice with 70% ethanol, air-dried, dissolved in TE buffer and stored at -20oC. The concentration of the DNA was measured with a spectra-photometer (SpectraMax M5). 2.2.2 Isolation of total RNA Approximately 50 zebrafish embryos were harvested; chorion was removed and embryos were collected in a 2 ml microfuge tube. Excess water was removed with a pipette. The embryos were frozen in liquid nitrogen and homogenized in 1 ml of TRIZOL® RNA isolation reagent (Invitrogen) with a plastic pestle. The insoluble material was removed by centrifugation at 12,000 x g for 10 minutes at 4oC. The aqueous phase was transferred to a fresh tube and RNA was precipitated with 2 volumes of isopropyl alcohol and centrifuged at 12,000 x g for 10 minute at 4oC. The RNA pellet was washed with 70% ethanol, air-dried and dissolved in 20 μl of 0.1% diethylpyrocarbonate (DEPC, Sigma) treated water. The RNA was stored at -80oC and concentration was measured with a spectra-photometer (SpectaMax M5). 2.3 Full-length cDNA sequence 2.3.1 Rapid amplification of complementary DNA ends (RACE) of zebrafish twist1b (formerly twist1) 17 Based on the then available 624 bp cDNA sequence of zebrafish twist1b (GenBank accession no. NM_130984 (25 April 2005)) (Figure 2.1), primers were designed for both 5’ and 3’ RACE experiments to identify the additional 5’ and 3’ cDNA sequences. The RACE experiments were performed using the SMART™ RACE cDNA amplification kit (BD Biosciences Clontech). Two non-overlapping reverse primers complementary to 5’ region of the incomplete cDNA sequence (Twist1R: 5’- GTC TCT CGT GCG CCA CAT AAC TG-3’ and Twist1Ra: 5’- GCT TCG GTT GTC GCC TGT CGA GC-3’) were designed for nested 5’ RACE experiment. Similarly, two non- overlapping primers complimentary to the 3’ region of the incomplete cDNA sequence (Twist1F: 5’GCC ACG ACC CGC AAT CTG-3’ and Twist1Fa: 5’-GTC CAT GTC AAC ATC TCA CTA ACGC-3’) were designed for the nested 3’ RACE experiment. Other primers that were provided by the kit and used were Universal Primer Mix (UPM), Nested Universal Primer (NUP) and Control TFR primer. The PCR conditions for the RACE experiments were 5 thermal cycles of 94oC for 10 sec and 72oC for 3 min, 5 thermal cycles of 94oC for 10 sec, 70oC for 20 sec and 72oC for 3 min followed by 5 thermal cycles of 94oC for 10 sec, 68oC for 20 sec and 72oC for 3 min. Amplified products were analyzed by agarose gel electrophoresis (Figure 2.2). Sequencing was carried out using BigDye® Terminator v3.1 cycle sequencing kit (on an ABI 3100 Genetic Analyzer (Applied Biosystems)). 18 NM_130984 (25-Apr-2005) 1 accgagcctc tgaccccatt ccgtcggact catttttgcc acgacccgca atctgagctt Twist1F 61 ttccagaggt gatgtttgag gaagaggcga tgcacgagga ctccagctct ccagagtctc 121 cggtggacag tctgggaaac agcgaggagg agctcgacag gcgacaaccg aagcgcgtca Twist1Ra 181 241 301 361 421 481 gcaggaaaaa agaggagcaa cgcagcgcgt tcgcctccct cgctcaaact tggactccaa acgcgccagc gaagtgcagc catggcgaac gcgcaaaatc cgcggcccgg gatgtccagc cgcaaaaacg aacagcagca gtgcgcgagc atccccacct tacattgact tgcagttatg ccgaggattc gcagcccgca gtcagaggac taccctcgga tcctctgtca tggcgcacga cgacagtccc atctctggag tcagtctctg caaactcagc ggtcctgcag gagactcagc acgcccggga gacctgcaga aacgaggcgt aaaatacaga agcgatgagc tacgcgtttt Twist1R 541 ctgtgtggag aatggagggc gcgtggtcca tgtcaacatc tcactaacgc acggatgcac Twist1Fa 601 gcgttgatgc agcatggtat gcga Figure 2.1: The incomplete cDNA sequence of zebrafish twist1b gene. GenBank accession number NM_130984 (25-Apr-2005). Primers used for RACE experiments are indicated by arrows. Start (ATG) and stop (TAA) codons are underlined. A 300bp Figure 2.2: M1 1 2 3 4 5 B 1 2 3 4 5 M2 500bp Agarose gel electrophoresis of 5’ and 3’ RACE experiment. (A) 5’RACE and (B) 3’RACE. M1 and M2 are 50bp and DNA ladder mix from 19 Fermentas respectively. The bands were amplified with Twist1Ra (A)/Twist1Fa (B) and NUP (lane 1), TFR and NUP (positive control) (lane 2), UPM primer only (lane 3), Twist1Ra (A)/Twist1Fa (B) only (lane 4). PCR blank is added as a negative control (lane 5). 2.3.2 Assembly of zebrafish twist1b full-length cDNA 5’UTR sequences obtained from 5’RACE is as follows: gagaaagccc tttcgtcgga aggaagaggc acagcgagga tccgtgacgc ctcagggaag gatgcacgag ggagctcgac aggaggagac ccacgacccg gactccagct aggcgacaac gcgctgagag ggaccgagcc tctgacccca caatctgagc ttttccagag gtgatgtttg ctccagagtc tccggtggac agtctgggaa cgaagc Twist1Ra Figure 2.3: 5’ UTR sequences of the zebrafish twist1b gene obtained from 5’RACE experiment. Arrow indicates the position of primer. Start (ATG) codon is underlined. 3’UTR sequences obtained from 3’RACE is as follows: gtccatgtca acatctcact aacgcacgga tgcacgcgtt tgatgcagca tgattctcgg Twist1Fa cctgaggagc aaagggaaaa catgggagca tgcttccgtc caacaaccag gaaggaaatc tatttattta attttaaata aaaa Figure 2.4: tgaactcact ttctggagcg ggaattacag cacaaaacag catggcgtca ggctcatatc ttgatgattg tgatgtaaat ggaaggagcg tcatgacgtc tcagatctgt agactcgctg tatttttttc agtgttaaca tcacaatgca atgtgtatat gctcaaaaca gttgcaagca gctgttgcga caggaaaaga tctgaaggaa ttttctttga gaatagatct tttctgcaat agggcgaaaa cttacagttg cggtgaatgt cgctcctgcg aacacacaca tcggtccaag ggtgtctaca aaaacatgat taaggattat tgaactacga ggaaaacatg cttctgacag ctcaacgaat aaaatacttt tgcattttct ttgaaataca 3’ UTR sequences of the zebrafish twist1b gene obtained from 3’RACE experiment. Arrow indicates the position of primer. Stop (TAA) codon is underlined. The complete sequence of the full-length cDNA of zebrafish twist1b was obtained and has been deposited in GenBank under accession no. DQ351987 (Figure 2.5). The 20 open reading frame (ORF) was translated using an online software (http://www.tw.expasy.org/). DQ351987: 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 gagaaagccc tttcgtcgga aggaagaggc acagcgagga gccgcaaaaa gcaacagcag acgtgcgcga tcatccccac ggtacattga gctgcagtta gcgcgtggtc ttctcggcct ggattataaa actacgacat aaacatgtgc ctgacagcaa aacgaatgaa atacttttat attttctatt aaatacaaaa Figure 2.5: 2.4 tccgtgacgc ctcagggaag gatgcacgag ggagctcgac cgccgaggat cagcagcccg gcgtcagagg cttaccctcg cttcctctgt tgtggcgcac catgtcaaca gaggagctga gggaaaattc gggagcagga ttccgtccac caaccagcat ggaaatcggc ttatttattg ttaaatatga a aggaggagac ccacgacccg gactccagct aggcgacaac tccgacagtc caatctctgg actcagtctc gacaaactca caggtcctgc gagagactca tctcactaac actcactgga tggagcgtca attacagtca aaaacagaga ggcgtcatat tcatatcagt atgattgtca tgtaaatatg gcgctgagag caatctgagc ctccagagtc cgaagcgcgt ccacgcccgg aggacctgca tgaacgaggc gcaaaataca agagcgatga gctacgcgtt gcacggatgc aggagcggct tgacgtcgtt gatctgtgct ctcgctgcag ttttttctct gttaacattt caatgcagaa tgtatatttt ggaccgagcc ttttccagag tccggtggac cagcaggaaa gaagaggagc gacgcagcgc gttcgcctcc gacgctcaaa gctggactcc ttctgtgtgg acgcgtttga caaaacaagg gcaagcactt gttgcgacgg gaaaagacgc gaaggaaaac tctttgatcg tagatctggt ctgcaataaa tctgacccca gtgatgtttg agtctgggaa aaacgcgcca aagaagtgca gtcatggcga ctgcgcaaaa ctcgcggccc aagatgtcca agaatggagg tgcagcatga gcgaaaataa acagttgtga tgaatgtgga tcctgcgctt acacacactc gtccaagaaa gtctacatgc acatgatttg The complete full-length cDNA sequence of the zebrafish twist1b gene. Start (ATG) and stop (TAA) codons are underlined. Polyadenylation signal is double-underlined. Genomic sequence of zebrafish twist1b An intron within the zebrafish twist1b gene was determined by polymerase chain reaction (PCR) amplification of genomic DNA using the gene-specific primer pair 5′ GTTTGATTCTTGGTATAACG-3′ and 5′-GATCTATTCTGCATTGTGAC- 3′. The PCR reaction mixture contained 100 ng of genomic DNA template, 200 μM of each deoxyribonucleotide triphosphate (dNTP), 0.2 μM of each primer, and 1 U of HotStarTaq™ DNA polymerase (Qiagen) in 1X supplied PCR buffer containing 1.5 mM 21 MgCl2. The amplification reaction consisted of a 15-min polymerase activation at 95°C, followed by 30 thermal cycles of 95°C denaturation for 45 sec, 50°C annealing for 1 min, and 72°C extension for 5 min. A final 5-min extension at 72°C completed the reaction. The amplified products were analyzed by gel electrophoresis. The amplified fragment was excised from the gel and extracted using GFXTM PCR, DNA and Gel Band Purification Kit (GE Healthcare). The extracted fragment was sequenced using BigDye® Terminator v3.1 cycle sequencing kit on an ABI 3100 Genetic Analyzer (Applied Biosystems). The genomic sequence of zebrafish twist1b has been deposited in GenBank under accession no. DQ191169. 2.5 RT-PCR Total RNA was extracted from embryos at various stages using TRIZOL® RNA isolation reagent (Invitrogen): 1-cell (0.2 hour post fertilization (hpf)), 8-cell (1.25 hpf), 64-cell (2 hpf), 1000-cell (3 hpf), shield (6 hpf), bud (10 hpf), 14-somite (14 hpf), 1 day post fertilization (dpf), 2 dpf and 3 dpf. Intron-spanning gene-specific primer pairs for twist1a, twist1b, twist2 and twist3 transcript detection were 5’- AGGTTCTACAGAGTGACGAGC-3’ / 5’-GCACAGGATTCGAACTAGAGG-3’, 5’TGTGGCGCACGAGAGACT-3’ / 5’-GATCTATTCTGCATTGTGAC-3’, 5’- CGCACGAGAGACTCAGTTAC-3’ / 5’-CCATACAGATAGCAGATAGCC-3’, and 5’CTGAATCCCGAACTCTGATCC-3’ / 5’-GTGTTACCCGTCACTGAAG-3’, respectively. As a control for equivalent starting amounts of RNA template, β-actin transcript expression was detected using the primer pair CGCACTGGTTGTTGACAACG-3’ / 5’-AGGATCTTCATGAGGTAGTC-3’. 5’Total 22 RNA was reverse transcribed using SuperScript™ III reverse transcriptase (Invitrogen), followed by PCR. The PCR reaction mixture contained 1 μl of cDNA as template, 200 μM of each dNTP, 0.2 μM of primer, and 1 U of HotStarTaq™ DNA polymerase (Qiagen) in 1X supplied PCR buffer containing 1.5 mM MgCl2. The amplification reaction consisted of a 15-min polymerase activation at 95°C, followed by 35 thermal cycles of 95°C denaturation for 45 sec, 60°C annealing for 1 min, and 72°C extension for 5 min. A final 5-min extension at 72°C completed the reaction. 2.6 Synthesis of RNA probes for in situ hybridization analysis 2.6.1 Identification of unique 3’UTR sequences of the zebrafish twist gene family Alignment of zebrafish twist gene family full-length cDNA was carried out using Vector NTI® Suite 7.0 to identify unique sequences used as probes for in situ hybridization. 2.6.2 Isolation of unique 3’UTR sequences of zebrafish twist gene family Zebrafish twist1a, twist1b, twist2 and twist3 3’UTR were amplified from reverse- transcribed RNA of 24 hpf GAAAACACGAGGACCAATG-3’ embryos / CTGAACTCACTGGAAGGAGC-3’ / using the primer pairs 5’- 5’-GAATTGTACTAAAGCTTTGTA-3’, 5’- 5’-GATCTATTCTGCATTGTGAC-3’, 5’- GAACGGACTGTTTACTTCCAC-3’ / 5’-CCATACAGATAGCAGATAGCC-3’ and 5’-CTGAATCCCGAACTCTGATCC-3’ / 5’-CGACATCTCATCCTATTAGCG- 3’respectively (Figure 2.6 to Figure 2.9) 23 EF620930 (cDNA sequence of twist1a): 1 61 121 181 241 301 361 421 481 541 601 661 721 ctcctctcaa cacaaagttg tgtatttcac aagcgagatg cagtctcagc acggtcgagc aaagtctagc gatggcgaac acgcaaaatc cgccgccagg gatggcaagt gatggagggc tttgttttta acactttacc cttggaatac cctcatgctg cccgaagagc aacagcgacg aagaaaaacg aacagcagca gtgcgcgagc atccccactt tacatcgatt tgtagttatg gcttggtcca atggtcaacc agactataag ctagtgatct gaataacgtc ccgcgcgaga gagagcccga gggaggattc acagccctca gacagaggac taccttccga ttctctgtca ttgctcacga tgtctgcatc cgtgagctgg agctccctaa tctccagaac cttattcgca ctcctccagc caggccacca cgatagctcg gtctttcgag gcagtcgctc taaactgagc ggttctacag gcgtttgagc tcactagtgt gaaaacacga cttttttcct acgaaacgta cgcgctttca tcccccgtgt aaaaggtgcg acccttggga gagctgcaga aacgaagcgt aaaatacaga agtgacgagc tacgcgttct gcagggaaac ggaccaatgc ttcaacctaa cgcgtggaat gcagagactt ctcccgcgga caaggaaaag aaagggggaa cgcagcgcgt ttgcggcttt cgctgaaact tggactccaa cggtttggag tttttcttgt taattccatc Ztwist1a-F 781 841 901 961 1021 ataatcttgg ccacggacag aatatgtgca taactttctg gaagcctcta ggaaaacggc cgaattgtca tatgcatgtt gaagaaagtg gttcgaatcc aaatgttcca tatggatttc tttttttttt aatttgcatt tgtgcaaata acagaggtca ctcccgagtc tttttttttt acaaggactg caaagcttta tggctgttac ttatgacgac ttggaagact tcgataagaa gtacaattct cgagagaagg gaatgttgga cagatgtgca aatgggaatt atttatttat Ztwist1a-R 1081 tgatgacaca ctttttgaaa tgaaagtaaa tgtatcaaat gtgttgaaat gcattattat 1141 tttttattac ttttgtaaat aaatgtgtat ttctgtaata aaaaaatgaa gaattttaag 1201 aaata Figure 2.6: cDNA sequence of zebrafish twist1a. Positions of primers are indicated by arrows and the 3’UTR region used for the synthesis of RNA probe for in situ hybridization is highlighted in grey. Start (ATG) and stop (TAG) codons are underlined. Polyadenylation signal is double-underlined. 24 DQ351987 (cDNA sequence of twist1b): 1 61 121 181 241 301 361 421 481 541 601 661 gagaaagccc tttcgtcgga aggaagaggc acagcgagga gccgcaaaaa gcaacagcag acgtgcgcga tcatccccac ggtacattga gctgcagtta gcgcgtggtc ttctcggcct tccgtgacgc ctcagggaag gatgcacgag ggagctcgac cgccgaggat cagcagcccg gcgtcagagg cttaccctcg cttcctctgt tgtggcgcac catgtcaaca gaggagctga aggaggagac ccacgacccg gactccagct aggcgacaac tccgacagtc caatctctgg actcagtctc gacaaactca caggtcctgc gagagactca tctcactaac actcactgga gcgctgagag caatctgagc ctccagagtc cgaagcgcgt ccacgcccgg aggacctgca tgaacgaggc gcaaaataca agagcgatga gctacgcgtt gcacggatgc aggagcggct ggaccgagcc ttttccagag tccggtggac cagcaggaaa gaagaggagc gacgcagcgc gttcgcctcc gacgctcaaa gctggactcc ttctgtgtgg acgcgtttga caaaacaagg tctgacccca gtgatgtttg agtctgggaa aaacgcgcca aagaagtgca gtcatggcga ctgcgcaaaa ctcgcggccc aagatgtcca agaatggagg tgcagcatga gcgaaaataa tgacgtcgtt gatctgtgct ctcgctgcag ttttttctct gttaacattt caatgcagaa gcaagcactt gttgcgacgg gaaaagacgc gaaggaaaac tctttgatcg tagatctggt acagttgtga tgaatgtgga tcctgcgctt acacacactc gtccaagaaa gtctacatgc Ztwist1-F 721 781 841 901 961 1021 ggattataaa actacgacat aaacatgtgc ctgacagcaa aacgaatgaa atacttttat gggaaaattc gggagcagga ttccgtccac caaccagcat ggaaatcggc ttatttattg tggagcgtca attacagtca aaaacagaga ggcgtcatat tcatatcagt atgattgtca Ztwist1-F 1081 attttctatt ttaaatatga tgtaaatatg tgtatatttt ctgcaataaa acatgatttg 1141 aaatacaaaa a Figure 2.7: cDNA sequence of zebrafish twist1b. Positions of primers are indicated by arrows and the 3’UTR region used for the synthesis of RNA probe for in situ hybridization is highlighted in grey. Start (ATG) and stop (TAA) codons are underlined. Polyadenylation signal is double-underlined. 25 NM_001005956 (cDNA sequence of twist2): 1 61 121 181 241 301 361 421 481 541 601 661 aaaaaaaatt agtaggggac acggatatta ctttaaaaaa gaccagcgag aaggaagtcg gagcccgagc acgcgagcgg ccccacgctc cattgatttc cagctacgtc gtggtcgatg aggaaaaaac gcgcgcagtg ctagtctaaa gaaatggaag gaggagctgg agcgaggaca agcactcagt caacggactc ccctcggata ctctgtcagg gcgcacgaga tctgcgtccc ttcctgcggg tcaggacttc ccacggaacc agagttctag acagacagca gcagcagccc ccttcgagga aatcgctgaa aactcagcaa tgctgcagag gactcagtta actagcagcg aggaaaaccg aggaggacag aaaagtggag ctctcccgtc gaaaaggttc gagctccgtc gctccagaac cgaagccttc gatccagacg cgacgagatg cgcgttttca agacgcgtcc tgccaagctc acaaaatccc atcgtatttt tccccagtgg gggaggaaaa aataaacgta cagcgcgtcc gcgtctttgc ctcaaactcg gacaacaaga gtgtggagga tgataatgcc tacttctgga caacaacaaa ctcctcgttt acagcctggt ggaggcaagg acaaaaagcc tggcgaacgt gcaaaatcat catccaggta tgtccagttg tggagggcgc gaacggactg 721 tttacttcca ctaattttga ggatgccaaa ggattattcg atgaacctct aaacctcagt Ztwist2-F 781 841 901 961 gacgtggcca acaataaaag caaatacatt aaggctatct aaggacattc ggacttttta taatcttttt gctatctgta agtggataca atatacttag cacaagttat tggaaactat tctacggact aggaaaagta tcttaaatgt atttcatgtc tgagcatcct gatgacgtct cgccatgatg tgtctctaaa catgaaggac tctgtgtcgt atagtacagg cagaagagtt Ztwist2-R 1021 tatatatata taagaacaaa aaaaaaaaaa aaaa Figure 2.8: cDNA sequence of zebrafish twist2. Positions of primers are indicated by arrows and the 3’UTR region used for the synthesis of RNA probe for in situ hybridization is highlighted in grey. Start (ATG) and stop (TAG) codons are underlined. 