Expression of the dnmt3 genes in zebrafish development: similarity to Dnmt3a and Dnmt3b

The zebrafish differs from mammals in that they have six dnmt3 genes as opposed to the two that can produce a catalytically active protein in mammals. Zebrafish also do not show evidence of genomic imprinting and lack the Dnmt3l gene necessary to that process in mammals. As such, they offer a unique opportunity to compare the two genetic situations in order to define the roles of the multiple genes in developmental gene methylation. To this end, we have analyzed the developmental expression of the six dnmt3 genes in zebrafish and find that they fall into two distinct patterns. The expression patterns of the dnmt6 and dnmt8 genes, which more closely resemble the mammalian Dnmt3a gene in sequence, also show an expression pattern that is more similar to the expression of Dnmt3a rather than Dnmt3b. Conversely, the other four dnmt3 genes in zebrafish (dnmt3, dnmt4, dnmt5, and dnmt7) show an expression pattern that is more similar to Dnmt3b. The dnmt6 and dnmt8 genes are also expressed in the adult zebrafish and in the brain in particular. In situ expression analyses show that the dnmt6 and/or dnmt8 genes also show tissue-specific differences in expression with those two genes being more ubiquitously expressed in the developing zebrafish than the other dnmt3 genes. Although differences in dnmt3 function may exist between mammals and fish, our results showing similar expression patterns between the genes in fish and mammals suggest that the six dnmt3 genes in the zebrafish may be analogous to the two Dnmt3 genes in mammals.     

Expression analysis of the family of 14-3-3 proteins in zebrafish development

14-3-3 proteins comprise a family of dimeric multi-functional proteins present in all eukaryotes, that are important in a whelm of ubiquitous biological processes. We have analyzed the genomic structure of all 14-3-3s from zebrafish comprising 11 genes and have analyzed their phylogeny. The gene family was cloned and its expression pattern in zebrafish embryogenesis was analyzed by whole mount in situ hybridization and microarray analysis with gene specific probes. We demonstrate that maternal mRNA of 14-3-3s is expressed evenly at the first cell division. At later stage all genes are expressed in a patterned way with, in most cases, intricate patterns in the developing brain. Our result shows distinct expression patterns of various genes. Microarray results show that differences in expression levels of highly similar 14-3-3 genes also occur in the adult stage.     

Evolution of trace amine associated receptor (TAAR) gene family in vertebrates: lineage-specific expansions and degradations of a second class of vertebrate chemosensory receptors expressed in the olfactory epithelium

The trace amine-associated receptors (TAARs) form a specific family of G protein-coupled receptors in vertebrates. TAARs were initially considered neurotransmitter receptors, but recent study showed that mouse TAARs function as chemosensory receptors in the olfactory epithelium. To clarify the evolutionary dynamics of the TAAR gene family in vertebrates, near-complete repertoires of TAAR genes and pseudogenes were identified from the genomic assemblies of 4 teleost fishes (zebrafish, fugu, stickleback, and medaka), western clawed frogs, chickens, 3 mammals (humans, mice, and opossum), and sea lampreys. Database searches revealed that fishes had many putatively functional TAAR genes (13-109 genes), whereas relatively small numbers of TAAR genes (3-22 genes) were identified in tetrapods. Phylogenetic analysis of these genes indicated that the TAAR gene family was subdivided into 5 subfamilies that diverged before the divergence of ray-finned fishes and tetrapods. In tetrapods, virtually all TAAR genes were located in 1 specific region of their genomes as a gene cluster; however, in fishes, TAAR genes were scattered throughout more than 2 genomic locations. This possibly reflects a whole-genome duplication that occurred in the common ancestor of ray-finned fishes. Expression analysis of zebrafish and stickleback TAAR genes revealed that many TAARs in these fishes were expressed in the olfactory organ, suggesting the relatively high importance of TAARs as chemosensory receptors in fishes. A possible evolutionary history of the vertebrate TAAR gene family was inferred from the phylogenetic and comparative genomic analyses.     

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.     

