Biochemical and Functional Characterization of the Three Zebrafish Transglutaminases 2.

Transglutaminase 2 (TG2) is a multifunctional protein widely distributed in various tissues and involved in many physiological and pathological processes. However, its actual role in biological processes is often controversial as TG2 shows different effects in these processes depending on its localization, cell type, or experimental conditions. We characterized the enzymatic and functional properties of TG2 proteins expressed in Danio rerio (zebrafish) to provide the basis for using this established animal model as a reliable tool to characterize TG2 functions in vivo. We confirmed the existence of three genes orthologous to human TG2 (zTGs2) in the zebrafish genome and their expression and function during embryonic development. We produced and purified the zTGs2s as recombinant proteins and showed that, like the human enzyme, zTGs2 catalyzes a Ca2+ dependent transamidation reaction that can be inhibited with TG2-specific inhibitors. In a cell model of human fibroblasts, we also demonstrated that zTGs2 can mediate RGD-independent cell adhesion in the extracellular environment. Finally, we transfected and selected zTGs2-overexpressing HEK293 cells and demonstrated that intracellular zTGs2 plays a very comparable protective/damaging role in the apoptotic process, as hTG2. Overall, our results suggest that zTGs2 proteins behave very similarly to the human ortholog and pave the way for future in vivo studies of TG2 functions in zebrafish.

Systematic expression profiling of neuropathy-related aminoacyl-tRNA synthetases in zebrafish during development.

Aminoacyl tRNA synthetases (ARSs) are a group of proteins, acting as transporters to transfer and attach the appropriate amino acids onto their cognate tRNAs for translation. So far, 18 out of 20 cytoplasmic ARSs are reported to be connected to different neuropathy disorders with multi-organ defects that are often accompanied with developmental delays. Thus, it is important to understand functions and impacts of ARSs at the whole organism level. Here, we systematically analyzed the spatiotemporal expression of 14 ars and 2 aimp genes during development in zebrafish that have not be previously reported. Not only in the brain, their dynamic expression patterns in several tissues such as in the muscles, liver and intestine suggest diverse roles in a wide range of development processes in addition to neuronal function, which is consistent with potential involvement in multiple syndrome diseases associated with ARS mutations. In particular, hinted by its robust expression pattern in the brain, we confirmed that aimp1 is required for the formation of cerebrovasculature by a loss-of-function approach. Overall, our systematic profiling data provides a useful basis for studying roles of ARSs during development and understanding their potential functions in the etiology of related diseases.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution.

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-ß 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

Multiple vitellogenins in zebrafish (Danio rerio): quantitative inventory of genes, transcripts and proteins, and relation to egg quality.

Scrutiny of the zebrafish (Danio rerio) genomic database confirmed eight functional vitellogenin (vtg) genes, each with one or two transcript variants, and the encoded Vtg polypeptides were structurally and functionally characterized in detail by in silico and experimental analyses. There were five type I (vtgs1, 4, 5, 6, and 7), two type II (vtg2 and vtg8), and one type III (vtg3) vtg gene(s) encoding three major types of Vtg protein based on subdomain structure (Vtg-I, Vtg-II, and Vtg-III, respectively). Among various tissues of mature zebrafish, transcripts of the eight vtg genes were detected by RNA-Seq only in liver and intestine, with liver being the main site of vtg expression. All vtg transcripts except vtg8 were also detected in mature female liver by RT-qPCR. The relative abundances of Vtg proteins and their variants were quantified by LC-MS/MS in the liver of mature females and in eggs. The Vtgs were generally several fold more abundant in eggs, but profiles of abundance of the 19 different forms of Vtg evaluated were otherwise similar in liver and eggs, suggesting that yolk protein composition is determined largely by hepatic Vtg synthesis and secretion. Based on transcript and protein levels, Vtg-I is, by far, the dominant type of Vtg in zebrafish, followed by Vtg-II and then Vtg-III. When relative abundances of the different forms of Vtg were evaluated by LC-MS/MS in egg batches of good versus poor quality, no differences in the proportional abundance of individual forms of Vtg, or of different Vtg types, attributable to egg quality were observed.

Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio).