26 NM_130985 (cDNA sequence of twist3) 1 61 121 181 241 301 361 421 481 541 601 661 721 781 ggggacaaac attgcatttg ccacagagct acacggagga gatccttccc tccaccggcg cgacaaacca tccgtcatcc agttcccgga acgccagcgc cttgccttcg cttcctctac tctggcccac catgtccgcc agccacatga atttctaaca cgcttcagtg ctgatcgtca attgaagaag ggcgcacgca tccctggaca tcatcgtctt cagcccttcg acacagtccc gacaagctga caggtcctgc gagaggctga actcactaaa cacacacaca acaaacccaa tttaactttg tgcgagagga agcaggagcg agcggctgac acccatcgaa cttcgtcctc aagacctcca tgaacgacgc gcaagatcca agagcgacga gctacgcctt catccacccg catacacaca agcaatggct acaccgctca acagacttgt gcgccccaat gggtcccaaa tctggctccc tctggtgccc cacgcaacga cttcgcctcc gatcctcaaa gatggacgcc ctccgtctgg ttcctcatct cacacacaca gtataactct caggtacagc ggagattttc aagtgtgcgg aaagagcccg aaacgtccca gtcgtgagtt gtgatcgcca ctcagaaaga ctggcctcca aagctggcca aggatggagg caaaaactga cactgcagag cacagagatc aggagcactg ctgaaagtgg ttgtggtttc tctcacaaga aaagaagctc cggtttctcc acgttcggga tcatccccac gatacatcga gctgcaacta gcgcctggtc atcccgaact 841 ctgatcctga ttttcatctc tatatccact ccacacggcc tccgactcac cctgccatga Ztwist3-F 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 ccccaccctg gtttcccaca cctaatggag agagcatttc tcagatcagt tgttctgagg gcttcattat attcctgctt taatgcttca gtggctttaa aagacattta atagcagggt ctgtttgagt cctaaagatg accccagagg ctttaccagc cacaagactg gttcctccat actcactaac gcagatgtga cagtgacggg agattcactc tgggggaatg gaaagatgct tcatgtgagt acatctttac acctctgacc acttcatgct aaacgcagga aatccagcac gttgactgtt cctgacctga gagtgattgt taacactgtg tattcctcta aggagctgta ctgcttcagg ggacttggtg agcagatcca tctctcactt tcatctacaa gagaaagagt tgagctgagt ctctggactc tggatcaagg gcttgtgttt taaaccgcca ttaaaagaga catcaataat attcagtcaa attataaata ggcagcggtc ttccaaatca gccgacagtt cttgaggagc ggaagtgact atgtttgggc cttcctccgc ctgctgtata gttcatgtaa ctttaactgc gatgtgtcgg acgtctgcaa tttctacaat gctaatagga agccttcgca ttcccctttc gcagatgagc gctgctgatc agatattgac tctttatatt ttagccattc aataatcaaa agaagtgtta agtacagagg acgtataaag aatgaagaag tgagatgtcg Ztwist3-R 1681 1741 1801 1861 gctaaacttg actcgatgag tcaataaaca aaaaaaaaaa Figure 2.9: gtgttggtga tagctaacag aacaataaga aaaaaaaaaa tggtgtgaaa actaagagtt tgaaaaaaaa aattaaaaaa ataaactgaa atgaagcgta cagcaggaga tacgctgatt atcaagagct tgaaaattca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa cDNA sequence of zebrafish twist3. Positions of primers are indicated by arrows and the 3’UTR region used for the synthesis of RNA probe for in situ hybridization is highlighted in grey. Start (ATG) and stop (TAA) codons are underlined. Polyadenylation signal is double-underlined. 27 The amplified products were analyzed by gel electrophoresis. Each amplified fragment was excised from the gel and extracted using GFXTM PCR, DNA and Gel Band Purification Kit (GE Healthcare). Amplified fragments of twist1b and twist3 were cloned into a pBlueScript®II KS(+) vector (Stratagene) and amplified fragments of twist1a and twist2 were cloned into pCR®2.1-TOPO vector (Invitrogen). The inserts region of the plasmid DNA were sequenced using BigDye® Terminator v3.1 cycle sequencing kit on an ABI 3100 Genetic Analyzer (Applied Biosystems) to confirm the clones. 2.6.3 Linearization of plasmid DNA For generation of antisense RNA probes, 3’UTR-twist1a and twist2 plasmid DNA were digested with EcoRV restriction enzyme; 3’UTR-twist1b and twist3 plasmid DNA were digested with HindIII restriction enzyme. For generation of sense RNA probes, 3’UTR-twist1a and twist2 plasmid DNA were digested with XbaI restriction enzyme; 3’UTR-twist1b and twist3 plasmid DNA were digested with BamHI restriction enzyme. The linearized products were analyzed by gel electrophoresis. RNase free reagents, tips and tubes are used subsequently. The linearized plasmids were purified once with phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged at 13,000 rpm for 5 mins and residual phenol was removed by chloroform:isoamyl alcohol(24:1) extraction. The aqueous phase was transferred to a fresh tube and precipitated with 0.1 volume of 3M sodium acetate (pH5.2), 2.5 volumes of cold absolute ethanol and 20 μg of glycogen (Invitrogen) and incubated at -20oC for 20 min. The solution was centrifuged at 13,000 rpm for 20 min. The supernatant was removed and DNA pellet was washed with 250 μl 28 of cold 70% ethanol and centrifuged at 13,000 rpm for 10 min. The supernatant was removed, pellet was air-dried and dissolved in 0.1% DEPC water. 2.6.4 RNA labeling with Digoxenin/Fluorescein RNA labeling kits (SP6/T7/T3)(Roche) One μg of linearized plasmid was transferred to a 200 μl PCR tube. The labeling reaction contained 2 μl of 10X NTP labeling mixture (Digoxenin or Fluorescein), 2 μl of 10X transcription buffer, 1μl of RNase inhibitor and 2 μl of RNA polymerase. DEPC treated water was used to top up the reaction to final volume of 20 μl. The mixture was mixed and incubated at 37oC for 2 hours. Plasmid DNA was degraded with 2 μl of DNase I (Roche) at 37oC for 1 hour. Antisense twist1b and twist3 RNA probes were synthesized using T7 RNA polymerase, while negative control sense twist1b and twist3 RNA probes were synthesized using T3 RNA polymerase. Antisense twist1a and twist2 RNA probes were synthesized using SP6 RNA polymerase and negative control sense twist1a and twist2 RNA probes were synthesized using T7 polymerase. 2.6.5 Purification of RNA probe The RNA probes were precipitated with 0.5 volume of 10M ammonium acetate, 3 volumes of cold absolute ethanol and 20 μg of glycogen (Invitrogen) at -80oC for 30 min or longer. The mixture was centrifuged at 13,000 rpm at 4oC for 30 min. The pellet was washed with 300 μl of cold 70% ethanol, air-dried and dissolved in 40-75 μl of 0.1% DEPC water. The RNA probes were further purified through the Chroma SpinTM-100 29 columns (Clontech). The RNA probes were kept at -80oC and the quality of the RNA probes were checked by gel electrophoresis. 2.7 Whole mount in situ hybridization 4 g paraformaldehyde (Sigma) was added into 100 ml of 1X PBST (1/10 dilution from 10X PBS (1st Base) and 0.1% Tween 20) to achieve final concentration of 4%. The fixative was incubated at 70oC until it is completely dissolved. Embryos younger than 24-somite stage were fixed with 4% paraformaldehyde and incubated overnight at room temperature prior to dechorionation. Embryos older than 24-somite stage were dechorionated prior to fixation. After fixation, the embryos were washed twice with 1X PBST to remove the fixative and dehydrated by three washes in absolute methanol for 5 min each. The dehydrated embryos were stored in absolute methanol at -20oC until next use. The embryos were rehydrated in 75% methanol/25% 1X PBST, followed by 50% methanol/50% 1X PBST, 25% methanol/75% 1X PBST and four washes with 100% 1X PBST, 5 min each. Proteinase K was used to digest protein on the embryos aiding in the penetration of RNA probes into tissues. Embryos at and older than 18-somite stage were treated with 5 or 20 μg/ml proteinase K prepared in 1X PBST at room temperature. The incubation time with proteinase K varied with the stage of the embryos. Embryos at 18-somite stage were treated with 5μg/ml proteinase K for 5 min; embryos at 24-hpf stage were treated 30 with 5 μg/ml proteinase K for 12 min; embryos at 48-hpf stage were treated with 5 μg/ml proteinase K for 25 min; embryos at 72-hpf were treated with 20 μg/ml proteinase K for 20 min. Proteinase K activity was inactivated by 20 min wash in 4% paraformaldehyde. The embryos were then washed in 1X PBST for five times, 5 min each. Prehybridization was performed by immersing the embryos in hybridization buffer (50-60% formamide (Merck), 5X SSC (150mM NaCl and 15mM sodium citrate), 500 μg/ml yeast RNA (sigma), 50μg/ml heparin (sigma) and 0.1% Tween 20, adjusted to pH 7.0 with 1M citric acid) for 4 hours at 55-68oC. The prehybridized embryos were transferred into fresh hybridization buffer containing 500 ng/ml labeled RNA probe. The embryos were incubated overnight at the same temperature used for prehybridization with gently shaking. The formamide concentration in the hybridization buffer and the hybridization temperature were used at the optimized condition. After hybridization, the embryos were washed at the hybridization temperature with 50% or 60% formamide/2X SSC, 37.5 or 45% formamide/2X SSC, 25% or 30% formamide/2X SSC, 12.5 or 15% formamide/2X SSC for 10 min each, followed by two washes in 0.2X SSC/0.1% Tween 20 for 30 min each. Embryos were then washed at room temperature with 0.15X SSC/1X PBST, 0.1X SSC/1X PBST and 0.05X SSC/ 1X PBST for 10 min each. The hybridized embryos were blocked in blocking buffer prepared with 2% Boehringer Blocking Reagent (Roche) and 150 mM NaCl in DEPC water for 2 hours. 31 After blocking, the embryos were incubated overnight at 4oC in 1:4000 dilution of antiDigoxenin (Roche) or 1:2500 dilution of anti-Fluorescein Fab (Roche) in blocking buffer. The embryos were washed four times with maleic acid buffer (150 mM maleic acid (Sigma) and 100 mM NaCl) for 20 min each. The embryos were washed three times in alkaline phosphatase buffer (100 mM Tris-HCl at pH9.5 (detection of anti-Dig AP Fab fragments) or pH8.2 (detection of antiFluorescein-AP Fab fragments), 50 mM MgCl2, 100 mM NaCl and 0.1% Tween 20) for 5 min each. The embryos were the incubated in the dark in the substrate solution for color signal development. There were three types of substrates used in this study: 4-Nitroblue tetrazolium chloride/ 5-Bromo-4-chloro-3-indolylphosphate (NBT/BCIP) and BM-Purple substrate (Roche) were used for the detection of anti-Dig-AP Fab fragments. The staining buffer for NBT/BCIP was prepared with 4.5 μl of NBT (100 mg/ml in dimethylformamide (Roche) and 3.5 μl of BCIP (50mg/ml in dimethylformamide (Roche) in 1 ml of alkaline phosphatase buffer. The staining buffer was replaced with freshlymade substrate solution upon turning purple. As for BM-purple substrate, it was added directly to stain the embryos. The other substrate, Fast Red (Roche) was used to detect anti-Fluorescein-AP Fab fragments. One tablet of Fast-red was dissolved in 4 ml of alkaline phosphatase buffer (100 mM Tris-HCl at pH8.2, 50 mM MgCl2, 100 mM NaCl and 0.1% Tween 20). The solution was centrifuged to remove the particles. In another tube, 40 μl of NAMP stock (Naphthol AS-MX phosphate (Sigma) prepared in dimethyl sulfoxide (50 ng/ml) was added into 4ml of alkaline phosphatase (pH8.2). The Fast Red staining solution was prepared by mixing the 4ml of Fast red solution and 4ml of alkaline 32 phosphatase with 40 μl of NAMP (50 mg/ml). The embryos were stained for not more than 48 hours in the dark at room temperature until the desired signal was developed. For double color in situ hybridization, both Dig-labeled and Fluorescein-labeled RNA probes were added into the hybridization buffer after the embryos were prehybridized. After blocking, the embryos were incubated with one antibody, either anti-Dig Fab fragments or anti-Fluorescein Fab fragments. After the first color detection was completed, the staining was stopped by washing the embryos three times in 1X PBST, 5 min each. The embryos were incubated in 0.1M Glycine (pH2.2 (Sigma)) for 1 hour to inactivate the first antibody. The embryos were washed three times in maleic acid buffer for 5 min each. After which, the embryos were blocked with blocking agent, followed by incubation with the second antibody. The embryos were washed and stained as mentioned above. When the color development was completed, the staining was terminated by washing the embryos thrice in 1X PBST for 5 min each and the embryos were re-fixed in 4% paraformaldehyde for half an hour and stored in 30% glycerol/1X PBST at 4oC. The table below describes the restriction enzymes and RNA polymerases used to generate RNA probes and in situ hybridization conditions used in this study. 33 Table 1: The synthesis of RNA probes and in situ hybridization conditions Gene name Restriction enzyme for RNA polymerase In situ hybridization linearization of plasmid conditions EcoRV (antisense) SP6 (antisense) 50% formamide, 60oC twist1a XbaI (sense) T7 (sense) HindIII (antisense) T7 (antisense) 50% formamide, 68oC twist1b BamHI (sense) T3 (sense) EcoRV (antisense) SP6 (antisense) 50% formamide, 60oC twist2 XbaI (sense) T7 (sense) EcoRV (antisense) T7 (antisense) 60% formamide, 68oC twist3 XbaI (sense) T3 (sense) SacII (Cfr42I) T7 50% formamide, 68oC pax2.1 BamHI T7 50% formamide, 68oC dlx2a NotI T7 50% formamide, 68oC wt1 XbaI T7 50% formamide, 60oC fli1a 2.8 Cryosection Zebrafish embryos were fixed in 4% paraformaldehyde/1X PBS for half an hour and washed three times in 1X PBS. Agar-sucrose solution (1.5% Bacteriological agar (Sigma) in 5% sucrose) was prepared and cooled to 40-50oC. An embryo was transferred with a drop of 1X PBS to a cap of 1.5 ml microfuge tube (as a mold for making agar block). Excess fluid was removed and agar-sucrose solution was added to cover the embryo. A syringe needle was used to manipulate the embryos to ensure even coating and correct position. The agar block was cooled to solidify. The agar blocked was removed, trimmed into a “pyramid” shape and immersed into 30% sucrose solution at 4oC overnight. A raised platform was made with a layer of tissue freezing medium to the cryostat chuck and left at -20oC for 1hr. A small drop of tissue freezing medium was added to the raised platform. The agar block was positioned onto the raised platform of the cryostat chuck and lowered into liquid nitrogen surface. The block/chuck was 34 equilibrated in the cryostat machine prior to sectioning for 30 min or longer. The agar block was cryosection at 10-15 μm thickness using a cryostat machine (Microtome cryostat HM525 (Microm International)). The sections were overlaid onto poly-lysine coated slides (Sigma) and heated at 42oC for 30 mins on a hot plate. The slides were left to air-dry overnight. The section slides were washed three times with 1X PBS, 5 min each. The sections were mounted in 50% glycerol/1X PBS solution by a large coverslip with nail polish. 2.9 Image processing All images were digitally captured using the MicroPublisher™ 5.0 color digital camera (QImaging™) and processed using Image-Pro® Plus version 5 software (Media Cybernetics). 2.10 Phylogenetic analysis 2.10.1 Alignment and phylogenetic tree Multiple sequence alignments of Twist protein/ Twist cDNA sequences were generated using Vector NTI® Suite 7.0. Inter-species sequence comparisons and similarity calculations were performed using the AlignX software in Vector NTI® Suite 7.0. Phylogenetic tree was generated by the Neighbor-Joining method (Saitou and Nei, 1987) using PHYLIP software, with default gap opening penalty of 10 and gap extension 35 penalty of 0.2, and with bootstrap analysis of 1000 replicates. Gaps that were present in the alignment were removed before phylogenetic tree generation. Phylogenetic analysis was performed with four zebrafish Twist peptides and 29 other Twist peptides from 11 species, based on GenBank cDNA sequences NM_000474 (human TWIST1), NM_057179 (human TWIST2), NM_053530 (rat Twist1), NM_021691 (rat Twist2), NM_011658 (mouse Twist1), NM_007855 (mouse Twist2), NM_204739 (chick Twist1), NM_204679 (chick Twist2), BK006265 (chick Twist3), NM_204084 (frog twist1), NM_001103209 (frog twist3), BK006281 (spotted green pufferfish twist1a), BK006282 (spotted green pufferfish twist1b), BK006283 (spotted green pufferfish twist2), NM_001104599 (fugu twist1a), NM_001104598 (fugu twist1b), NM_001104600 (fugu twist2), BK006268 (medaka twist1a), BK006269 (medaka twist1b), BK006270 (medaka twist2), BK006271 (medaka twist3a), BK006272 (medaka twist3b), BK006276 (stickleback twist1a), BK006277 (stickleback twist1b), BK006278 (stickleback twist2), BK006279 (stickleback twist3a), BK006280 (stickleback twist3b), EF620930 (zebrafish twist1a), NM_130984 (zebrafish twist1b), NM_001005956 (zebrafish twist2), NM_130985 (zebrafish twist3), AF097914 (Japanese lancelet twist), and ABW34714 (sea urchin twist). 