Expression of unconventional myosin genes during neuronal development in zebrafish

Neuronal migration and growth cone motility are essential aspects of the development and maturation of the nervous system. These cellular events result from dynamic changes in the organization and function of the cytoskeleton, in part due to the activity of cytoskeletal motor proteins such as myosins. Although specific myosins such as Myo2 (conventional or muscle myosin), Myo1, and Myo5 have been well characterized for roles in cell motility, the roles of the majority of unconventional (other than Myo2) myosins in cell motility events have not been investigated. To address this issue, we have undertaken an analysis of unconventional myosins in zebrafish, a premier model for studying cellular and growth cone motility in the vertebrate nervous system. We describe the characterization and expression patterns of several members of the unconventional myosin gene family. Based on available genomic sequence data, we identified 18 unconventional myosin- and 4 Myo2-related genes in the zebrafish genome in addition to previously characterized myosin (1, 2, 3, 5, 6, 7) genes. Phylogenetic analyses indicate that these genes can be grouped into existing classifications for unconventional myosins from mouse and man. In situ hybridization analyses using EST probes for 18 of the 22 identified genes indicate that 11/18 genes are expressed in a restricted fashion in the zebrafish embryo. Specific myosins are expressed in particular neuronal or neuroepithelial cell types in the developing zebrafish nervous system, spanning the periods of neuronal differentiation and migration, and of growth cone guidance and motility.     

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.     

Expression profiles of ndel1a and ndel1b, two orthologs of the NudE-Like gene, in the zebrafish

NudE-Like (NDEL1/NUDEL), through its interaction with LIS1 and DISC1, has been implicated in the etiology of neurological disorders such as lissencephaly and schizophrenia, respectively. Subsequently, a large portion of the research done on the function of NDEL1 has been specifically targeted to its role in brain development while ignoring its function in other developing and adult tissues. To begin a more global exploration of NDEL1's function, this study characterizes the developmental expression pattern of the NDEL1 orthologs in the zebrafish embryo. Our bioinformatic analyses identified two NDEL1 orthologs in the zebrafish, ndel1a and ndel1b. ndel1a is expressed predominantly in the anterior central nervous system (CNS), trigeminal ganglia, and eyes while ndel1b is expressed in the developing somites and, later, in the CNS. In addition to the spatial differences in their expression patterns, these genes are also individually regulated in their temporal expression. Both are expressed maternally but at later time-points there are subtle differences. ndel1a expression is lost between 6 and 12 hpf but then increases to a higher, near steady state, level from 72 to 120 hpf. ndel1b expression decreases from 3 to 36 hpf and subsequently increases from 36 to 120 hpf. The non-overlapping expression patterns of these two orthologs may indicate that they have split the functional role of the one NDEL1 gene present in mammalian species. The temporal and spatial regulation of these two orthologs will aid in the characterization of the multiple functions of this gene in both the developing and mature organism.     

Characterization of the retinoic acid receptor genes raraa, rarab and rarg during zebrafish development

Retinoic acid signaling is important for patterning the central nervous system, paired appendages, digestive tract, and other organs. To begin to investigate retinoic acid signaling in zebrafish, we determined orthologies between zebrafish and tetrapod retinoic acid receptors (Rars) and examined the expression patterns of rar genes during embryonic development. Analysis of phylogenies and conserved syntenies showed that the three cloned zebrafish rar genes include raraa and rarab, which are co-orthologs of tetrapod Rara, and rarg, which is the zebrafish ortholog of tetrapod Rarg. We did not, however, find an ortholog of Rarb. RNA in situ hybridization experiments showed that rarab and rarg, are maternally expressed. Zygotic expression of raraa occurs predominantly in the hindbrain, lateral mesoderm, and tailbud. Zygotic expression of rarab largely overlaps that of raraa, except that in later stages rarab is expressed more broadly in the brain and in the pectoral fin bud and pharyngeal arches. Zygotic expression of zebrafish rarg also overlaps the other two genes, but it is expressed more strongly in the posterior hindbrain beginning in late somitogenesis as well as in neural crest cells in the pharyngeal arches. Thus, these three genes have largely overlapping expression patterns and a few gene-specific expression domains. Knowledge of these expression patterns will guide the interpretation of the roles these genes play in development.     

Zebrafish promoter microarrays identify actively transcribed embryonic genes

We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.     

Coordinate embryonic expression of three zebrafish engrailed genes.

We have identified three genes, expressed in zebrafish embryos, that are members of the engrailed gene family. On the basis of sequence comparisons and analyses of their expression patterns, we suggest that two of these genes, eng2 and eng3, are closely related to the En-2 gene of other vertebrates. The third gene, eng1, is probably the zebrafish homolog of En-1. Subsets of cells at the developing junction between the midbrain and hindbrain express three different combinations of these genes, revealing a previously unknown complexity of this region of the CNS. Other cells, for example, jaw and myotomal muscle precursors, express two of the three genes in combinations which, in the myotomal muscles, change during development. Cells in the developing hindbrain and fins express only a single engrailed gene. We propose that the fates and patterning of these cells may be regulated by the coordinate expression of particular combinations of these closely related homeoproteins.     

Expression of Dlx genes during the development of the zebrafish pharyngeal dentition: evolutionary implications

In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.     

Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio)

Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.