BACKGROUND: SLC22 protein family is a member of the SLC (Solute carriers) superfamily of polyspecific membrane transporters responsible for uptake of a wide range of organic anions and cations, including numerous endo- and xenobiotics. Due to the lack of knowledge on zebrafish Slc22 family, we performed initial characterization of these transporters using a detailed phylogenetic and conserved synteny analysis followed by the tissue specific expression profiling of slc22 transcripts. RESULTS: We identified 20 zebrafish slc22 genes which are organized in the same functional subgroups as human SLC22 members. Orthologies and syntenic relations between zebrafish and other vertebrates revealed consequences of the teleost-specific whole genome duplication as shown through one-to-many orthologies for certain zebrafish slc22 genes. Tissue expression profiles of slc22 transcripts were analyzed using qRT-PCR determinations in nine zebrafish tissues: liver, kidney, intestine, gills, brain, skeletal muscle, eye, heart, and gonads. Our analysis revealed high expression of oct1 in kidney, especially in females, followed by oat3 and oat2c in females, oat2e in males and orctl4 in females. oct1 was also dominant in male liver. oat2d showed the highest expression in intestine with less noticeable gender differences. All slc22 genes showed low expression in gills, and moderate expression in heart and skeletal muscle. Dominant genes in brain were oat1 in females and oct1 in males, while the highest gender differences were determined in gonads, with dominant expression of almost all slc22 genes in testes and the highest expression of oat2a. CONCLUSIONS: Our study offers the first insight into the orthology relationships, gene expression and potential role of Slc22 membrane transporters in zebrafish. Clear orthological relationships of zebrafish slc22 and other vertebrate slc22 genes were established. slc22 members are mostly highly conserved, suggesting their physiological and toxicological importance. One-to-many orthologies and differences in tissue expression patterns of zebrafish slc22 genes in comparison to human orthologs were observed. Our expression data point to partial similarity of zebrafish versus human Slc22 members, with possible compensatory roles of certain zebrafish transporters, whereas higher number of some orthologs implies potentially more diverse and specific roles of these proteins in zebrafish.

Transcriptional factors smad1 and smad9 act redundantly to mediate zebrafish ventral specification downstream of smad5.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that play crucial roles during embryonic development and cell fate determination. Nuclear transduction of BMP signals requires the receptor type Smad proteins, Smad1, Smad5, and Smad9. However, how these Smad proteins cooperate in vivo to regulate various developmental processes is largely unknown. In zebrafish, it was widely believed that the maternally expressed smad5 is essential for dorso-ventral (DV) patterning, and the zygotically transcribed smad1 is not required for normal DV axis establishment. In the present study, we have identified zygotically expressed smad9, which cooperates with smad1 downstream of smad5, to mediate zebrafish early DV patterning in a functional redundant manner. Although knockdown of smad1 or smad9 alone does not lead to visible dorsalization, double knockdown strongly dorsalizes zebrafish embryos, which cannot be efficiently rescued by smad5 overexpression, whereas the dorsalization induced by smad5 knockdown can be fully rescued by overexpression of smad1 or smad9. We have further revealed that the transcription initiations of smad1 and smad9 are repressed by each other, that they are direct transcriptional targets of Smad5, and that smad9, like smad1, is required for myelopoiesis. In conclusion, our study uncovers that smad1 and smad9 act redundantly to each other downstream of smad5 to mediate ventral specification and to regulate embryonic myelopoiesis.

nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary.

Female zebrafish have a prolific reproductive capacity, suggesting that a germline stem cell (GSC) population drives oocyte production. However, a zebrafish female GSC population has yet to be identified. Adult stem cells are defined by their ability to both self-renew and differentiate, and by their localization to a stem cell niche. We show here that mitotic and early meiotic germ cells are present in the adult ovary and that the zebrafish homolog of the conserved vertebrate GSC marker, nanos2, is expressed in a subset of pre-meiotic oogonia in the adult gonad. We propose that these nanos2(+) cells are GSCs. Importantly, we find that mitotic, nanos2(+), and early meiotic germ cells localize to the germinal zone, thus identifying this region as the probable ovarian GSC niche in zebrafish. nanos3, which encodes a conserved RNA-binding protein, is known to be required for the continued production of oocytes in the zebrafish. Although mammalian homologs of nanos3 are expressed in early spermatogonia, no study has defined the role of nanos3 in the regulation of vertebrate GSCs. Here we demonstrate that nanos3 function is required for the maintenance of GSCs, but not for their specification, and propose that nanos2 and nanos3 are partially redundant in this role.

Phylogenetic analysis and expression of zebrafish transient receptor potential melastatin family genes.