2.10.2 Calculation of genetic distances Coding cDNA sequences of zebrafish, medaka, stickleback and fugu twist1a and twist1b used were taken from GenBank and the accession numbers were as above. Kimura 2-parameter nucleotide distances and Tajima-Nei corrected number of 36 substitutions per site at third codon positions were computed using MEGA 4.0 software (Tamura et al., 2007). 2.10.3 Comparative synteny analysis The map locations of the orthologous genes in human and zebrafish were obtained from NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/projects/mapview/), Human genome view (Build 36.3, March 2008) and Zebrafish genome view (Zv 7, July 2008) respectively. The map locations of Medaka twist1a (BK006268), twist1b (BK006269), twist2 (BK006270), twist3a (BK006271) and twist3b (BK006272) were determined using the search engine on the Ensembl Medaka Blast View (http://www.ensembl.org/Oryzias_latipes/blastview ). Other orthologous gene locations in Medaka were obtained from the Ensembl Oryzias latipes genome website (http://www.ensembl.org/Oryzias_latipes/index.html) (Ensembl release 50, July 2008). 37 Chapter 3: Results _____________________________________________________________ 3.1 Characterization of zebrafish full-length cDNA of twist1b Using the incomplete cDNA sequence of twist1b, 5’ and 3’ RACE experiments was performed. The complete full-length cDNA of the zebrafish twist1b was obtained. The 1,151 bp full-length cDNA of zebrafish twist1b comprises of a 113 bp 5’UTR, a 516 bp ORF encoding a predicted 171 amino acid protein, and a 522 bp 3’UTR. The deduced amino acid sequence has a bHLH domain (Gln78 to Ser134) and a WR motif (Glu150 to Gly163). The sequence has been deposited to GenBank under the accession number DQ351987 using the old name of twist1. 38 DQ351987: 1 gagaaagccc tccgtgacgc aggaggagac gcgctgagag ggaccgagcc tctgacccca 61 tttcgtcgga ctcagggaag ccacgacccg caatctgagc ttttccagag gtgatgtttg M F 121 aggaagaggc gatgcacgag gactccagct ctccagagtc tccggtggac agtctgggaa E E E A M H E D S S S P E S P V D S L G 181 acagcgagga ggagctcgac aggcgacaac cgaagcgcgt cagcaggaaa aaacgcgcca N S E E E L D R R Q P K R V S R K K R A 241 gccgcaaaaa cgccgaggat tccgacagtc ccacgcccgg gaagaggagc aagaagtgca S R K N A E D S D S P T P G K R S K K C 301 gcaacagcag cagcagcccg caatctctgg aggacctgca gacgcagcgc gtcatggcga S N S S S S P Q S L E D L Q T Q R V M A 361 acgtgcgcga gcgtcagagg actcagtctc tgaacgaggc gttcgcctcc ctgcgcaaaa N V R E R Q R T Q S L N E A F A S L R K 421 tcatccccac cttaccctcg gacaaactca gcaaaataca gacgctcaaa ctcgcggccc I I P T L P S D K L S K I Q T L K L A A 481 ggtacattga cttcctctgt caggtcctgc agagcgatga gctggactcc aagatgtcca R Y I D F L C Q V L Q S D E L D S K M S 541 gctgcagtta tgtggcgcac gagagactca gctacgcgtt ttctgtgtgg agaatggagg S C S Y V A H E R L S Y A F S V W R M E 601 gcgcgtggtc catgtcaaca tctcactaac gcacggatgc acgcgtttga tgcagcatga G A W S M S T S H * 661 ttctcggcct gaggagctga actcactgga aggagcggct caaaacaagg gcgaaaataa 721 ggattataaa gggaaaattc tggagcgtca tgacgtcgtt gcaagcactt acagttgtga 781 actacgacat gggagcagga attacagtca gatctgtgct gttgcgacgg tgaatgtgga 841 aaacatgtgc ttccgtccac aaaacagaga ctcgctgcag gaaaagacgc tcctgcgctt 901 ctgacagcaa caaccagcat ggcgtcatat ttttttctct gaaggaaaac acacacactc 961 aacgaatgaa ggaaatcggc tcatatcagt gttaacattt tctttgatcg gtccaagaaa 1021 atacttttat ttatttattg atgattgtca caatgcagaa tagatctggt gtctacatgc 1081 attttctatt ttaaatatga tgtaaatatg tgtatatttt ctgcaataaa acatgatttg 1141 aaatacaaaa a Figure 3.1: 3.2 Full-length cDNA sequence of zebrafish twist1b (in lowercase) and its deduced amino acid sequence (in UPPERCASE single letter code). The ATG start and TAA stop codon are underlined, and the polyadenylation signal is double-underlined. The bHLH domain is highlighted in light grey and the WR motif is highlighted in dark grey. Genomic organization of zebrafish twist1b The zebrafish twist1b gene, as in its orthologs in other organisms has a dual-exon genomic structure. A 884 bp intron was amplified and the sequence was generated by 39 sequencing. The genomic DNA sequence of twist1b has been deposited to GenBank under the accession number DQ191169 (Figure 3.2). DQ191169: 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 gtagttgtat tttgtcatga atcactattc ttctttgaca caacagcgtg cgtttgattc ttacatcact ctataagatc cgcgctgaga gcaatctgag tctccagagt ccgaagcgcg cccacgcccg gaggacctgc ctgaacgagg agcaaaatac cagagcgatg agctacgcgt cgcacggatg aaaacttgca gttagattga aaataattat tgaatttaaa atatagattg cccatcaaac atgttttcac ataattttaa taatgtagaa cagattttta aataatctgg ctttttatga cttgaataat aattaatgca cttcgttttt caagggcgaa cacttacagt gacggtgaat gacgctcctg aaaacacaca gatcggtcca ctggtgtcta ataaaacatg tcacagtaca tgaatcagtc tgacatgtca caaaaaatgg cctatgttgt ttggtataac cagcttcttg ctctgaactt gggaccgagc cttttccaga ctccggtgga tcagcaggaa ggaagaggag agacgcagcg cgttcgcctc agacgctcaa agctggactc tttctgtgtg cacgcgtttg cttgaatatc ttatgaaaca tcgttgtatt gaaacaaatt tttgcgactt tcgttcttga gtgttttggt atgtttggcc ctaaattaaa aaaacatatt tgttatttat actgttgtct gtttttttat taataattat ttagattctc aataaggatt tgtgaactac gtggaaaaca cgcttctgac cactcaacga agaaaatact catgcatttt atttgaaata tctgcatctg tacaattaat tgaatggatg ctttcaattc gtttatttta ggaacccttt tccaatcgca cccttcgagt ctctgacccc ggtgatgttt cagtctggga aaaacgcgcc caagaagtgc cgtcatggcg cctgcgcaaa actcgcggcc caagatgtcc gagaatggag atgcagcatg aagggttttg gtttcacatt tgttttttaa aaataatgtt accttaaaca aaatctcatg ggctccataa ataataaaga acatatttaa caaaatttaa aaacaatctt agtaataatt ttactgtata tagataaaca ggcctgagga ataaagggaa gacatgggag tgtgcttccg agcaacaacc atgaaggaaa tttatttatt ctattttaaa caaaaa agtctaaaat ttgtcagcgc atggatgctc tattttaatg ttttaaatga ggatcgcggt ggccagccca tgagaaagcc atttcgtcgg gaggaagagg aacagcgagg agccgcaaaa agcaacagca aacgtgcgcg atcatcccca cggtacattg agctgcagtt ggcgcgtggt gtatgcgatg gataaccagt cacgttgtag ggtaagtgat cagcttaatt cgtaaataca aaaatggttg tcctcaactt atgagtagtt gcatacttta aataccgaat ttccacttca taaaagcagt aaataagtac ttgcatcaat gctgaactca aattctggag caggaattac tccacaaaac agcatggcgt tcggctcata tattgatgat tatgatgtaa atcagcagat aagtctcagt atttagatat catttcaatg agcgctttga cccctcccgc aaaagttagc ctccgtgacg actcagggaa cgatgcacga aggagctcga acgccgagga gcagcagccc agcgtcagag ccttaccctc acttcctctg atgtggcgca ccatgtcaac caaaactttt caaaagtgtg acaattatac tgcaaacaat tgtttgttaa atgtgattta atatcacaac ttgaagtttc gtgtccaaac tgctttaaat ttgcacctaa gacaaaccag caatggtagc tgttattgtt gtctaaacta ctggaaggag cgtcatgacg agtcagatct agagactcgc catatttttt tcagtgttaa tgtcacaatg atatgtgtat catcagttca atttcgttgg atttggctat atattgacat ttggctcaga ctctcccttc tccttccaga caggaggaga gccacgaccc ggactccagc caggcgacaa Exon 1 ttccgacagt gcaatctctg gactcagtct ggacaaactc tcaggtcctg cgagagactc atctcactaa taaaggctca gacacaatac atactctgga ttatatgggc aattcagccc tctatattta Intron 1 attgattttc tcaagtttta ttttgtctgc gtagctaatt aatgcttcat tatttacaga tattttatca tagtaaaata aaatattttc cggctcaaaa tcgttgcaag gtgctgttgc Exon 2 tgcaggaaaa tctctgaagg cattttcttt cagaatagat attttctgca 40 Figure 3.2: 3.3 Genomic DNA sequence of zebrafish twist1b. Exons are enclosed by brackets. The ATG start and TAA stop codons are underlined, and the polyadenylation signal is double-underlined. Alignment of TWIST family peptides The alignment of the zebrafish twist family amino acid sequences against those from human, mouse, rat, chick, Xenopus, medaka, stickleback, fugu, green spotted pufferfish, sea urchin and lancelet revealed a high degree of inter-species sequence conservation (Figure 3.3). 3.4 Comparison and alignment of zebrafish twist gene family Comparison of the coding cDNA sequences showed that zebrafish twist genes are highly conserved and a higher similarity can be observed in the bHLH region and the WR domain (Table 2). Table 2: Nucleotide identity of the coding region, bHLH domain, and WR domain of the zebrafish twist genes. Coding cDNA Dr1a Dr1b Dr2 bHLH region only WR domain only Dr1b Dr2 Dr3 Dr1b Dr2 Dr3 Dr1b Dr2 Dr3 81 76 53 85 83 74 81 81 86 76 55 52 86 80 77 83 83 83 With the knowledge that the zebrafish twist genes have high coding nucleotide similarity, an alignment was performed on the full-length cDNA of the zebrafish twist 41 gene family, to identify unique regions that can be used as specific probes for in situ hybridization to minimize cross-hybridization (Figure 3.4). 42 Mm1 Rn1 Hs1 Gg1 Xt1 Tn1a Tr1a Ga1a Ol1a Dr1a Dr1b Ol1b Ga1b Tr1b Tn1b Bb Lv Ga3a Ol3a Ol3b Ga3b Xt3 Gg3 Dr3 Dr2 Ol2 Tr2 Ga2 Tn2 Gg2 Hs2 Rn2 Mm2 Consensus (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) ------------------------MMQDVSSSPVSPADDSLSNSEEEPDRQQPA-----SGKRGARKRRSSRRSAGGSAGPGG-ATGGGIGGGDEPGSPAQGKRGKKS-AGGGGGGGAGGGG ------------------------MMQDVSSSPVSPADDSLSNSEEEPDRQQPA-----SGKRGARKRRSSRRSAGGSAGPGG-ATGGGIGGGDEPGSPAQGKRGKKS-AGGGGGAGGGGGG ------------------------MMQDVSSSPVSPADDSLSNSEEEPDRQQPP-----SGKRGGRKRRSSRRSAGGGAGPGG-AAGGGVGGGDEPGSPAQGKRGKKS-AGCGGGGGAGGGG -----------------------MMQQDESNSPVSPADDSLSNSEEEPDRQQLP-----SAKRGGRKRRSSRR-----------SAGGAVGAADEPCSPAQGKRGKK----CGAGAGGGGGG ------------------------MMQEESSSPVSP-VDSLSNSEEELDRQ--------HSKRGCRKKRSTRKS------------------PEDPDSPISVKRNKK---------------------------------MSEENLGEESGSSPVSP-VDSLSNSEGELDR---------QPKRSGRKRRPSRKN------------------GEDSDSPTPGKRGKK---------------------------------MSEENLGEESGSSPVSP-VDSLSNSEGELER---------QPKRSGRKRRPSRKN------------------GEDSDSPTPGKRGKK---------------------------------MSEENLGEESSSSPVSP-ADSLSNSEGELDR---------QPKRCGRKKRPSRRN------------------GEDSDSPTPGKRGKK---------------------------------MSEENLGEDSASSPVSP-VDSVSNSEGELDR---------PPKRCGRKRRPSRKN------------------GEDSDSPTPGKRGKK----------------------------------MPEEPARDSSSSPVSP-ADSLSNSDGEPDR---------PPKRCARKRRSSKKN------------------GEDSDSSTLGKRGKK---------------------------------MFEEEAMHEDSSSPESP-VDSLGNSEEELDRR--------QPKRVSRKKRASRKN------------------AEDSDSPTPGKRSKK-------------------------------------------MREENSPPMDSAGNSEEETERQ--------LPRRGARKRRIARRSA------------DEDDEADMESSTCPGKKKCRK-SCEEGGGSAGSGS -----------------------------MREEDSSPMDSAGNSEEENDRQ--------RPRRGARKRRATRRRAG----------DDEEEEEEDIEIPSPEKKQCRK-SCDAGGGSAGSG-----------------------------MRDEDCSPMDSAGNSEEETERQ--------LPRRGARKRRPTRRSS------------GGEEEEGDTESPSPGTKKCRK-SSEGGGGSAGSGG -----------------------------MREEDSSPMDSAGNSEEETERL--------LPRRGARKRRPTRRSS------------GEEEEEGDTESPSSGKKKCRK-SSEGGGGSAGSAG -----MAADVVTYTTLETFSHEDLVKPDPDASPLADHSSSGPEAEEDPSR----------SKRYERKRRYSKSSA------------SSDDGSTGGGSVGGGKSGKN--------------MVHAPKMNHDNVMTNGGGGMKYLNGRCDDLDVKREPQLDLREHFELEDDPM--------KQHRYGRKRRYRQSEGE--------DECQLGDSSDDGSSTGSGSLSHK----------------------------------------MREEVSCTNSPEGGLGASEEELERGSKK-----TSQAGNRKRSPYPKKD------------SLVQAEESSTGSPTSLLPTGP-KKVKKSPMTVVSL --------------------------MREEVSCTSAPEGGMGASEEELERGSKK-----SLQQGNRKRSPYPKKE-------------S--LSQAEESCTGSPSSLLP-SGPKRPKKSPTAV ------------------MRKEQASRNEHPEERVAVSEGKTGKPFSNTCPVVVA-----TPGLTGRKRQMGSHTEDDLTPVTP-PSSVDNQVKDGDGFQVYSPSGPKRLKRSPQHPSSSSGP ------------------MREEEQSNEDHPEGGVVSSEENTVRPPSNTCPVVVA-----TPGVTGRKRHTGSHPEDHVTTVTPTTSAHNGKTKPVEDIQIHALTGPKRHKRSSQDRSPSSGT --------------------------MKEEGVCPDSPEGSMVTSEEEADRQ---------QRKAIRKRTILVGKP----------------SEGRVPLSPPCKRNKR----------------------------------------MKEENMCPESPEGSLVTSEEEGERL---------HKKCLRKRGQAGKAL-----------------EDGGAASPQGKRCKR----------------------------------------MREEQTCGDFPESGILPIEEEQERRPNKCAVVVSPPAGARKRLTGPKKE------------PVSQDDKPSLDNPSNLAPKRPKRSSPSSSSSSSSS --------------------------MEESSSSPVSPVDSLVTSEEELDRQ---------QKRFGRKRRQGRKSS-----------------EDSSSPSSVNKRNKKP---------------------------------------MEEGSGSPVSPVDSLVTSEEELDRQ---------QKRFARKRRHSKKFS----------------EDSSGSSPGPVKRAKKP---------------------------------------MEEGSSSPVSPVDSLVTSEEELDRQ---------QKHFSKKRRHSKKSS----------------EDSSGSSPGRVKRGKKA---------------------------------------MEEGSSCPGSPVDSLGTSEEELDRQ---------QKRFPAKRRQSKKSS----------------GDSSGSSPGPVKRAKKP---------------------------------------MEEGSSSPVSPVDSLVTSEEELDRQ---------QKRFAKKRRHSKKSS----------------EDSSGSSPGPLKRGKK----------------------------------------MEESSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPNPGKRGKK----------------------------------------MEEGSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPTPGKRGKK----------------------------------------MEEGSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPTPGKRGKK----------------------------------------MEEGSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPTPGKRGKK--------------EE SS SSPVDSL SEEELDRQ PKR GRKRR SRKSS DD SP GKRGKK 43 Mm1 Rn1 Hs1 Gg1 Xt1 Tn1a Tr1a Ga1a Ol1a Dr1a Dr1b Ol1b Ga1b Tr1b Tn1b Bb Lv Ga3a Ol3a Ol3b Ga3b Xt3 Gg3 Dr3 Dr2 Ol2 Tr2 Ga2 Tn2 Gg2 Hs2 Rn2 Mm2 Consensus (92) (92) (92) (80) (57) (61) (61) (61) (61) (60) (62) (73) (74) (73) (73) (81) (92) (79) (76) (99) (100) (57) (56) (85) (57) (58) (58) (58) (57) (54) (54) (54) (54) (123) Figure 3.3: GGGGGSSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH-----GG---SSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH-----G----SSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGA------------G----SSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH----------SSSTGSSPQSFEELQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH----------SN--SSSPQSFEDIQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SN--SSSPQSFEDIQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SS--SSSPQSFEDLQSQRVMANVRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMSS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SS--SSSPQSFEELQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKLSS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SSNSSNSPQSFEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH----------CSNSSSSPQSLEDLQTQRVMANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMSS-CSYVAHERLSYAFSVWRMEGAWSMSTSH-----E----ASSSSSPARSFDDLQTQRVMANIRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWAMSTSH-----D----SETSSSPAPSFDDLQTQRVMANVRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLESDELDARGTS-CSYVAHERLSYAFSVWRMGGSWSLSTTTH---------SEGSSSPELSFDDLQTQRVLANIRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLESDELDGRGTS-CSYVAHERLSYAFSVWRMGGAWSLSTTSH---------SEGSSSPELSFDDLQTQRVLANIRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDGRGTS-CSYVAHERLSYAFSVWRMGGAWSLSTTSH----------RKKTSKAESFEDLQNQRVLANVRERQRTQSLNEAFSSLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLRSDDTDTKMASSCSYVAHERLSYAFSVSRQELYFGLGAPSSGPNHF -------IRRKGPQSFEELQNQRVLANVRERQRTQSLNDAFANLRKIIPTLPSDKLSKIQTLKLASRYIDFLFQVLKSDEEDQKMVGSCTYMAHERLSYAFSVWRMEGAWNSMASHR----AP---TSLGPRLDQPFEDLHSQRVIANVRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQILKLASRYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWAMSTSH-----VSLAPTSLGPRSEPPFEELHSQRVIANVRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQILKLASRYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWAMSTSH-----SLSPVPANSPGGLSALEDPHAQRVIANIRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLYQVLQSDQMDSKLAG-CNYLAHERLSYAFSVWRMEGAWSSMSAGH----SL---SPGPDPSPGDLEDPHGQRVIANIRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLYQVLQNDEMDTKLAG-CNYLAHERLSYAFSVWRMEGAWSTMSAGH-------------SPHIETFEDVHTQRIIANVRERQRTQSLNDAFAELRKIIPTLPSDKLSKIQTLKLASRYIDFLYQVLQSDELDHKIAS-CNYLAHERLSYAFSVWRMEGAWSMSTTH--------------SPVPQSFEDVHTQRVIANVRERQRTQSLNDAFAELRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDHKITS-CNYLAHERLSYAFSVWRMEGAWSMSASH-----LVPVVSSVSPVPGQPFEDLHTQRVIANVRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQILKLASRYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWSMSATH-------------SPSSTQSFEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SPSSTQSYEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDSKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SPSSSQSYEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SPSSNQSYEELQNQRCLANVRERQRTQSLNEAFSSLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH------------ASPSSSQSYEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SSPSSQSYEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDSKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------GSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------GSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------GSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-----S S S QSFEELQTQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS CSYVAHERLSYAFSVWRMEGAWSMSASH Alignment of predicted Twist proteins. Zebrafish Twist1a (Dr1a), Twist1b (Dr1b), Twist2 (Dr2) and Twist3 (Dr3) predicted amino acid sequences were aligned with human (Hs), rat (Rn), mouse (Mm), chick (Gg), frog (Xt), fugu (Tr), medaka (Ol), stickleback (Ga), pufferfish (Tn), lancelet (Bb) and sea urchin (Lv) Twist proteins. The consensus bHLH and WR domains are highlighted in blue and red, respectively. 44 Dr1a Dr1b Dr2 Dr3 Consensus (1) (1) (1) (1) (1) 1 100 -------------------------------------------------------------------CTCCTCTCAAACACTTTACCAGACTATAAGAGC -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AAAAAAAATTAGGAAAAAACTTCC GGGGACAAACAGCCACATGACACACACACACATACACACACACACACACACACTGCAGAGATTGCATTTGATTTCTAACAACAAACCC-AAAGCAATGGC T T TC AACAA AAACC GAAAA AAT GC Dr1a Dr1b Dr2 Dr3 Consensus (34) (1) (25) (100) (101) 101 200 TCCCTAACTTTTTTCCTTTCAA-CCTAACACAAAGTTGCTT-GGAATACCTAGTGATCTTCTCCAGAACACGAAACGTACG--CGTGGAATTGTATTTCA ---------------GAGA-AAGCCCTCCGTGACGCAGGAG-GAGACGCGCTGAGAG--------------GGACCGAG----CCTCTGACCCCATTTCG TGCGGGAGGAAAACCGTGCCAAGCTCTACTTCTGGAAGTAG-GGGACGCGCGCAGTGTCAGGACTTCAGGAGGACAGACAAAATCCCCAACAACAAAACG TGTATAACTCTCACAGAG--ATCCCACAGAGCTCGCTTCAGTGTTTAACTTTGACACCGCTCACAGGTACAGCAGGAGCACTGACACGGAGGACTGATCG TGC TAACT T ACCGTG CAAGCCCTACATCTCGCTGCAG GGGACGCGTTGAGAGC ACAG A AGGACCGACA CCTCGGAC ACATTTCG Dr1a Dr1b Dr2 Dr3 Consensus (130) (66) (124) (198) (201) 201 300 CCCTCATGCTGGAATAACGTCCTTATTCGCACGCGC-----TTTCAGCAGAGACTTAAGCGA-GATGCCCGAAGAGCCCGCGCGAGACTCCTCCAGCTCC ---TCGGACTCAGGGAAGCCACG-ACCCGCAATCTG-----AGCT----TTTCCAGAGGTGATGTTTGAGGAAGAGGCGATGCACGAGGACTCCAGCTCT GA-TATTACTAGTCTAAACCACGGAACCAAAAGTGG-----AGATCGTATTTTCTCCTCGTTTCTTTAAAAAAGAA---ATGGAAGAGAGTTCTAGCTCT ---TCATGCGAGAGGAACAGACTTGTGGAGATTTTCCTGAAAGTGGGATCCTTCCCATTGAAGAAGAGCAGGAGCGGCGCCCCAATAAGTGTGCGGTTGT TCATGCTAGAGTAACCCACTTATCCGCAATTTG AGTT G ATTTTCTCATGGGATGTTTGCAGAAGAGGCGATGCAAGAGG CTCCAGCTCT Dr1a Dr1b Dr2 Dr3 Consensus (224) (153) (215) (295) (301) 301 400 CCCGTGTCT-CCCGCGGACA---------GTCTCAGCAACAGCGACGGAGAGCCCGACAGG---CCACCAAAAAGGTGCG-CAAGGAAAAGACGGTCGAG CCAGAGTCT-CCGGTGGACA---------GTCTGGGAAACAGCGAGGAGGAGCTCGACAGGCGACAACCGAAGCGCGTCA-GCAGGAAAAAACGCGCCAG CCCGTCTCC-CCAGTGGACA---------GCCTGGTGACCAGCGAGGAGGAGCTGGACAG---ACAGCAGAAAAGGTTCG-GGAGGAAAAGGAG-GCAAG GGTTTCTCCACCGGCGGGCGCACGCAAGCGGCTGACGGGTCCCAAAAAAGAGCCCGTCTCACAAGACGACAAACCATCCCTGGACAACCCATCGAATCTG CCCGTGTCT CCGGTGGACA GTCTGGGGAACAGCGAGGAGGAGCTCGACAGGC ACAACCGAAACGGTTCG GGAGGAAAAGACG GCCAG Dr1a Dr1b Dr2 Dr3 Consensus (310) (242) (300) (395) (401) 401 500 CAAGAAAAACGG-------GGAGGATTCCGATAGCTCGACCCTTGGGAAAA--------GGGGGAAAAAGTCT-AGCAACAGCAGCAACAGCCCT-CAGT CCGCAAAAACGC-------CGAGGATTCCGACAGTCCCACGCCCGGGAAGA--------GGAGCAAGAAGTGC-AGCAACAGCAGCAGCAGCCCG-CAAT GAAGGAAGTCGAG------CGAGGACAGCAGCAGCCCGAGCTCCGTCAATA--------AACGTAACAAAA---AGCCG-AGCCCGAGCAGCACT-CAGT GCTCCCAAACGTCCCAAAAGAAGCTCTCCGTCATCCTCATCGTCTTCTTCGTCCTCTCTGGTGCCCGTCGTGAGTTCGGTTTCTCCAGTTCCCGGACAGC GCAGAAAAACG GGAGGATTCCGACAGCCCGACCCTCGTGAA A GG GCAAGAAGTG AGCAGCAGCAGCAGCAGCCCT CAGT Dr1a Dr1b Dr2 Dr3 Consensus (393) (325) (381) (495) (501) 501 600 CTTTCGAGGAGCTGCAGACGCAGCGCGTGATGGCGAACGTGCGCGAGCGACAGAGGACGCAGTCGCTCAACGAAGCGTTTGCGGCTTTACGCAAAATCAT CTCTGGAGGACCTGCAGACGCAGCGCGTCATGGCGAACGTGCGCGAGCGTCAGAGGACTCAGTCTCTGAACGAGGCGTTCGCCTCCCTGCGCAAAATCAT CCTTCGAGGAGCTCCAGAACCAGCGCGTCCTGGCGAACGTACGCGAGCGGCAACGGACTCAATCGCTGAACGAAGCCTTCGCGTCTTTGCGCAAAATCAT CCTTCGAAGACCTCCACACGCAACGAGTGATCGCCAACGTTCGGGAACGCCAGCGCACACAGTCCCTGAACGACGCCTTCGCCTCCCTCAGAAAGATCAT CTTTCGAGGAGCTGCAGACGCAGCGCGTGATGGCGAACGTGCGCGAGCG CAGCGGACTCAGTCGCTGAACGAAGCGTTCGCGTCTTTGCGCAAAATCAT Dr1a Dr1b Dr2 Dr3 Consensus (493) (425) (481) (595) (601) 601 700 CCCCACTTTACCTTCCGATAAACTGAGCAAAATACAGACGCTGAAACTCGCCGCCAGGTACATCGATTTTCTCTGTCAGGTTCTACAGAGTGACGAGCTG CCCCACCTTACCCTCGGACAAACTCAGCAAAATACAGACGCTCAAACTCGCGGCCCGGTACATTGACTTCCTCTGTCAGGTCCTGCAGAGCGATGAGCTG CCCCACGCTCCCCTCGGATAAACTCAGCAAGATCCAGACGCTCAAACTCGCATCCAGGTACATTGATTTCCTCTGTCAGGTGCTGCAGAGCGACGAGATG CCCCACCTTGCCTTCGGACAAGCTGAGCAAGATCCAGATCCTCAAACTGGCCTCCAGATACATCGACTTCCTCTACCAGGTCCTGCAGAGCGACGAGATG CCCCACCTTACCTTCGGATAAACTGAGCAAGATCCAGACGCTCAAACTCGCCTCCAGGTACATTGATTTCCTCTGTCAGGTCCTGCAGAGCGACGAGCTG 45 Dr1a Dr1b Dr2 Dr3 Consensus (593) (525) (581) (695) (701) 701 800 GACTCCAAGATGGCAAGTTGTAGTTATGTTGCTCACGAGCGTTTGAGCTACGCGTTCTCGGTTTGGAGGATGGAGGGCGCTTGGTCCATGTCTGCATCTC GACTCCAAGATGTCCAGCTGCAGTTATGTGGCGCACGAGAGACTCAGCTACGCGTTTTCTGTGTGGAGAATGGAGGGCGCGTGGTCCATGTCAACATCTC GACAACAAGATGTCCAGTTGCAGCTACGTCGCGCACGAGAGACTCAGTTACGCGTTTTCAGTGTGGAGGATGGAGGGCGCGTGGTCGATGTCTGCGTCCC GACGCCAAGCTGGCCAGCTGCAACTATCTGGCCCACGAGAGGCTGAGCTACGCCTTCTCCGTCTGGAGGATGGAGGGCGCCTGGTCCATGTCCGCCACTC GACTCCAAGATGTCCAGTTGCAGTTATGTGGCGCACGAGAGACTGAGCTACGCGTTTTC GTGTGGAGGATGGAGGGCGCGTGGTCCATGTCTGCATCTC Dr1a Dr1b Dr2 Dr3 Consensus (693) (625) (681) (795) (801) 801 900 ACTAGTGTGCAGGGAAACTT-TTTCTTGTTTTGTTTTTAATGGTCAACCCGTGAGCTGGGAAAACACG-AGGACCAATGCTAATTCCA-TCATAATCTTACTAACG--CACGGATGCAC-GCGTTTGATGCAGCATGATTCTCGGCCTGAGGAGCTGAACTCACTGGAAGGAGCGGCTCAAAACAAGGGCGAAAATAAACTAGCA--GCGAGACGCGT-CCTGATAATGCCGAA-------CGGACTGTTTACTTCCACTAATTTTGAGGAT---------------GCCAAA----ACTAAAC---ATCCACCCGTTCCTCATCTCAAAAACTGAATCCCGAACTCTGATCCTGATTTTCATCTCTATATCCACTCCACACGGCCTCCGACTCACC ACTAGCG CAGGGACGCGT CCTCTTGTTGCAGAATGAATC CGGACTGTTGAGCTGAACTAACTCT AGGATC ACTC AAAC TCCAAATCA Dr1a Dr1b Dr2 Dr3 Consensus (789) (721) (751) (892) (901) 901 1000 GGGGAAAACGGCAAATGT---TCCAACAGAG----GTCATGGCTGT--TACCGAGAGAAGG---CCACGGACAGCGAATTGTCATATGGATTT-----CC GGATTATAAAGGGAAAATT-CTGGAGCGTCAT---GACGTCGTTGC---AAGCACTTACAG---TTGTGAACTACGACATGGGAGCAGGAATTACAGTCA GGATTATTCGATGAACCT--CTAAACCTCAGT---GACGTGGCCA----AAGGACATTCAG---TGGATACAT-CTAC---------GGACTT------CTGCCATGACCCCACCCTGCCTAAAGATGACCTCTGACCTCTCTCACTTTTCCAAATCAAGCCTTCGCAGTTTCCCACAACCCCAGAGGACTT------GGGTTATACGGCGAACCT CTAAAGCTGAGT GACGTGGCTG TAAGGACATACAG TCGCGGACT CGACATG CA AGGACTT C Dr1a Dr1b Dr2 Dr3 Consensus (872) (811) (822) (985) (1001) 1001 1100 TCCCGAGTCTTATG--ACGACGAATGTTGGAAATATGTGCATATGCATGTTTTTTTTTTTTTTTTTTTTTTTTGGAAGACTCAGATGTGCATA----ACT GATCTGTGCTGTTGCGACGGTGAATGTGGAAAACATGTGCTTCCG--TCCACAAAACAGAGACTCGCTGCAGGAAAAGACGCTCCTGCGCTTCTGACAGC GAGCA-TCCTCATG--AAGGACACAAT----AAAAGGGACTT-----TTTAATATACTTAGA---GGAAAAGTAGATGACG-TCTTCTGTGTC-GTCAAA CATGCTTCATCTAC--AAGCCGACAGTTTTCCCCTTTCCCTAATG---GAGCTTTACCAGCAAACGCAGGAGAGAAAGAGT--CTTGAGGAGC--GCAGA GATC TCCTCTTG ACGGCGACTGTTG AAACATGTGCTTATG TGTACTTTACTTAGA TCGCTG AGTGGAAGACTCTCTTGTGCATC G CAGA Dr1a Dr1b Dr2 Dr3 Consensus (966) (909) (905) (1076) (1101) 1101 1200 TTCTGGAAGAAAGTG--AATTTGCATTACAAGGACTGTC--GATAAGAAAATGGGAA--TTGAAGCCT-CTAGTTCGAATCCTGTGCAAATACAAAGCTT AACAACCAGCATGGCGTCATATTTTTTTCTCTGAAGGAA--AACACACACACTCAACGAATGAAGGAA-ATCGGCTCATATCAGTGTTAACATTTTCTTT TACATTTAATCTTTT-TCACAAGTTATTCT-TAAATGTC--GCCATGATGATAGTAC--AGGAAGGCT-ATCTGCT---ATCTGTATGGAAACTATATTT TGAGCAGAGCATTTCCACAAGACTGAATCCAGCACTGAGCTGAGTGGAAGTGACTGCTGCTGATCTCAGATCAGTGTTCCTCCATGTTGACTGTTCTCTG TACA AGCATTTC TCATATGTTTTTCTATGACTGTC GACA GAAGATAGTAC ATGAAGGCT ATCGGTT A ATCTGTGTTGACACTTT TTT Dr1a Dr1b Dr2 Dr3 Consensus (1059) (1006) (995) (1176) (1201) 1201 1300 TA---GTACAATTCTATTTATTTATTGATGACACACTTTTTGAAATGAAAGTAAATGTATCAAATGTGTTGAAATGCATTATTATTTTTTATTACTTTTG GATCGGTCCAAGAAAATACTTTTATTTATTTATTGATGATTGTCACAATGCAGAATAGATCTGGTGTCT--ACATGCATTTTCTATTTTAAATA-TGATG CATGTCTGTCTCTAAACAGAAGAGTTTATATATATATAAGAACAAAAAAAAAAAAAAAAA---------------------------------------GACTCATGTTTGGGCAGATATTGACTGTTCTGAGGACTCACTAACCCTGACCTGATGGATCAAGGCTTC----CTCCGCTCTTTATATTGCTTCATTATG GAT GTGTATGTAAATATATTTATTTAT TAT GATTATTGAAACAAAACAAAATGGATCAAGTGT T A ATGCATT TTTATTTT ATTA TTATG Dr1a Dr1b Dr2 Dr3 Consensus (1156) (1103) (1055) (1272) (1301) 1301 1400 TAAATAAATGTGTATTTCTGTAATAAAAAAATGAAGAATTTTAAGAAATA-------------------------------------------------TAAATATGTGTATATTTTCTGCAATAAAACATGATTTGAAATACAAAAA-----------------------------------------------------------------------------------------------------------------------------------------------------CAGATGTGAGAGTGATTGTGCTTGTGTTTC-TGCTGTATATTAGCCATTCATTCCTGCTTCAGTGACGGGTAACACTGTGTAAACCGCCAGTTCATGTAA TAAATATGTGTGTATTT TG A TAAAACATGATGTATATTA AAAT 46 Dr1a Dr1b Dr2 Dr3 Consensus (1206) (1152) (1055) (1371) (1401) Dr1a Dr1b Dr2 Dr3 Consensus (1206) (1152) (1055) (1471) (1501) Dr1a Dr1b Dr2 Dr3 Consensus (1206) (1152) (1055) (1571) (1601) Dr1a Dr1b Dr2 Dr3 Consensus (1206) (1152) (1055) (1671) (1701) Dr1a Dr1b Dr2 Dr3 Consensus (1206) (1152) (1055) (1771) (1801) Dr1a Dr1b Dr2 Dr3 Consensus (1206) (1152) (1055) (1871) (1901) Figure 3.4: 1401 1500 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AATAATCAAATAATGCTTCAAGATTCACTCTATTCCTCTATTAAAAGAGACTTTAACTGCAGAAGTGTTAGTGGCTTTAATGGGGGAATGAGGAGCTGTA 1501 1600 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CATCAATAATGATGTGTCGGAGTACAGAGGAAGACATTTAGAAAGATGCTCTGCTTCAGGATTCAGTCAAACGTCTGCAAACGTATAAAGATAGCAGGGT 1601 1700 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TCATGTGAGTGGACTTGGTGATTATAAATATTTCTACAATAATGAAGAAGCTGTTTGAGTACATCTTTACAGCAGATCCAGGCAGCGGTCGCTAATAGGA 1701 1800 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TGAGATGTCGGCTAAACTTGGTGTTGGTGATGGTGTGAAAATAAACTGAAATGAAGCGTACAGCAGGAGAACTCGATGAGTAGCTAACAGACTAAGAGTT 1801 1900 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TACGCTGATTATCAAGAGCTTGAAAATTCATCAATAAACAAACAATAAGATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1901 1930 ---------------------------------------------------------------------------------------AAAAAAAAAAAATTAAAAAAAAAAAAAAAA Alignment of zebrafish full-length twist cDNAs. Start (ATG) and stop (TAA/TAG) codons are highlighted in black. Identical regions are highlighted in grey. The consensus bHLH and WR domains are highlighted in blue and red, respectively. Sequences used for synthesis of antisense RNA in situ hybridization probes are underlined. Sequences were obtained from GenBank: twist1a (EF620930), twist1b (DQ351987), twist2 (NM_001005956) and twist3 (NM_130985). 47 3.5 Identification and confirmation of the true orthologs of zebrafish twist genes 3.5.1 Comparison of zebrafish twist gene family with other species A detailed comparison of the coding cDNA and amino acid sequence of the zebrafish twist gene family against other species was performed (Table 3). Table 3: Comparison of zebrafish twist gene sequences with TWIST sequences from other species Coding nucleotides/amino acid similarity (%) Zebrafish Zebrafish Zebrafish Zebrafish twist1a twist1b twist2 twist3 Human TWIST1 60/66 62/63 59/63 61/51 Mouse Twist1 58/65 61/61 58/60 61/50 Rat Twist1 59/66 62/63 59/62 61/52 Chick Twist1 66/70 68/69 65/68 59/54 77/84 80/82 77/86 57/55 Xenopus twist1 Fugu twist1a 84/88 82/87 78/82 53/51 84/90 83/87 78/83 54/50 Medaka twist1a Stickleback twist1a 78/85 78/86 73/78 56/53 Tetradon twist1a 81/86 79/85 73/52 56/53 Fugu twist1b 64/61 63/63 64/64 58/55 Medaka twist1b 63/61 63/65 63/64 58/61 65/62 62/58 59/52 Stickleback twist1b 60/58 Tetradon twist1b 61/59 64/62 62/61 60/52 Human TWIST2 70/73 74/74 80/86 54/53 Mouse Twist2 70/73 73/74 78/86 53/53 Rat Twist2 70/73 73/74 78/86 54/53 75/86 49/54 Chick Twist2 68/73 69/75 Fugu twist2 68/72 71/75 84/90 51/54 Medaka twist2 69/74 72/75 84/90 52/54 49/58 Chick Twist3 66/73 73/72 77/76 64/69 71/69 74/75 48/59 Xenopus Twist3 Medaka twist3a 50/57 61/57 65/61 71/70 68/65 Stickleback twist3a 55/53 63/56 58/58 Medaka twist3b 48/41 50/42 48/44 64/51 Stickleback twist3b 52/49 52/47 52/50 66/60 The comparison table clearly revealed that zebrafish twist1a and twist1b have the highest percentage of coding cDNA and amino acid sequence similarity compared to 48 other species TWIST1 and zebrafish twist2 has the highest percentage of coding cDNA and amino acid sequence similarity compared to other species TWIST2. Interestingly, zebrafish twist1 genes (twist1a and twist1b) have a higher percentage similarity to Twist2 of other species compared to Twist1 of other species. In addition, zebrafish twist1a, twist1b and twist2 showed higher sequence similarity to chick and Xenopus Twist3 compared to zebrafish twist3. However, zebrafish twist3 is highly homologous to medaka and three spined stickleback twist3a. 3.5.2 Phylogenetic analysis To understand the relationships of the zebrafish twist genes to each other and to those of a non-chordate, a cephalochordate, and other vertebrates, a phylogenetic tree was constructed using multiple alignments of homologous proteins belonging to human (Homo sapiens / Hs), rat (Rattus norvegicus / Rn), mouse (Mus musculus / Mm), chick (Gallus gallus / Gg), frog (Xenopus tropicalis / Xt), zebrafish (Danio rerio / Dr), medaka (Oryzias latipes / Ol), fugu (Takifugu rubripes / Tr), spotted green pufferfish (Tetraodon nigroviridis / Tn); stickleback (Gasterosteus aculeatus / Ga), Japanese lancelet (Branchiostoma belcheri / Bb), and sea urchin (Lytechinus variegatus / Lv). The Twist proteins clustered into three clades, with the zebrafish Twist1 (Twist1a and Twist1b), Twist2 and Twist3 peptides falling into different clades (Figure 3.5). Interestingly, zebrafish Twist1b did not cluster with the Twist1b of the other fish species medaka, fugu, pufferfish, and stickleback (Figure 3.5A and 3.5B). 49 Tn1a 999 601 Tr1a Ol1a 901 Ga1a 434 Dr1a 270 Dr1b A II Tn1 I Rn1 809 Mm1 818 Hs1 998 Gg 687 Xt1 767 735 960 932 918 489 976 Rn Ga3 Ol3 98 L B Gg 91 35 99 Gg 84 97 40 Xt 96 83 Mm 93 Dr 96 I Tn 100 48 73 II 53 100 Hs 92 43 76 68 90 99 60 27 Dr1 Dr1 Ga1 Ol1 Tn1 Tr1 99 46 81 Xt Ga3b 1000 Ol3b Tr1b 998 Tn1b 982 Ga1b 1000 Ol1b Dr 100 Ol3a 1000 Ga3a 530 Dr3 Xt3 843 Gg3 Ga3 Ol1 99 Tr2 928 Tn2 Mm2 354 Hs2 997 Rn2 967 Gg2 Ol3 Ga1 Tr1 Ga2 467 Ol2 Dr2 839 405 B Tr III Ol Ga 80 G I Rn Hs Mm Bb Lv Figure 3.5: Rooted cladogram (A) and unrooted radial tree (B) of Twist proteins generated by the neighbor-joining method, with bootstrap values shown. The cladogram is rooted using sea urchin (Lv) as an outgroup. Zebrafish Twist1 (Dr1a and Dr1b), Twist2 (Dr2) and Twist3 (Dr3) proteins are clustered into the three major clades. Twist protein members from human (Hs), rat (Rn), mouse (Mm), chick (Gg), frog (Xt), fugu (Tr), medaka (Ol), stickleback (Ga), pufferfish (Tn), lancelet (Bb) and sea urchin (Lv) were used to produce the phylogenetic trees. 