BACKGROUND: The transient receptor potential melastatin (TRPM) gene family belongs to the superfamily of nonselective TRP ion channels. TRP channels are cellular sensors, detecting a multitude of inputs, including temperature, light, chemical, and mechanical stimuli. Recent studies revealed diverse roles during development, linking TRP channels to differentiation, proliferation, cell motility, cell death, and survival. A detailed description of this gene family in the zebrafish is still missing. RESULTS: Phylogenetic analysis revealed 11 trpm genes in the zebrafish genome. The zebrafish orthologs of mammalian TRPM1 and TRPM4 are duplicated and quadruplicated, respectively, and TRPM8, a cold sensitive channel has been lost in zebrafish. Whole-mount in situ hybridization experiments revealed dynamic expression pattern of trpm genes in the developing embryo and early larva. Transcripts were mainly found in neural cell clusters, but also in tissues involved in ion homeostasis. CONCLUSIONS: Our results suggest a role of TRPM channels in sensory information processing, including vision, olfaction, taste, and mechanosensation. An involvement in developmental processes is likely, as some trpm genes were found to be expressed in differentiating cells. Our data now provide a basis for functional analyses of this gene family of ion channels in the vertebrate model organism Danio rerio.

Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development.

BACKGROUND: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. RESULTS: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. CONCLUSIONS: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.

Suppressor of cytokine signaling 1 regulates embryonic myelopoiesis independently of its effects on T cell development.

Suppressor of cytokine signaling 1 (SOCS1) has been shown to play important roles in the immune system. It acts as a key negative regulator of signaling via receptors for IFNs and other cytokines controlling T cell development, as well as Toll receptor signaling in macrophages and other immune cells. To gain further insight into SOCS1, we have identified and characterized the zebrafish socs1 gene, which exhibited sequence and functional conservation with its mammalian counterparts. Initially maternally derived, the socs1 gene showed early zygotic expression in mesodermal structures, including the posterior intermediate cell mass, a site of primitive hematopoiesis. At later time points, expression was seen in a broad anterior domain, liver, notochord, and intersegmental vesicles. Morpholino-mediated knockdown of socs1 resulted in perturbation of specific hematopoietic populations prior to the commencement of lymphopoiesis, ruling out T cell involvement. However, socs1 knockdown also lead to a reduction in the size of the developing thymus later in embryogenesis. Zebrafish SOCS1 was shown to be able to interact with both zebrafish Jak2a and Stat5.1 in vitro and in vivo. These studies demonstrate a conserved role for SOCS1 in T cell development and suggest a novel T cell-independent function in embryonic myelopoiesis mediated, at least in part, via its effects on receptors using the Jak2-Stat5 pathway.

Mouse and zebrafish Hoxa3 orthologues have nonequivalent in vivo protein function.

Hox genes play evolutionarily conserved roles in specifying axial position during embryogenesis. A prevailing paradigm is that changes in Hox gene expression drive evolution of metazoan body plans. Conservation of Hox function across species, and among paralogous Hox genes within a species, supports a model of functional equivalence. In this report, we demonstrate that zebrafish hoxa3a (zfhoxa3a) expressed from the mouse Hoxa3 locus can substitute for mouse Hoxa3 in some tissues, but has distinct or null phenotypes in others. We further show, by using an allele encoding a chimeric protein, that this difference maps primarily to the zfhoxa3a C-terminal domain. Our data imply that the mouse and zebrafish proteins have diverged considerably since their last common ancestor, and that the major difference between them resides in the C-terminal domain. Our data further show that Hox protein function can evolve independently in different cell types or for specific functions. The inability of zfhoxa3a to perform all of the normal roles of mouse Hoxa3 illustrates that Hox orthologues are not always functionally interchangeable.

Functional prediction and physiological characterization of a novel short trans-membrane protein 1 as a subunit of mitochondrial respiratory complexes.