50 3.5.3 Calculation of genetic distances The genetic distance between twist1a and twist1b among these fish was compared using the Kimura 2-parameter model of nucleotide substitution, transitions and transversions. The calculated Kimura value between zebrafish twist1a and twist1b was 0.26, whereas it was almost double in the fugu, stickleback, pufferfish and medaka (0.46, 0.47, 0.55 and 0.55 respectively). These values suggest a higher nucleotide substitution/mutation rate between the twist1a and twist1b sequences in the other fish species compared to the zebrafish. The Tajima-Nei-corrected number of substitutions per site at third codon positions between twist1a and twist1b sequences of zebrafish, fugu, pufferfish, stickleback and medaka were also determined. Since most third-codon position substitutions do not result in an amino acid change, mutations at these positions are assumed to be under little or no selective pressure and the calculated mutation rates are thus more likely to reflect neutral evolution. The Tajima-Nei value of 0.718 (± 0.118) for zebrafish twist1a and twist1b was lower compared to fugu (1.113, ±0.248), stickleback (0.794, ±0.145), pufferfish (1.243, ±0.273) and medaka (0.992, ±0.183). These results again suggest a lower substitution/mutation rate between the twist1a and twist1b genes in the zebrafish compared with the other four fish species. 51 3.5.4 Comparative synteny analysis Zebrafish twist1a (EF620930) is located on linkage group (LG) 16 at ~13.484 Mb, while twist1b (NM_130984) is located on LG19 at ~ 4.245 Mb, based on the Zv7 assembly, July 2007 release (http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=7955). The comparative synteny analysis showed that many genes on human chromosome 7, including TWIST1 (chromosome 7p21 (~19.1 MB), were found to be duplicated on zebrafish LG16 and LG19 (Table 4). Medaka twist1a (BK006268) is located on LG16 at ~12.859 Mb, while twist1b (BK006269) is located on LG11 at ~8.931 Mb, based on the HdrR assembly, July 2008 release (http://www.ensembl.org/Oryzias_latipes/index.html). The comparative synteny analysis of the chromosomal regions around zebrafish twist1a (LG16) with medaka twist1a (LG16) and twist1b (LG11) showed a significant higher number of genes in medaka LG16 compared to LG11 (Table 5). Conversely, the comparative synteny analysis of the chromosomal regions around zebrafish twist1b (LG19) against medaka twist1a and twist1b revealed comparatively a higher number of genes in medaka LG11 compared to LG16 (Table 6). 52 Table 4. Comparative synteny analysis of chromosomal regions around zebrafish twist1a and twist1b and human TWIST1. D.rerio LG16            hdac9b (17.0Mb)  twist1a (13.5Mb)    sp8l (13.9Mb)        dfna5 (15.4Mb)  cbx3b (15.8Mb)    hoxa11b (16.0Mb)  hoxa10b (16.0Mb)  hoxa9b (16.0Mb)      hoxa2b (16.0Mb)    tax1bp1 (16.0Mb)  creb5 (16.3Mb)  cpvl (16.4Mb)  tbx20 (34.3Mb)  sept7 (49.7Mb)    psma2 (32.9Mb)  nacad (22.2Mb)  ddc (5.0Mb)      abcb4 (21.1Mb)  adam22 (33.0Mb)  sri (3.9Mb)  cldn12 (33.1Mb)                  Hsa Chr 7  GLCCI1 (7.9Mb)  NXPH1 (8.5Mb)  BZW2 (16.5Mb)  TSPAN13 (16.5Mb)  AGR2 (16.8Mb)  HDAC9 (18.7Mb)  TWIST1 (19.1Mb)  TMEM196 (19.6Mb)  SP8 (20.8Mb)  TRA2A (23.5Mb)  NPY (24.3Mb)  MPP6 (24.5Mb)  DFNA5 (24.6Mb)  CBX3 (26.1Mb)  SNX10 (26.3Mb)  HOXA11 (27.3Mb)  HOXA10 (27.3Mb)  HOXA9 (27.3Mb)  HOXA5 (27.3Mb)  HOXA3 (27.3Mb)  HOXA2 (27.3Mb)  HOXA1 (27.2Mb)  TAX1BP1 (27.6Mb)  CREB5 (28.5Mb)  CPVL (29.1Mb)  TBX20 (35.2Mb)  SEPT7 (35.8Mb)  ELMO1 (37.1Mb)  PSMA2 (42.9Mb)  NACAD (45.0Mb)  DDC (50.4Mb)  FKBP9L (55.5Mb)  GRB10 (50.6Mb)  ABCB4 (86.7Mb)  ADAM22 (87.4Mb)  SRI (87.5Mb)  CLDN12 (89.8Mb)  PEX1 (91.8Mb)  COL1A2 (93.7Mb)  GNGT1 (93.7Mb)  CASD1 (93.8Mb)  SGCE (94.1Mb)  ASB4 (94.9Mb)  SHFM1 (96.2Mb)  ASNS (97.2Mb)  D.rerio LG19  glcci1 (16.1Mb)  nxph1 (34.9Mb)  bzw2 (27.9Mb)  tspan13 (28.6Mb)  agr2 (28.6Mb)  hdac9a (4.2Mb)  twist1b (4.2Mb)  tmem196 (45.6Mb)  sp8 (45.5Mb)  tra2a (11.0Mb)  npy (10.9Mb)  mpp6 (10.9Mb)    cbx3a (46.0Mb)  snx10 (46.0Mb)  hoxa11a (10.5Mb)  hoxa10ap (10.5Mb)  hoxa9a (10.5Mb)  hoxa5a (10.5Mb)  hoxa3a (10.5Mb)    hoxa1a (10.5Mb)            elmo1 (31.5Mb)  psma2 (32.5Mb)      fkbp9 (39.5Mb)  grb10 (14.0Mb)          pex1 (31.5Mb)  col1a2 (35.4Mb)  gngt1 (35.1Mb)  casd1 (37.7Mb)  sgce (37.7Mb)  asb4 (37.9Mb)  shfm1 (38.0Mb)  asns (16.4Mb)  53 Table 5. Comparative synteny analysis of chromosomal regions around zebrafish twist1a and medaka twist1a and twist1b. O. latipes LG16  dctn3 (24.1Mb)  ctnnb1 (11.1Mb)    bat5 (17.7Mb)  nt5c3 (23.3Mb)  ddc (9.8Mb)  entpd3 (9.8Mb)  m6pr (17.0Mb)  atp1a3 (17.8Mb)  gsk3a (16.2Mb)  ceacam1 (2.4Mb)  usp5 (17.5Mb)  tex10 (14.6Mb)  epn1 (16.8Mb)  u2af2 (16.7Mb)  ntd5 (16.5Mb)  rpp38 (14.8Mb)  gapdh (17.2Mb)  twist1a (12.8Mb)  sp8 (12.8Mb)  rxrb (12.6Mb)  dfna5 (13.3Mb)  hoxa13b (13.1Mb)  hoxa11b (13.1Mb)  hoxa10b (13.1Mb)  hoxa9b (13.1Mb)  hoxa2b (13.1Mb)  cpvl (13.0Mb)  ctsk (24.5Mb)  hapln2 (23.9Mb)  mef2d (23.7Mb)  tmem67 (14.6Mb)      mcl1 (24.6Mb)  styk1 (17.0Mb)  ptpn6 (17.3Mb)  psma2 (11.6Mb)  cldn12 (14.9Mb)  thbs3 (29.3Mb)  tbx20 (29.1Mb)  hfe2 (29.4Mb)  sh3bp5 (23.6Mb)  snip1 (20.9Mb)  otud7b (26.8Mb)  dnase2 (26.3Mb)  gapdhs (11.4Mb)    pbx2 (26.3Mb)  etfb (22.4Mb)  ubr5 (2.4Mb)  D. rerio LG16  dctn3 (1.0Mb)  ctnnb1 (2.1Mb)  ndufb9 (2.3Mb)  bat5 (3.6Mb)  nt5c3 (4.9Mb)  ddc (5.0Mb)  entpd3 (5.1Mb)  m6pr (6.3Mb)  atp1a3b (7.5Mb)  gsk3a (7.8Mb)  ceacam1 (8.3Mb)  usp5 (8.4Mb)  tex10 (9.3Mb)  epn1 (9.4Mb)  u2af2a (9.5Mb)  ntd5 (10.3Mb)  rpp38 (10.9Mb)  gapdh (12.9Mb)  twist1a (13.5Mb)  sp8l (13.9Mb)  rxrbb (14.9Mb)  dfna5 (15.50Mb)  hoxa13b (16.0Mb)  hoxa11b (16.0Mb)  hoxa10b (16.0Mb)  hoxa9b (16.0Mb)  hoxa2b (16.0Mb)  cpvl (16.4Mb)  ctsk (20.3Mb)  hapln2 (20.5Mb)  mef2d (20.6Mb)  tmem67 (22.2Mb)  gdf6a (25.0Mb)  ptpru (26.5Mb)  mcl1b (26.6Mb)  styk1 (27.5Mb)  ptpn6 (28.5Mb)  psma2 (32.9Mb)  cldn12 (33.0Mb)  thbs3a (33.9Mb)  tbx20 (34.3Mb)  hfe2 (37.5Mb)  sh3bp5 (39.5Mb)  snip1 (40.4Mb)  otud7b (42.8Mb)  dnase2 (43.3Mb)  gapdhs (43.6Mb)  ubin (44.4Mb)  pbx2 (45.4Mb)  etfb (46.2Mb)  ubr5 (47.2Mb)  O. latipes LG11    ctnnb1 (8.4Mb)  ndufb8 (0.9Mb)            atp1a3 (16.0Mb)  gsk3a (16.2Mb)                  twist1b (8.9Mb)  sp8 (8.8Mb)  rxrb (15.1Mb)  dfna5 (10.7Mb)                      gdf6 (16.8Mb)  ptpru (17.8Mb)                            ubin (22.0Mb)        54 Table 6. Comparative synteny analysis of chromosomal regions around zebrafish twist1b and medaka twist1a and twist1b. O. latipes LG16  mcl1 (24.6Mb)        pbx2 (26.3Mb)    gnb3 (17.4Mb)  atp1a3 (17.8Mb)  twist1a (12.8Mb)  flot1 (17.6Mb)    ptp4a3 (6.8Mb)  rxrb (12.6Mb)  cnot3 (16.3Mb)          crabp2 (15.8Mb)    vamp1 (17.3Mb)  ing4 (17.2Mb)  u2af2 (16.7Mb)        MF‐hoxa11b (13.1Mb)  hoxa10b (13.1Mb)  hoxa9b (13.1Mb)    npy (13.2Mb)  rab5a (27.7Mb)      grb10 (9.7Mb)  thbs3 (29.3Mb)      glcci1 (28.3Mb)  spag1 (1.9Mb)  sox4 (7.8Mb)      ndrg1 (6.7Mb)          azin1 (2.5Mb)        psma2 (11.6Mb)            D. rerio LG19  mcl1a (0.1Mb)  msto1 (0.3Mb)  ehmt2 (0.3Mb)  hnrpr (0.6Mb)  pbx2 (0.7Mb)  edn1 (1.0Mb)  gnb3 (2.5Mb)  atp1a3a (3.5Mb)  twist1b (4.2Mb)  flot1 (4.5Mb)  psmb9a (4.8Mb)  ptp4a3 (4.8Mb)  rxrba (4.9Mb)  cnot3 (5.0Mb)  skiv2l (5.0Mb)  gtf2h4 (5.0Mb)  pygo2 (5.3Mb)  mylip (5.5Mb)  crabp2b (6.7Mb)  oprd1 (6.8Mb)  vamp1 (7.1Mb)  ing4 (7.2Mb)  u2af2b (8.0Mb)  vps52 (9.5Mb)  slc39a7 (9.7Mb)  mrpl3 (10.0Mb)  hoxa11a (10.5Mb)  hoxa10ap (10.5Mb)  hoxa9a (10.5Mb)  cbx3a (11.0Mb)  npy (11.0Mb)  rab5a (11.5Mb)  mbp (12.6Mb)  gdf6b (13.8Mb)  grb10 (14.0Mb)  thbs3b (15.2Mb)  txnip (15.2Mb)  eya3 (15.6Mb)  glcci1 (16.1Mb)  spag1 (17.3Mb)  sox4a (19.5Mb)  yars (28.0Mb)  mrps18b (28.5Mb)  ndrg1 (28.6Mb)  col1a2 (30.0Mb)  dlx6a (30.1Mb)  rrm2b (30.5Mb)  rpl30 (30.5Mb)  azin1 (30.6Mb)  anp32e (30.7Mb)  elmo1 (31.5Mb)  trps1 (32.5Mb)  psma2 (32.5Mb)  nxph1 (35.0Mb)  gngt1 (35.2Mb)  col1a2 (35.4Mb)  sgce (37.7Mb)  shfm1 (38.0Mb)  O. latipes LG11    msto1 (19.2Mb)  ehmt2 (4.4Mb)  hnrpr (5.6Mb)    edn1 (18.9Mb)    atp1a3 (16.0Mb)  twist1b (8.9Mb)    psmb9 (15.3Mb)    rxrb (15.1Mb)  cnot3 (14.1Mb)  skiv2l (4.4Mb)  gtf2h4 (21.1Mb)  pygo2 (0.3Mb)  mylip (23.3Mb)  crabp2 (5.8Mb)  oprd1 (18.3Mb)  vamp1 (14.9Mb)      vps52 (2.1Mb)  slc39a (13.9Mb)  mrpl3 (8.5Mb)  hoxa11a (10.5Mb)  hoxa10a (10.5Mb)  hoxa9a (10.5Mb)  cbx3a (10.4Mb)  npy (9.8Mb)  rab5a (22.1Mb)  mbp (18.7Mb)  gdf6 (16.8Mb)      txnip (21.2Mb)  eya3 (19.2Mb)    spag1 (6.0Mb)    yars (26.6Mb)  mrps18b (21.0Mb)  ndrg1 (8.4Mb)  col1a2 (9.5Mb)  dlx6 (9.8Mb)  rrm2b (23.6Mb)  rpl30 (23.5Mb)  azin1 (22.6Mb)  anp32e (3.3Mb)  elmo1 (7.7Mb)  trps1 (7.4Mb)    nxph1 (20.7Mb)  gngt1 (9.4Mb)  col1a2 (9.5Mb)  sgce (9.5Mb)  shfm1 (9.8Mb)  55     sp8 (12.8Mb)      fkbp9 (39.5Mb)  angpt1 (42.2Mb)  sp8 (45.5Mb)  cbx3 (46.0Mb)  snx10 (46.0Mb)  fkbp9 (21.8Mb)  angpt1 (20.3Mb)  sp8 (8.8Mb)  cbx3 (10.4Mb)  snx10 (13.2Mb)  Zebrafish twist2 (NM_001005956) is located on LG9 at ~39.145 Mb on the zebrafish Zv7 genome assembly. The zebrafish twist2 chromosomal segment is syntenic to the human and medaka twist2 locus on chromosome 2 and LG21 respectively (Table 7). 56 Table 7. Comparative synteny analysis of chromosomal regions around twist2 from zebrafish, human and medaka. O. latipes LG21  dlx1 (24.1Mb)  slc9a2 (11.7Mb)    rab20 (28.4Mb)  tmem41a (22.1Mb)  fzd7 (18.9Mb)  als2 (18.9Mb)    gdap2 (0.9Mb)  tbx15 (1.8Mb)  txndc9 (11.6Mb)  arl5a (6.0Mb)  ccnt2 (29.7Mb)  ndufa10 (30.4Mb)  cxcr7 (26.8Mb)    ebi2 (9.4Mb)      ercc1 (21.7Mb)  olig2 (6.7Mb)  gabpa (5.2Mb)  dirc2 (6.5Mb)  ptplb (6.0Mb)  ddx18 (0.4Mb)  mcm3ap (18.4Mb)  pms1 (18.5Mb)  asnsd1 (18.7Mb)  sestd1 (8.0Mb)    twist2 (28.7Mb)  ofd1 (26.2Mb)  ttc21b (23.4Mb)  prpf40a (22.3Mb)  pou1f1 (19.9Mb)  D. rerio LG9  dlx1a (3.0Mb)  slc9a2 (5.8Mb)  fev (7.3Mb)  rab20 (7.5Mb)  tmem41a (9.4Mb)  fzd7a (10.3Mb)  als2 (10.5Mb)  nrp2b (11.2Mb)  gdap2 (14.3Mb)  tbx15 (14.4Mb)  txndc9 (15.2Mb)  arl5a (16.3Mb)  ccnt2 (16.7Mb)  ndufa10 (16.8Mb)  cxcr7b (17.4Mb)  acvr2a (18.9Mb)  ebi2 (25.0Mb)  sf3b1 (26.0Mb)  rftn2 (26.2Mb)  ercc1 (26.6Mb)  olig2 (26.8Mb)  gabpa (29.1Mb)  dirc2 (31.5Mb)  ptplb (32.2Mb)  ddx18 (32.6Mb)  mcm3ap (32.7Mb)  pms1 (34.9Mb)  asnsd1 (35.1Mb)  sestd1 (37.2Mb)  ube2e3 (37.7Mb)  twist2 (39.2Mb)  ofd1 (43.6Mb)  ttc21b (45.0Mb)  prpf40a (45.6Mb)  pou1f1 (47.6Mb)  Hsa chr2  DLX1 (172.7Mb)  SLC9A2 (102.7Mb)  FEV (219.5Mb)      FZD7 (202.6Mb)  ALS2 (202.3Mb)  NRP2 (206.3Mb)      TXNDC9 (1.0Mb)  ARL5A (152.4Mb)  CCNT2 (135.4Mb)  NDUFA10 (240.6Mb)  CXCR7 (237.2Mb)  ACVR2A (148.4Mb)    SF3B1 (198.0Mb)  RFTN2 (198.2Mb)            DDX18 (0.1Mb)    PMS1 (0.2Mb)  ASNSD1 (190.2Mb)  SESTD1 (179.7Mb)  UBE2E3 (181.6Mb)  TWIST2 (239.5Mb)    TTC21B (166.5Mb)  PRPF40A (153.2Mb)    Zebrafish twist3 is located on LG23 at ~0.105 Mb on the zebrafish Zv7 genome assembly. In medaka, there are two twist3 genes, twist3a on LG5 and twist3b on LG7. Comparative synteny analysis revealed that the zebrafish LG23 region around twist3 shows a greater number of shared genes with medaka LG7 compared with medaka LG5 (Table 8). 57 Table 8. Comparative synteny analysis of chromosomal regions around zebrafish twist3 and medaka twist3a and twist3b. O. latipes LG5  twist3a (30.2Mb)    mitf (5.2Mb)              csrp1 (13.1Mb)              eif4b (30.3Mb)  foxp1 (5.0Mb)  eif4e3 (21.0Mb)  ppp4r2 (21.2Mb)  ndrg3 (26.8Mb)                      trpc4ap (26.8Mb)      slmap (33.5Mb)      tkt (3.7Mb)      adnp (27.0Mb)    dhrs3 (32.5Mb)  phc2 (13.8Mb)  slc2a1 (13.9Mb)                            D. rerio LG23  twist3 (0.1Mb)  ctnnbl1 (0.2Mb)  mitfb (0.8Mb)  plcg1 (1.5Mb)  zhx3 (1.5Mb)  mip2 (1.6Mb)  spata2 (2.6Mb)  tnnc1 (3.3Mb)  slc9a8 (3.6Mb)  csrp1 (4.2Mb)  c20orf14 (5.0Mb)  slco4a1 (5.1Mb)  gata5 (5.4Mb)  tm9sf4 (5.5Mb)  tcea2 (6.4Mb)  adrm1b (7.2Mb)  eif4b (8.0Mb)  foxp1a (8.2Mb)  eif4e3 (8.3Mb)  ppp4r2a (8.4Mb)  ndrg3b (11.2Mb)  ddx23 (11.8Mb)  cacnb3a (11.9Mb)  adcy6 (12.0Mb)  ard1a (12.2Mb)  smc1a (13.3Mb)  fbxo44 (14.6Mb)  rnd1 (15.6Mb)  prim1 (15.7Mb)  hsd17b10 (16.2Mb)  ssr4 (16.4Mb)  trpc4apb (16.6Mb)  nsfl1c (16.6Mb)  pard6b (16.9Mb)  slmap (17.5Mb)  emd (17.7Mb)  atp2b3b (17.7Mb)  tkt (17.9Mb)  tnnc (18.4Mb)  dpm1 (18.5Mb)  adnp (18.5Mb)  her12 (19.9Mb)  dhrs3 (20.1Mb)  phc2 (20.5Mb)  slc2a1 (20.6Mb)  eno1 (21.1Mb)  samd11l (21.4Mb)  her9 (21.8Mb)  pa2g4b (23.7Mb)  hnf4a (24.3Mb)  ada (24.4Mb)  gdi1 (24.5Mb)  lzic (25.1Mb)  clstn1 (25.1Mb)  ube4b (25.2Mb)  kif1b (25.3Mb)  pgd (25.3Mb)  casz1 (25.7Mb)  O. latipes LG7  twist3b (1.7Mb)  ctnnbl1 (1.3Mb)  mitf (20.7Mb)  plcg1 (15.0Mb)  zhx3 (15.1Mb)  mip (19.4Mb)  spata2 (15.4Mb)  tnnc1 (18.1Mb)  scl9a8 (15.7Mb)    c20orf14 (15.8Mb)  slco4a1 (5.4Mb)  gata5 (5.1Mb)  tm9sf4 (5.0Mb)  tcea2 (27.2Mb)  adrm1 (22.2Mb)    foxp1 (20.7Mb)  eif4e3 (20.7Mb)  ppp4r2 (20.7Mb)    ddx23 (19.5Mb)  cacnb3 (19.5Mb)  adcy6 (19.6Mb)  ard1a (11.8Mb)  smc1a (12.1Mb)  fbxo44 (21.0Mb)  rnd1 (19.5Mb)  prim1 (19.4Mb)  hsd17b10 (26.6Mb)  ssr4 (11.8Mb)    nsfl1c (5.6Mb)  pard6b (4.4Mb)    emd (10.7Mb)  atp2b3 (4.6Mb)  tkt (7.3Mb)  tnnc1 (18.1Mb)  dpm1 (4.4Mb)  adnp (4.4Mb)  her12 (7.7Mb)        eno1 (10.6Mb)  samd11 (10.3Mb)  her9 (10.2Mb)  pa2g4 (12.5Mb)  hnf4a (25.9Mb)  ada (25.8Mb)  gdi1 (25.5Mb)  lzic (10.0Mb)  clstn1 (10.0Mb)  ube4b (10.0Mb)  kif1b (9.9Mb)  pgd (9.9Mb)  casz1 (9.7Mb)  58     arf3 (2.3Mb)    pfkm (2.9Mb)                itga5 (23.9Mb)    bin2 (23.9Mb)  racgap1 (31.4Mb)    rarg (28.0Mb)              src (12.8Mb)  mafb (26.9Mb)  dhh (27.0Mb)  wnt10b (27.1Mb)  arf3a (27.1Mb)  phf8 (27.1Mb)  pfkm (27.2Mb)  asb8 (27.2Mb)  atf7a (27.2Mb)  dip2b (27.3Mb)  ptges3 (27.7Mb)  uts2a (28.8Mb)  cd63 (30.9Mb)  taf8 (31.2Mb)  itga5 (32.3Mb)  dazap2 (32.5Mb)  bin2 (32.5Mb)  racgap1 (32.5Mb)  sars (32.8Mb)  rarga (33.6Mb)  hoxc13a (33.7Mb)  hoxc12a (33.7Mb)  hoxc11a (33.7Mb)  cbx5 (33.9Mb)  copz1 (34.0Mb)  csad (34.1Mb)  src (38.1Mb)  mafb (38.2Mb)  dhh (12.5Mb)  wnt10b (12.5Mb)  arf3 (12.5Mb)  phf8 (12.0Mb)  pfkm (11.4Mb)  asb8 (11.4Mb)  atf7 (11.4Mb)  dip2b (11.5Mb)  ptges3 (19.4Mb)  uts2 (21.4Mb)  cd63 (22.6Mb)  taf8 (25.5Mb)  itga5 (22.9Mb)  dazap2 (22.8Mb)  bin2 (22.7Mb)  racgap1 (22.8Mb)  sars (3.5Mb)    hoxc13a (12.8Mb)  hoxc12a (12.8Mb)  hoxc11a (12.9Mb)  cbx5 (13.0Mb)  copz1 (13.0Mb)  csad (13.0Mb)  src (25.4Mb)  mafb (15.3Mb)  3.6 Embryonic expression patterns of the zebrafish twist gene family 3.6.1 RT-PCR analysis To detect the presence of the four zebrafish twist transcripts at different embryonic stages, an RT-PCR analysis of total RNA extracts using gene-specific intronspanning amplification primers was performed (Figure 3.6). 59 twist1a 37 193 510 twist1b 472 30 113 516 twist2 492 34 175 467 250 twist3 191 600 Figure 3.6: 16 879 Gene structure of twist1a, twist1b, twist2 and twist3 showing positions of intron-spanning primers (arrows) for RT-PCR and extent of unique 3’UTR probe targets (blue underline) for in situ hybridization. Empty boxes represent untranslated regions. Black boxes represent coding regions. GenBank accession numbers of zebrafish twist1a, twist1b, twist2 and twist3 are NM_001017820, NM_130984, NM_001005956 and NM_130985, respectively. Zebrafish twist1b and twist3 transcripts could be detected from as early as the 1cell stage, indicating the presence of maternally expressed and deposited transcripts in the early embryo prior to zygotic transcript expression (Figure 3.7). In contrast, zebrafish twist1a and twist2 transcripts were detected only at or after the 1-K cell stage, suggesting that twist1a and twist2 transcripts are expressed exclusively from the zygote (Figure 3.7). 60 M 1cell 8cell 64cell 1Kcell shield bud 14som 1dpf 2dpf 3dpf No RNA twist1a 476bp twist1b 516bp twist2 372bp twist3 520bp β-actin 561bp Figure 3.7: 3.6.2 RT-PCR of zebrafish twist genes. Using RT-PCR, twist1b and twist3 are detectable at the 1-cell stage, indicating the presence of maternally deposited transcripts. Zebrafish twist1a and twist2 are detectable only from the 1K-cell stage onwards, when zygotic transcription begins. In situ hybridization analysis The observations from RT-PCR analysis were further confirmed by whole-mount in situ hybridization analysis (Figure 3.8A-D). Given the high nucleotide identity between the coding regions of the four zebrafish twist genes, especially in the highly conserved bHLH and WR domains (Figure 3.4 & Table 2), RNA hybridization probes were synthesized to target unique 3’UTR of each transcript (Figure 3.6). During gastrulation (5.25-10 hpf), twist1b and twist3 transcripts are ubiquitously expressed (Figure 3.9B, D, F, H), whereas there was no detectable expression of twist1a 61 (Figure 3.9A, E). Interestingly, twist2 was specifically expressed in the organizer and the axial mesoderm at the shield and bud stage respectively (Figure 3.9C, G). During early segmentation, twist1a transcripts were found to be concentrated in the premigratory neural crest cells (Figure 3.10A & E). At around 2-somite stage, zebrafish twist1b expression was detected in the head mesenchyme and intermediate mesoderm (Figure 3.10B). Expression in the intermediate mesoderm was confirmed by two-color hybridization with pax2.1 molecular marker for intermediate mesoderm (Majumdar et al., 2000) (Figure 3.10B). Expression of twit1b in the developing somites was first detected in all somites of the five-somite stage embryo in a clearly segmental pattern (Figure 3.10F). Expression of twist2 was observed in the axial mesoderm (Figure 3.10C & G). Zebrafish twist3 was expressed in the somites (Figure 3.10D & H). 62 twist1b twist1a A Figure 3.8: B twist2 C twist3 D Expression of zebrafish twist genes during the cleavage period, 16-cell stage (A-D). Lateral (A-D) views are shown. Zebrafish twist1b and twist3 are maternally transcribed (B & D respectively) and no twist1a and twist2 expression was detected at the 16-cell stage (A & C respectively). 63 twist1b twist1a A B twist2 twist3 C D or E F G H am Figure 3.9: Expression of zebrafish twist genes during the gastrula period. The shield (A-D) and bud (E-H) stages are shown. Lateral (A-H) views are shown. During the gastrula stage, twist1b and twist3 are homogeneously expressed (B, D, F, H) while twist2 is specifically expressed in the organizer (C) and later in the axial medsoderm (G). No expression was detected for twist1a during this period (A, E). am, axial mesoderm; or, organizer. 64 twist1b twist1a A B twist2 twist3 D C hm pnc so im am 5S twist1b/pax2.1 E 2S H G F 5S 5S hm pnc so am im 5S sc im 5S 5S 5S Figure 3.10: Expression of zebrafish twist genes during the early segmentation period (2-5 somite stage). Lateral (A-D) and dorsal (E-H) views are shown. At the 5-somite stage, twist1a transcript is detected in the premigratory neural crest cells (A & E). At the 2-somite stage, twist1b and pax2.1 double color hybridization showed that twist1b is expressed in the intermediate mesoderm (B). At the 5-somite stage, expression of twist1b is also observed in the head mesenchyme and sclerotome (F). Expression of twist2 is observed in the axial mesoderm (C & G) and twist3 expression is detected in the somite (D & H). am, axial mesoderm; hm, head mesenchyme; im, intermediate mesoderm; pnc, premigratory neural crest cells; sc, sclerotome; so, somites. 65 Later in the segmentation period (11-22 hpf), both distinct and overlapping expressions was observed in the zebrafish twist gene family. At 14-somite stage, twist1a transcripts were detected in the neural crest cells (Figure 3.11A & E) and also in the sclerotome by late somitogenesis (Figure 3.12A & B). At the 18-somite stage, expression of twist1a was also observed in the neural crest cells that delaminates from the neural tube at the dorsal region of the somites (Figure 3.12A) Zebrafish twist1b was expressed in the head mesenchyme, intermediate mesoderm, sclerotome and caudal gut (Figure 3.11B & F, 3.13A-C). Double-color in situ hybridization with dlx2a revealed that twist1b is expressed in the cranial neural crest derived head mesenchyme (Figure 3.13A). The expression in the intermediate mesoderm declined through early somitogenesis to the 18-somite stage and double-color in situ hybridization with wt1, a molecular marker for glomerulus showed that twist1b is not expressed in the glomerulus (Figure 3.15K) The expression in the somite was restricted to the anterior portion of each formed somite (Figure 3.14A). Analysis of sagittal cryosections further revealed that twist1b expression was concentrated in the sclerotome, with stronger expression throughout the anterior region of each formed somite (Figure 3.14B & C). This pattern of expression was maintained until the 11-somite stage (14 hpf) (Figure 3.14D). With continuing somitogenesis, twist1b expression appeared to shift from the anterior to the more posterior somites. From the 14-somite stage onward, twist1b expression decreased in the 66 more anterior somites and was concentrated around the more posterior somites (Figure 3.13E-I). Zebrafish twist2 was expressed in the presumptive vasculature and axial mesoderm (Figure 3.11C & G). With progressing somitogenesis, its expression in the axial mesoderm was greatly reduced, and was restricted to the caudal notochord by the 18-somite stage (18 hpf) (Figure 3.12D). Two-color in situ hybridization, using fli1a as a molecular marker for the presumptive vasculature, confirmed localization of twist2 in this tissue (Figure 3.12C-E). Expression of twist3 was observed in the head mesenchyme adjacent to the forebrain region (Figure 3.11D & 3.13D) as well as in the somites (Figure 3.11H). 67 twist1b twist1a twist2 B A twist3 D C hm hm cn F E im nc H G im sc so pv Figure 3.11: Expression of zebrafish twist genes during mid-somitogenesis (14-somite stage). Lateral (A-D) and dorsal (EH) views are shown. During somitogenesis, twist1a is expressed in the premigratory neural crest cells (A, E), twist1b is expressed in the head mesenchyme, intermediate mesoderm and sclerotome (B, F), twist2 is expressed in the presumptive vasculature and caudal notochord (C, G), , and twist3 is expressed in the head mesenchyme and somites (D, H). cn, caudal notochord; hm, head mesenchyme; im, intermediate mesoderm; nc, neural crest cells; pv, presumptive vasculature; sc, sclerotome; so, somites. 68 A B sc sc nc 18S twist1a twist1a C D pv pv cn cn twist2/fli1a E 18S 14S twist2/fli1a 18S twist2/fli1a 18S Figure 3.12: Zebrafish twist expression along the trunk. Lateral (A, C & D), dorsal (B) and sagittal section (E) views are shown. At 18-somite stage, expression of twist1a is detected in the sclerotome and neural crest delaminated from the neural tube (arrowhead) (A & B). Zebrafish twist2 is expressed in the presumptive vasculature (C, D & E)), as shown by colocalization with molecular marker fli1a. cn, caudal notochord; nc, neural crest; pv, presumptive vasculature; sc, sclerotome. 69 twist1b/dlx2a A B 14S 16S twist1b/dlx2a D C twist3/dlx2a hm hm hm hm fb fb 18S 18S Figure 3.13: Zebrafish twist1b and twist3 expression during somitogenesis. Ventral flat mount (A), lateral (B) and transverse section (C-D) views are shown. Zebrafish twist1b and dlx2a double color hybridization shows twits1b expression in the cranial neural crest-derived head mesenchyme (A). At the 16-somite stage, twist1b expression is detected in the head mesenchyme (arrowheads) and caudal gut (arrow) (B). Zebrafish twist1b (C) and twist3 (D) are not expressed in the forebrain but in the head mesenchyme adjacent to forebrain. 70 A 6S B C 9S 10S D 11S 14S F 14S no E no G 18S H 18S I 21S no Figure 3.14: Zebrafish twist1b expression in the somites. Embryos are shown with anterior to the left (A, B, D, F, H & I). Dorsal flat mount (A, D, F), transverse section (C, E & G), sagittal section (B), and lateral flat mount (H & I) views are shown. During earlier stages of somitogenesis (A-D), twist1b transcripts are strongly expressed in the sclerotome (arrows). No expression is observed in the posterior half of the somite. At the 14-somite stage (E & F), weak expression is restricted to the anterior portion of the successively more posterior somites. During late somitogenesis (G – I), weak twist1b expression is confined to the last seven somites from the tail bud, again, only in the anterior portion of each somite. 71 At the prim-5 stage (24-hpf), twist1a was expressed in the sclerotome, delaminated neural crest cells from the neural tube, and weakly in the fin bud (Figure 3.15A, E & I). Zebrafish twist1b transcripts were localized in the head mesenchyme between the eye and diencephalon, cranial neural crest stream 1 to 3, posterior somites, and the tail bud (Figure 3.15B, F & J). Two-color in situ hybridization with wnt1, a marker for glomerulus showed that twist1b is not expressed in the glomerulus (Figure 3.15K). Zebrafish twist2 transcripts were observed in the hypochord, dorsal aorta and caudal notochord (Figure 3.15C, G & L). Strong expression of twist3 was detected in the head mesenchyme, fin bud, and tail bud, with weak expression in the somites (Figure 3.15D, H & M). By the long-pec stage (48hpf), transcripts of all four zebrafish twist genes were detected in the pharyngeal arches (Figure 3.16A-H). In addition, twist1a and twist3 transcripts were also observed in the pectoral fins, specifically in the actinotrichs for twist1a (Figure 3.16I) and in the proximal (strong) and distal (weak) endochondral disc for twist3 (Figure 3.16K). Expression of twist1a was also detected in the head mesenchyme, fin fold and intermediate cell mass (Figure 3.16E & J). Weak expression of twist2 was detected in the dorsal aorta (Figure 3.16C). 72 twist1a A twist1b D C B fb twist3 twist2 fb fb fb E nc F sc s2 H G s3 fb s1 cn tb hm tb K J I M L nc no no s1 s2 s3 no gl sc twist1a hc da twist1b twist1b/wt1 twist2 twist3 Figure 3.15: Expression of zebrafish twist genes during the prim-5 stage. Dorsal (A-D), lateral (E-H) and transverse section (IL) views are shown. At 24 hpf, twist1a is expressed in the sclerotome, neural crest cells delaminated from the neural tube and fin bud (A, E and I). Zebrafish twist1b is expressed in the neural-crest derived head mesenchyme (s1, s2 & s3), tail bud and three preceding somites (B, F). No expression is observed along the trunk (J) and in the glomerulus (K), as shown by double color in situ with wt1. twist2 expression is observed in the hypochord, dorsal aorta and caudal notochord (C, G, and L). twist3 is expressed in the head mesenchyme, fin bud, and tail bud, with low expression in the somites (D, H, and M). Dotted lines denote the location of the transverse sections. cn, caudal notochord; da, dorsal aorta; fb, fin bud; ff, fin fold; gl, glomerulus; hc, hypochord; hm, head mesenchyme; nc, neural crest; no, notochord; s1 – s3, neural crest streams 1 through 3; sc, sclerotome; tb, tail bud. 73 twist1a A fb twist3 twist2 twist1b B C da pa fb D pa fb fb ff E G F icm pa I pa H pa pa 48hp 36hp K J fb ed no twist1a at hm twist1a twist3 Figure 3.16: Expression of zebrafish twist genes during the long-pec stage. Dorsal (A-D), lateral (E-H), transverse section (J), and flat mount (I, K) views are shown. At 48 hpf, all four twist genes are expressed in the pharyngeal arch (A-H). Only twist1a and twist3 transcripts are detected in the fin buds (A, E, D, H, I, K). twist1a is also expressed in the head mesenchyme (arrow), fin fold and intermediate cell mass (E, J), and twist2 is weakly expressed in the dorsal aorta (C, G). Dotted lines denote the location of the transverse sections. at, actinotrichs; da, dorsal aorta; ed, endochondral disc; fb, fin bud; ff, fin fold; hm, head mesenchyme; icm, intermediate cell mass; pa, pharyngeal arches; no, notochord; tb, tail bud. 74 At the protruding mouth stage (72hpf), all four zebrafish twist transcripts continued to be observed in the pharyngeal arches (Figure 3.17A-H). Additionally, twist1a was expressed in the head mesenchyme, heart valve, pectoral fin, fin fold and intermediate cell mass (Figure 3.17A & E). Expression in the heart was not observed for the other twist genes (Figure 3.17B-D). Zebrafish twist1b was also expressed in the olfactory placode (Figure 3.17F). Expression of twist3 was also detected in the pectoral fin (Figure 3.17D & H). 75 twist1a twist1b ff A B twist3 twist2 D C icm pa pa pa hv E F pa H G pf pf pa of hm pf Figure 3.17: pa pa pa pf Expression of zebrafish twist genes during the hatching period. Lateral (A-D) and dorsal (E-H) views are shown. At 72hpf, all four twist genes are expressed in the pharyngeal arch (A-H). Zebrafish twist1a and twist3 are expressed in the pectoral fins (E, H). twist1b is also found to be expressed in the olfactory placode (F), and twist1a is expressed in the head mesenchyme, heart valve (arrow head), fin fold and intermediate cell mass (A, E). Insets show enlarged heart views. ff, fin fold; hm, head mesenchyme; hv, heart valve; icm; intermediate cell mass; of, olfactory placode; pa, pharyngeal arch; pf, pectoral fin. 76 Chapter 4: Discussion _____________________________________________________________ 4.1 The zebrafish twist gene family The zebrafish twist gene family consists of four members: twist1a, twist1b, twist2 and twist3 (Gitelman, 2007). All four members are highly conserved transcription factors that shared the bHLH domain and the WR motif. Within the conserved bHLH domain and WR motif, high nucleotide similarity of up to 86% is observed. Regions outside the conserved domains are also relatively similar (52-81% similarity) (Table 2). Prior to the Gitelman (2007) publication, there were three members of the zebrafish twist gene family reported in GenBank (Accession no: NM_130984 for twist1, NM_130985 for twist2 and NM_001005956 for dermo1). However, there was some confusion in the identity of the zebrafish orthologs as the namings by Gitelman (2007) were different from GenBank. The first step in identifying and confirming the true zebrafish orthologs was to compare them against the sequences in other species. Zebrafish twist1a and twist1b show a higher sequence similarity to Twist1 in other species compared to zebrafish twist2 and twist3 with other species Twist1. This suggests that zebrafish twist1a and twist1b are coorthologs of mammalian Twist1. Interestingly, zebrafish twist1a and twist1b have higher similarity to mammalian Twist2 compared to mammalian Twist1. Nevertheless, the ortholog of human TWIST2 is zebrafish twist2 because zebrafish twist2 has higher similarity (compared to zebrafish twist1a and twist1b with Twist2 of other species) and the alignment also showed that zebrafish twist2 peptide is highly similar to mammalian 77 Twist2 peptide (Figure 3.4 and Table 3). The high sequence similarity of zebrafish twist1a and twist1b to mammalian TWIST2 suggests that the functions of zebrafish twist1a and twist1b could be more similar to the functions of mammalian TWIST2 compared to the functions of mammalian TWIST1. Based on the amino acid analysis, TWIST2 is highly conserved not just in mammals but also in chick suggesting conserved functions. Interestingly, zebrafish twist3 has a lower sequence similarity to chick and Xenopus Twist3 than zebrafish twist1 and twist2. However, the sequence similarity of zebrafish twist3 compared to the other Acanthopterygiian twist3 (medaka and stickleback) is significantly higher compared to zebrafish twist1 or twist2. 