Mitochondrial respiration is mediated by a set of multisubunit assemblies of proteins that are embedded in the mitochondrial inner membranes. Respiratory complexes do not only contain central catalytic subunits essential for the bioenergetic transformation, but also many short trans-membrane subunits (sTMs) that are implicated in the proper assembly of complexes. Defects in sTMs have been discovered in some human neurodegenerative diseases. Here we identify a new subunit that we named Stmp1 and have characterized its function using both computational and experimental approaches. Stmp1 is a short trans-membrane protein, and sequence/structure analysis revealed that it shares common features like the small size, presence of a single or two TM region, and a COOH-terminal charged region, as many typical sTMs of respiratory complexes. In situ hybridization and RT-PCR assays showed that the Stmp1 expression is ubiquitous throughout zebrafish embryogenesis. In adults, Stmp1 expression was highest in the brain compared with muscle and liver. In zebrafish larvae (3-5 days postfertilization), antisense morpholino oligonucleotide-mediated knockdown of the Stmp1 gene (Stmp1-MO) resulted in a series of mild morphological defects, including abnormal shape of head and jaw and cardiac edema. Larvae injected with the Stmp1-MO had negligible responses to touch stimuli. By ventilation frequency analysis we found that Stmp1-MO-injected zebrafish displayed a severe dysfunction of ventilatory activities when exposed to hypoxic conditions, suggesting a defective mitochondrial activity induced by the loss of Stmp1. Phylogenetic profiling of known respiratory sTMs compared with Stmp1 revealed that all defined sTMs from four respiratory complexes have restricted or variable phyletic distribution, indicating that they are products of evolutionary innovations to fulfill lineage-related functional requirements for respiratory complexes. Thus, being present in animals, filasterea, choanoflagellida, amoebozoa, and plants, Stmp1 may have evolved to confer a new or complementary regulation of respiratory activities.

Rnf11-like is a novel component of NF-κB signaling, governing the posterior patterning in the zebrafish embryos.

RING finger protein 11 (RNF11) is a novel regulator of immunity and cell survival via ubiquitination process in mammalian cells whereas its vertebrate embryonic roles are undefined. Here, we are reporting the isolation, expression and functional roles of an RNF11 orthologue, Rnf11-like in zebrafish embryos. Zebrafish Rnf11-like is composed of 154 amino acids containing RING-H2-finger domain in the C-terminal region and PY-motif. Spatiotemporal expression patterns of rnf11-like indicate that rnf11-like is expressed maternally and zygotically throughout embryogenesis. However, rnf11-like transcripts are present specifically in the presomatic mesoderm (PSM), and later in the brain and retina. Knock-down of Rnf11-like using rnf11-like-specific morpholino causes cell death and developmental defects in the posterior somites, elevating transcripts of NF-κB target gene, ikk1, a negative regulator of NF-κB signaling. All these findings indicate that Rnf11-like is an essential component of NF-κB signaling pathway for specification of the posterior somites in zebrafish embryos.

The Enhancer of split transcription factor Her8a is a novel dimerisation partner for Her3 that controls anterior hindbrain neurogenesis in zebrafish.

BACKGROUND: Neurogenesis control and the prevention of premature differentiation in the vertebrate embryo are crucial processes, allowing the formation of late-born cell types and ensuring the correct shape and cytoarchitecture of the brain. Members of the Hairy/Enhancer of Split (Hairy/E(spl)) family of bHLH-Orange transcription factors, such as zebrafish Her3, 5, 9 and 11, are implicated in the local inhibition of neurogenesis to maintain progenitor pools within the early neural plate. To better understand how these factors exert their inhibitory function, we aimed to isolate some of their functional interactors. RESULTS: We used a yeast two-hybrid screen with Her5 as bait and recovered a novel zebrafish Hairy/E(spl) factor--Her8a. Using phylogenetic and synteny analyses, we demonstrate that her8a evolved from an ancient duplicate of Hes6 that was recently lost in the mammalian lineage. We show that her8a is expressed across the mid- and anterior hindbrain from the start of segmentation. Through knockdown and misexpression experiments, we demonstrate that Her8a is a negative regulator of neurogenesis and plays an essential role in generating progenitor pools within rhombomeres 2 and 4--a role resembling that of Her3. Her8a co-purifies with Her3, suggesting that Her8a-Her3 heterodimers may be relevant in this domain of the neural plate, where both proteins are co-expressed. Finally, we demonstrate that her8a expression is independent of Notch signaling at the early neural plate stage but that SoxB factors play a role in its expression, linking patterning information to neurogenesis control. Overall, the regulation and function of Her8a differ strikingly from those of its closest relative in other vertebrates--the Hes6-like proteins. CONCLUSIONS: Our results characterize the phylogeny, expression and functional interactions involving a new Her factor, Her8a, and highlight the complex interplay of E(spl) proteins that generates the neurogenesis pattern of the zebrafish early neural plate.

Co-orthology of Pax4 and Pax6 to the fly eyeless gene: molecular phylogenetic, comparative genomic, and embryological analyses.