4.2 Phylogenetic relationships of the twist genes in fish Using peptide sequences from 33 homologous Twist proteins belonging to 12 different species, a clear phylogenetic clustering of the vertebrate Twist proteins into 3 major clades was observed, each of which contained both teleost and tetrapod orthologs. Zebrafish Twist1a (Dr1a) and Twist1b (Dr1b) fall within clade I, together with Twist1a from medaka, stickleback, fugu, and pufferfish (Figure 3.6). However, Twist1b from medaka, stickleback, fugu, and pufferfish did not cluster within clade I, but instead grouped together with the Twist3 proteins within clade III in the rooted and unrooted trees. These observations reflect the very high degree of peptide sequence similarity between Twist1a and Twist1b in zebrafish, a member of the superorder Ostariophysi, whereas the Twist1b peptides in the other four fish species, which all belong to the 78 superorder Acanthopterygii, exhibit greater peptide similarity with Twist3 than with Twist1a (Figure 3.4). While the Twist1b peptides of the acanthopterygii are tightly clustered together, their presence within clade III raises questions regarding their true phylogenetic relationships. A recent phylogenetic analysis of a similar set of Twist peptides, using multiple phylogenetic reconstruction methods, placed the acanthopterygian Twist1b peptides outside of the 3 major clades (Gitelman, 2007). The author postulated that this outgroup was likely an artifact of long-branch attraction, and suggested that their placement within clade I was the most parsimonious choice. I now provide evidence from the analysis of conserved synteny between the zebrafish and medaka twist1a and twist1b chromosomal regions that despite the observed phylogenetic grouping of the acanthopterygian Twist1b, these genes are the true orthologs of ostariophysian twist1b, as Germanguz et al. (2007) and Gitelman (2007) had predicted. The comparative synteny studies also demonstrate that the ostariophysian and acanthopterygian twist1a and twist1b genes are co-paralogs and co-orthologs of mammalian Twist1/TWIST1. Thus, although phylogenetic analysis algorithms provide reasonable estimations of phylogenetic and evolutionary relationships of gene families among species, ambiguities may arise which could be resolved through other methods such as comparative analysis of conserved synteny. 4.3 Genetic distance analysis of twist1a and twist1b among the fishes The Kimura and Tajima-Nei analyses of the nucleotide substitution rates between twist1a and twist1b cDNA sequences are consistent with the observed closer evolutionary 79 relationship among the four acanthopterygian fish as compared to the ostariophysian zebrafish. They also account for the observed phylogenetic clustering of the acanthopterygian Twist1b peptides into clade III. Given the substantial Twist1b peptide sequence difference between zebrafish and the other four fish species, twist1b expression and functional observations made in the zebrafish may not necessarily be extrapolatable to other species. Germanguz et al. (2007) have hypothesized that Twist1b in medaka, stickleback, fugu, and pufferfish may be involved in building the interarcual cartilage, a cephalic neural crest derivative that defines all four fish. It would be interesting to see if the medaka twist1b expression profile has indeed diverged significantly from zebrafish. 4.4 Comparative synteny analyses A detailed comparative synteny analysis was performed to confirm the zebrafish orthologs of human TWIST1 (Table 4). The comparative table clearly showed that zebrafish twist1a and twist1b are co-orthologs of the human TWIST1 as the genes around human TWIST1, on chromosome 7, were found to be duplicated or located on either LG16 or LG19 where zebrafish twist1a and twist1b are located respectively (Table 4). Both genes are linked to the hoxa gene cluster, whose duplication in ray-finned fishes was inferred to occur by whole genome duplication (Amores et al., 1998). Thus zebrafish twist1a and twist1b are co-paralogs and co-orthologs of human TWIST1 that most likely arose through a fish-specific genome duplication event. Due to some confusion in the naming of the zebrafish LG16 and LG19 located twist1 co-paralogs in the genomic databases, a more in-depth comparative synteny analysis of the zebrafish and medaka twist1a and twist1b chromosomal regions was 80 performed to resolve their orthologies. Comparison of the chromosomal region around zebrafish twist1a (on LG16) against the chromosomal regions around medaka twist1a (on LG16) and twist1b (on LG11), revealed that a large number of genes were present on both zebrafish LG16 and medaka LG16. Although there were some genes that were in common between zebrafish LG16 and medaka LG11, these were comparatively fewer (Table 5). When the chromosomal region around zebrafish twist1b (on LG19) was compared against the chromosomal regions around medaka twist1a and twist1b, a large number of genes were observed to be present on both zebrafish LG19 and medaka LG11 region, with fewer shared genes between zebrafish LG19 and medaka LG16 (Table 5 & 6). These observations provide further confirmation that the twist1a and twist1b genes arose from a fish-specific genome duplication event and are true co-paralogs. Furthermore, the results indicate that the zebrafish LG16 and LG19 twist1 genes are orthologous to the medaka LG16 and LG11 twist1 genes, respectively. The comparative synteny analysis of the zebrafish twist2 chromosomal segment clearly revealed that zebrafish twist2 is the ortholog of human and medaka Twist2 as it shows chromosomal synteny with the human and medaka twist2 loci on chromosome 2 and LG21, respectively (Table 7). Zebrafish twist3 has no known mammalian ortholog, and lacks chromosomal synteny with any of the other twist loci in mammals, consistent with its placement in a clade separate from twist1 or twist2. The comparative synteny analysis of zebrafish twist3 with medaka twist3a and twist3b show that the sole zebrafish twist3 gene is orthologous to both medaka twist3a and twist3b, although there are more shared genes 81 between the zebrafish twist3 chromosomal region and the medaka twist3b chromosomal region (Table 8). The two medaka twist3 genes appear to have arisen through a chromosomal or genome duplication event, as suggested by the presence of multiple shared genes between LG5 and LG7. Unlike twist1, however, the number of twist3 genes is variable, even within the acanthopterygii. There are two copies in medaka and stickleback, one in zebrafish, and none in fugu and pufferfish. Combining the data from our phylogenetic and comparative synteny analyses, a model for the evolutionary history of the twist genes has been reconstructed (Figure 4.1). This model assumes the occurrence of three genome duplication events in the evolutionary history of the teleosts. The first two events occurred prior to the divergence of the teleost ancestors the ray-finned fishes (Actinopterygii) from the tetrapod ancestors the lobe-finned fishes (Sarcopterygii). In the first duplication event, the ancestral Twist gene probably gave rise to the Twist3 gene and a Twist1/Twist2 ancestral gene. This latter ancestral gene then split to give rise to Twist1 and Twist2 during the second duplication event. Subsequently during the teleost-specific third duplication event, which occurred approximately 350 million years ago (Ravi and Venkatesh, 2008), the twist1 and twist3 genes were duplicated into twist1a and twist1b and into twist3a and twist3b, respectively. The absence of a complete set of 8 twist genes in any of the fish lineages that have been examined suggests that some of the duplicated genes were lost over time through functional redundancy. 82 D Tr1a Tn1a Ol1a Ga1a Dr1a Dr1b Ga1b Ol1b Tn1b Tr1b Twist1 Mm1 Rn1 Hs1 Gg1 Xt1 Tr2 Tn2 Ol2 Ga2 D Dr2 Mm2 Rn2 D Twist2 Hs2 Gg2 Ol3a Ga3a D Dr3 Ol3b Ga3b Twist3 Gg3 Xt3 Bb Lv Figure 4.1: A model for the evolutionary history of twist genes. Circle with a 'D' in the middle represent gene or genome duplication. 83 4.5 Comparison of zebrafish twist family expression patterns with other species. 4.5.1 Zebrafish twist1a and twist1b genes Zebrafish twist1a and twist1b are co-orthologs of mammalian Twist1. Interestingly, the expression patterns of this duplicated gene revealed shared and unique expression sites. Zebrafish twist1b is present as a maternal transcript in early zebrafish embryos prior to the mid-blastula transition; this observation is consistent with mouse and Xenopus Twist1 (Stoetzel et al., 1995; Stoetzel et al., 1998). The transcripts of zebrafish twist1a were first detected at 1K-cell stage by RT-PCR (Figure 3.8). This observation is similar in Drosophila, where twist is expressed as a zygotic transcript (Thisse et al., 1987; Thisse et al., 1988). During early somitogenesis from the 2-somite to the 10-somite stage, zebrafish twist1b is expressed in the intermediate mesoderm, also known as the presumptive pronephric duct. The 2-5 somite stage coincides with the first of 4 stages in the development of the zebrafish pronephros, when the intermediate mesodermal cells become committed and a nephrogenic field is established. The other 3 stages are between the 6-somite and prim-5 stages, when the pronephric duct and nephron primordia are formed, from the prim-5 to high-pec stages, when cell patterning events take place leading to the formation of the pronephric glomeruli and tubules, and from the high-pec to hatching (long-pec) stages, when the pronephros becomes functional with the formation of the glomerular capillary tuft and onset of blood filtration (Solnica-Krezel, 2002). Lee et al. (2000) reported that expression of human TWIST1 and/or DERMO1 (TWIST2) protein has been observed in the renal collecting and secretory tubules. Interestingly, there have been reports of patients with SCS who had renal tubular 84 dysfunction or agenesis (Russo et al., 1991; Oktenli et al., 2002), although the relationship between the renal condition and the TWIST1 gene in these patients remains to be proven. Two color in situ hybridization of twist1b and wt1, a marker for glomerulus, showed the absence of twist1b expression in the glomerulus. The presence of twist1b transcripts in the intermediate mesoderm only during stage 1 and early stage 2 of zebrafish pronephros development suggests that twist1b may be important in the commitment of the undifferentiated mesodermal cells to a nephrogenic fate. The localized expression of twist1a and twist1b to the sclerotome of the developing somites is consistent with similar observations in mouse, Xenopus, chick and medaka (Table 10) (Hopwood et al., 1989; Wolf et al., 1991; Tavares et al., 2001; Yasutake et al., 2004). In zebrafish, the sclerotome is located at the ventromedial region of each somite and gives rise to connective tissue and vertebral cartilages. The presence of twist1a and twist1b transcripts in the sclerotome suggests a role in somitogenesis and vertebral cartilage formation. Knockdown studies in the medaka fish reveal that twist1a is not involved in the migration of sclerotomal cells but in regulating the differentiation of the sclerotomal cells into the vertebral column (Yasutake et al., 2004). Furthermore, cervical spine abnormalities have been described in patients with SCS, including fusion of the vertebral bodies and posterior elements (Anderson et al., 1997; Trusen et al., 2003). Together, these observations strongly suggest that tight regulation of TWIST1 gene expression is necessary for normal development of vertebral bodies. The zebrafish cranial neural crest cells appear to be specified at an early stage and migrate in three distinct streams (Schilling and Kimmel, 1994). Stream 1 will eventually give rise to the first or mandibular branchial/pharyngeal arch, stream 2 will form the 85 second or hyoid arch, and stream 3 will give rise to the 5 ceratobranchial arches (Lumsden et al., 1991; Piotrowski and Nusslein-Volhard, 2000). Consistent with Twist1 expression in mouse and Xenopus, zebrafish twist1a and twist1b was expressed at the neural crest cells and later in the pharyngeal arches (Hopwood et al., 1989; Fuchtbauer, 1995). Twist1-/- knockout mouse embryos showed merging of the first and second branchial arches (Soo et al., 2002), suggesting that Twist1 activity is required for proper migration of the different streams of neural crest cells and differentiation of the branchial arches. 4.5.2 Zebrafish twist2 Consistent with the observation of Dermo-1 (Twist2) expression in the mouse, zebrafish twist2, whose transcript was detected at the 1K-cell stage, is present as a zygotic transcript (Li et al., 1995). During the pharyngula period, twist2 is also expressed in the pharyngeal arches. The expression of zebrafish twist2 in the pharyngeal arches is consistent with the expression of mouse twist2 (Li et al., 1995). Studies performed in mouse showed that Twist2 was expressed in osteoblastic cells and it may act as a negative regulator of the osteoblast differentiation, i.e. to inhibit osteoblast maturation and maintain the cells in a preosteoblast phenotype (Tamura and Noda, 1999; Lee et al., 2000). I was unable to detect the expression in osteoblastic cells because I was only looking at early embryonic expression up to 72 hpf and the osteoblast cells are not formed yet. 86 4.5.3 Zebrafish twist3 The Twist3 gene is absent in mammals but found in Xenopus, chick, medaka, stickleback and zebrafish (Gitelman, 2007). No expression data of the Twist3 gene is available in other species. 4.6 Shared and unique expression sites of the zebrafish twist genes 4.6.1 Importance of using unique 3’UTR sequences as riboprobes Two reports in the 1990s used zebrafish twist1 (twist1b) as a molecular marker for the sclerotome and the axial mesoderm (Halpern et al., 1995; Morin-Kensicki and Eisen, 1997). However, in this study, the expression in the axial mesoderm is from zebrafish twist2 and not zebrafish twist1b. It is possible that the apparent twist1b expression in axial mesoderm could have arisen from use of a riboprobe that included coding sequences, which could have cross-hybridized to twist2 transcript. As mentioned above and shown in the results section, the zebrafish twist genes have high sequence similarity especially in the coding region. Thus, in this study, the design of unique riboprobes is of great importance and 3’UTR sequences were chosen to be used as riboprobes to exclude and minimize any possible cross-hybridization among the family members. 4.6.2 Comparison of zebrafish twist genes expression sites with other publications The expression patterns of the zebrafish twist gene family have been previously described by Rauch (2003) and Thisse and Thisse (2004) in the ZFIN database, and 87 recently by Germanguz et al (2007). Comparing my in situ results and those previously reported, I found differences in the expression patterns and the differences could be attributed by the targeting RNA probes used for in situ hybridization. Previously reported expression profiles were performed using either expressed sequence tags (ESTs) or full-length cDNA, either exclusively or in combination with shorter gene-specific probe outside of conserved domains. In profiling of the twist gene expression, hybridization probes targeting unique 3’ UTR sequences of each of the four twist genes were utilized to completely avoid the highly homologous bHLH and WR domains (Table 2). Members of the twist gene family are highly homologous and this present study may provide a good example to illustrate that the use of probes targeting at different sequences of highly homologous gene family members can give rise to different expression profiles. Zebrafish twist1a and twist1b are co-orthologs of mammalian Twist1. Using both RT-PCR and whole-mount in situ hybridization analyses, maternal transcripts of zebrafish twist1b are detected in early zebrafish embryos prior to the mid-blastula transition. In contrast, zebrafish twist1b expression was detected only at the gastrula period by two previous groups (Rauch, 2003; Germanguz et al., 2007). Using RT-PCR, zebrafish twist1a transcripts is detected at 1K-cell stage and subsequenct in situ hybridization shows its expression in the neural crest cells at the bud stage (10hpf). This zygotic expression pattern of zebrafish twist1a is consistent with previously published data (Germanguz et al., 2007; Gitelman, 2007). Similarly, zebrafish twist2 transcripts are detected at 1K-cell stage by RT-PCR and previous reports in zebrafish also show that twist2 is zygotically transcribed (Thisse and Thisse, 2004; Germanguz et al., 2007; Gitelman, 2007). For zebrafish twist3, its expression is detected from as early as in the 1- 88 cell zygote stage (0.2 hpf) by both RT-PCR and in situ hybridization, strongly suggest that it is a maternal transcript. In contrast, this was not previously reported and the twist3 expression is only detected from the 13-somite stage (16 hpf) onwards (Germanguz et al., 2007; Gitelman, 2007). During early somitogenesis, Twist1 is expressed in the lateral plate mesoderm in mouse, chick and Xenopus (Hopwood et al., 1989; Wolf et al., 1991; Tavares et al., 2001). Similarly, previous report also showed that zebrafish twist1a (early- to midsomitogenesis) and twist3 (mid-somitogenesis) are expressed in the lateral plate mesoderm Germanguz et al. (2007). However, I did not detect any expression in the lateral plate mesoderm except for zebrafish twist1a and twist3 in the fin bud, which is derived from the posterior lateral plate mesoderm (Table 8). A previous report also found twist1a, twist1b, and twist3 expression in the pectoral fin bud (Germanguz et al., 2007; Gitelman, 2007). Contrastingly, I observed pectoral fin bud expression only for twist1a and twist3 and this observation corroborates with Rauch (2003) data. I also detected twist1b expression in the intermediate mesoderm until late somitogenesis and this was confirmed by two-color WISH using pax2.1 molecular marker. Zebrafish twist1a and twist1b is expressed in the sclerotome and twist3 in the somite. I also detected twist1b expression in the caudal gut at around the 16-somite stage (17hpf). However, a previous study showed zebrafish twist1b expression in the myotome and ventral gut (Germanguz et al., 2007). Besides the expression in the somites or sclerotome expression, I also detected twist1a expression in the intermediate cell mass during the pharyngula period (24-48 hpf), which was not reported previously. 89 Additionally, zebrafish twist1a, twist1b, and twist2 were previously reported to be expressed in the blood vessels of the developing brain at around 36 hpf (Germanguz et al., 2007), but I did not observe this. In contrast, I found that only zebrafish twist2 is expressed in the presumptive vasculature along the body from the 14- to 18-somite stage (16-18 hpf), and I further confirmed this localization by two-color in situ hybridization with fli1a, a molecular marker for presumptive vasculature. This expression has not been previously reported. Furthermore, I observed that twist2 expression in the axial mesoderm during mid-somitogenesis was concentrated not in the tail bud as previously reported (Germanguz et al., 2007; Gitelman, 2007), but in the caudal notochord. The Twist genes are also found to be expressed in the pharyngeal arches in mouse, chick and Xenopus (Hopwood et al., 1989; Wolf et al., 1991; Li et al., 1995; Scaal et al., 2001; Tavares et al., 2001). Similarly, we also observed expression of all the four members of the zebrafish twist gene family in the pharyngeal arches which corroborates with previously reported data for zebrafish twist gene family (Thisse and Thisse, 2004; Germanguz et al., 2007). Table 9 summarizes the essential similarities and differences in twist expression patterns between this study and the previous data reported in Germanguz et al (2007) and Gitelman (2007). It is not entirely clear why there are substantive differences in twist expression sites observed between my study and those previously reported but I note that the differences may be due to the different segments of cDNA used as probes by the different groups. 90 Table 9: Expression domains of the four zebrafish twist genes Period  Stage (time)  Tissue  Present study; Yeo et al. (2007)  Dr1a  Dr1b  Dr2  Dr3  (LG16)  (LG19)  (LG9)  (LG23)  Germanguz et al. / Gitelman (2007)  Dr1a  Dr1b  Dr2  Dr3  (LG16) (LG19) (LG9)  (LG23) Cleavage  Zygote (0 hpf)  ‐                  1K‐cell (3 hpf)  ‐                  Shield (6 hpf)  Whole embryo  Areas lateral to embryonic axis  Organizer  Whole embryo  Axial mesoderm  Anterior neural plate & mesodermal tissues                                                                                                  Whole embryo  Head mesenchyme  Axial mesoderm / Chordamesoderm  Intermediate mesoderm  Lateral plate mesoderm  Cephalic neural crest / Premigratory crest cells  Cranial/cephalic neural crest derived head  mesenchyme   Neural rod  Axial mesoderm / Chordamesoderm  Intermediate mesoderm  Lateral plate mesoderm  Premigratory crest cells  Somite  Sclerotome  Myotome  Tail bud  Cranial/cephalic neural crest derived head  mesenchyme  Olfactory placode  Dorsal forebrain  Migratory neural crest cells  Neural crest cells (delaminated from neural tube)  Intermediate mesoderm  Lateral plate mesoderm  Nephric duct/pronephric duct  Somite  Sclerotome  Myotome  Presumptive vasculature  Caudal gut (16‐somite)  Ventral gut (15‐somite)  Caudal notochord  Tail bud  Forebrain/Midbrain/Hindbrain area  Pharyngeal arches   Nephric duct/ pronephric duct  Hypochord  Somite  Sclerotome  Myotome  Pectoral fin bud  Tail bud                                                                                                                                                                                                                                                                                                                                                                                                                        a                         a                                                                                                                                                           Head mesenchyme (cranial neural crest derived)  Brain (forebrain, midbrain, hindbrain)  Neural crest cells (delaminated from neural tube)  Dorsal aorta                                                  Blastula  Gastrula  Bud (10 hpf)  Segmentation  2‐somite (10.7 hpf)  5‐10 somite (11.5‐14 hpf)  13‐18 somite (15.5‐18 hpf)  24 somite  Pharyngula  Prim‐5 (24 hpf)  Not analyzed                  91 30 hpf    36 hpf    Long‐pec (48 hpf)    Hatching  Protruding mouth (72 hpf)    a                                                                                                 Not reported  Head mesenchyme  Olfactory placode  Pharyngeal arch  Pectoral fin  Fin fold  Heart valve  Intermediate cell mass                                                          Not reported  Not analyzed  Not analyzed      a                                                         Hypochord  Nephric duct/pronephric duct  Somite  Sclerotome  Pectoral fin bud  Caudal notochord  Tail bud  Nephric duct/pronephric duct  Tail bud  Vasculature of developing forebrain / midbrain  Olfactory placode  Dorsal aorta  Cranial neural crest derivatives (pharyngeal arch)  Pectoral fin bud  Dorsal fin bud  Head mesenchyme  Pharyngeal arch  Pectoral fin  Fin fold  Intermediate cell mass                                dorsal somite 4.7 Evolutionary fates of the zebrafish twist gene family The distinct and overlapping expression sites that I observed for the different members of the twist gene family indicate that twist gene members may perform both redundant and non-redundant functions during embryonic development. The functions of each gene member in a gene family may evolve over time through non-, neo-, sub- and/or synfunctionalization (Postlethwait et al., 2004; Gitelman, 2007; Roth et al., 2007). For example, the mouse twist1 expression domains in the nasal placode and cells of the limb bud are partitioned between zebrafish twist1b (olfactory placode) and zebrafish twist1a and twist3 (pectoral fin) (Table 10). However, until the definitive expression patterns of these genes are confirmed, it would not be easy to make meaningful conclusions about the evolutionary changes that have occurred in the various zebrafish twist genes. For example, Gitelman (2007) attributed the unique expression of zebrafish twist1b in the 92                               myotome as a neofunctionalization event. However, I have not been able to reproduce the twist1a expression in the myotome in our study. Also, if indeed none of the zebrafish twist genes are expressed in the lateral plate mesoderm, this may represent a nonfunctionalization event in the fish lineage, or conversely a neofunctionalization of Twist1 in the tetrapod lineage, which exhibits lateral plate mesoderm expression in mouse, chick and Xenopus. However, I note that zebrafish twist1a is expressed in the intermediate cell mass, a derivative of the lateral plate mesoderm (Table 10). Additional detailed and careful expression profiles in other species are necessary in order to construct a comprehensive understanding of the evolution of the vertebrate, and teleost, twist genes. Nevertheless, what is clear is that despite the still significant degree of peptide similarity among the four zebrafish twist genes, their regulatory control has diverged to the point that there is now minimal overlap of their developmental expression profiles, and thus minimal functional redundancy among them. 93 Table 10: Twist expression sites in selected species. Mm1a  Mm2b  Lateral plate mesoderm              Head mesenchyme (neural crest derived)              Intermediate mesoderm  Somite  Caudal gut  Gg2d  Xl1e  Dr1a  Dr1bf  Dr2  Dr3                                                                      Neural crest cells                    Olfactory placode                    Pharyngeal arch/branchial arch                                      Axial mesoderm/caudal notochord   Tail bud                    Pectoral fin bud / limb buds                    Hypochord                     Dorsal aorta                    Fin fold /body wall                    Presumptive vasculature                    Heart valve                                    Intermediate cell mass  a Gg1c  b   c d e f (Wolf et al., 1991);  (Li et al., 1995);  (Tavares et al., 2001);  (Scaal et al., 2001);  (Hopwood et al., 1989);  (Yeo et al., 2007)  x 94 Chapter 5: Conclusion ________________________________________________________________________ In this study, I have clarified and confirmed the evolutionary orthology of the four zebrafish twist genes with their mammalian counterparts and other fish species by nucleotide similarity comparison, phylogenetic analysis as well as comparative synteny analysis. 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Mol Biol Evol 25:131-143. 101 102 [...]... analysis of the chromosomal regions flanking the zebrafish, medaka, and human twist genes showed that the zebrafish twist1 a and twist1 b are coparalogs and co-orthologs of human TWIST1 Furthermore, zebrafish twist1 a and twist1 b are orthologous to medaka twist1 a and twist1 b, respectively, despite the different phylogenetic clusterings of zebrafish and medaka twist1 b The orthology of zebrafish twist2 to... structure, and map location of the zebrafish twist genes 2 Sort out the confusion in evolutionary orthology of the zebrafish twist genes with their mammalian counterparts through comparative gene structure and linkage synteny analyses 3 Characterize and compare the developmental and tissue-specific expression profiles of the zebrafish twist genes during embryogenesis and in adult zebrafish 4 Compare expression. .. and developmental expression patterns of the zebrafish twist gene family (reference in press) xii Abstract _ Four members of the twist gene family (twist1 a, 1b, 2 and 3) are found in the zebrafish, and they are thought to have arisen through three rounds of gene duplication, two of which occurred prior to the tetrapod-fish split Phylogenetic analysis groups most of the. .. structures, including the forebrain and area of the nasal placodes, the diencephalon and the optical vesicles, the rhombencephalon and around the otic vesicles Furthermore, a high level of expression was observed in the branchial arches In the trunk, Twist1 expression is detected in the sclerotome and somatopleura In addition, Twist1 expression is also found in the posterior limb buds and tail, the mesenchyme... 2007) Phylogenetic and gene expression studies are invaluable tools that aid in our understanding of the regulation and function of conserved genes and gene families such as the TWIST gene family Together, they provide important clues to the evolutionary events and functional changes that have occurred in these genes in different species 14 1.5 Aims In this study, I aimed to: 1 Determine the complete... (Samonte and Eichler, 2002) In the TWIST gene family, gene duplication is observed TWIST1 and TWIST3 genes are found to be duplicated in some species Duplication of this Twist1 gene (twist1 a and twist1 b) has been observed specifically in Actinopterygii (ray-fined fishes) (Gitelman, 2007) In stickleback and medaka, there are also two copies of the twist3 gene (Gitelman, 2007) 1.4.1 Evolutionary fates of duplicate... Zebrafish twist1 a and twist1 b were expressed in the sclerotome and twist3 in the somite during the segmentation period Zebrafish twist1 b and twist3 were found to be present as maternal transcript Many expression sites were unique Transcripts of twist1 a were detected specifically in the premigratory neural crest cells during early somitogenesis and in the heart valve at the hatching period Zebrafish twist1 b... and medaka TWIST2 /twist2 , was also confirmed Finally, zebrafish twist3 showed greater chromosomal synteny to medaka twist3 b than to the medaka twist3 a Based on these results, a model for the evolutionary history of the twist genes has been reconstructed I also performed a comprehensive developmental expression analysis of all four twist genes All four genes were expressed in the pharyngeal arches Zebrafish. .. synfunctionalization, one copy of the duplicate gene acquires a unique expression domain of the other and hence, all unique functions of this gene can be found on one copy of the duplicates Therefore, the other copy of the duplicate gene becomes redundant and leads to gene loss (Figure 1.2) (Gitelman, 2007) If both copy of the duplicate gene are retained due to their remaining unique functions, function shuffling 13... expressed in the intermediate mesoderm during segmentation period and in the olfactory placode at the hatching period Zebrafish twist2 expression was observed in the organizer at the shield stage, presumptive vasculature during the segmentation period, and in the hypochord and dorsal aorta during the prim-5 stage Zebrafish twist1 a and twist3 were expressed in the fin bud, with twist3 expression concentrated ... pattern with other species 83 4.5.1 Zebrafish twist1 a and twist1 b genes 83 4.5.2 Zebrafish twist2 85 4.5.3 Zebrafish twist3 86 4.6 Shared and unique expression sites of the zebrafish twist genes 86... proteins generated by the neighbor-joining method Figure 3.6: Gene structure of twist1 a, twist1 b, twist2 and twist3 Figure 3.7: RT-PCR of zebrafish twist genes Figure 3.8: Expression of zebrafish twist. .. medaka, and human twist genes showed that the zebrafish twist1 a and twist1 b are coparalogs and co-orthologs of human TWIST1 Furthermore, zebrafish twist1 a and twist1 b are orthologous to medaka twist1 a