The functional equivalence of Pax6/eyeless genes across distantly related animal phyla has been one of central findings on which evo-devo studies is based. In this study, we show that Pax4, in addition to Pax6, is a vertebrate ortholog of the fly eyeless gene (and its duplicate, twin of eyeless [toy] gene, unique to Insecta). Molecular phylogenetic trees published to date placed the Pax4 gene outside the Pax6/eyeless subgroup as if the Pax4 gene originated from a gene duplication before the origin of bilaterians. However, Pax4 genes had only been reported for mammals. Our molecular phylogenetic analysis, including previously unidentified teleost fish pax4 genes, equally supported two scenarios: one with the Pax4-Pax6 duplication early in vertebrate evolution and the other with this duplication before the bilaterian radiation. We then investigated gene compositions in the genomic regions containing Pax4 and Pax6, and identified (1) conserved synteny between these two regions, suggesting that the Pax4-Pax6 split was caused by a large-scale duplication and (2) its timing within early vertebrate evolution based on the duplication timing of the members of neighboring gene families. Our results are consistent with the so-called two-round genome duplications in early vertebrates. Overall, the Pax6/eyeless ortholog is merely part of a 2:2 orthology relationship between vertebrates (with Pax4 and Pax6) and the fly (with eyeless and toy). In this context, evolution of transcriptional regulation associated with the Pax4-Pax6 split is also discussed in light of the zebrafish pax4 expression pattern that is analyzed here for the first time.

Cone arrestin confers cone vision of high temporal resolution in zebrafish larvae.

Vision of high temporal resolution depends on careful regulation of photoresponse kinetics, beginning with the lifetime of activated photopigment. The activity of rhodopsin is quenched by high-affinity binding of arrestin to photoexcited phosphorylated photopigment, which effectively terminates the visual transduction cascade. This regulation mechanism is well established for rod photoreceptors, yet its role for cone vision is still controversial. In this study we therefore analyzed arrestin function in the cone-dominated vision of larval zebrafish. For both rod (arrS ) and cone (arr3 ) arrestin we isolated two paralogs, each expressed in the respective subset of photoreceptors. Labeling with paralog-specific antibodies revealed subfunctionalized expression of Arr3a in M- and L-cones, and Arr3b in S- and UV-cones. The inactivation of arr3a by morpholino knockdown technology resulted in a severe delay in photoresponse recovery which, under bright light conditions, was rate-limiting. Comparison to opsin phosphorylation-deficient animals confirmed the role of cone arrestin in late cone response recovery. Arr3a activity partially overlapped with the function of the cone-specific kinase Grk7a involved in initial response recovery. Behavioral measurements further revealed Arr3a deficiency to be sufficient to reduce temporal contrast sensitivity, providing evidence for the importance of arrestin in cone vision of high temporal resolution.

Characterization of drCol 15a1b: a novel component of the stem cell niche in the zebrafish retina.

There is a clear need to develop novel tools to help improve our understanding of stem cell biology, and potentially also the utility of stem cells in regenerative medicine. We report the cloning, functional, and bioinformatic characterization of a novel stem cell marker in the zebrafish retina, drCol 15a1b. The expression pattern of drCol 15a1b is restricted to stem cell niches located in the central nervous system, whereas other collagen XVs are associated with muscle and endothelial tissues. Knocking down drCol 15a1b expression causes smaller eyes, ear defects, and brain edema. Microscopic analysis reveals enhanced proliferation in the morphant eye, with many mitotic nuclei located in the central retina, together with a delayed differentiation of the mature retinal cell types. Besides, several markers known to be expressed in the ciliary marginal zone display broader expression areas in morpholino-injected embryos, suggesting an anomalous diffusion of signaling effectors from the sonic hedgehog and notch pathways. These results indicate that drCol 15a1b is a novel stem cell marker in the central nervous system that has a key role in homing stem cells into specialized niches in the adult organism. Moreover, mutations in the hCol 18a1 gene are responsible for the Knobloch syndrome, which affects brain and retinal structures, suggesting that drCol 15a1b may function similarly to mammalian Col 18a1. Thus, our results shed new light on the signaling pathways that underlie the maintenance of stem cells in the adult organism while helping us to understand the role of extracellular matrix proteins in modulating the signals that determine stem cell differentiation, cell cycle exit and apoptosis.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Involvement of Prop1 homeobox gene in the early development of fish pituitary gland.

When mutated in mammals, paired-like homeobox Prop1 gene produces highly variable pituitary phenotypes with impaired regulation of Pit1 and eventually defective synthesis of Pit1-regulated pituitary hormones. Here we have identified fish prop1 orthologs, confirmed their pituitary-specific expression, and blocked the splicing of zebrafish prop1 transcripts using morpholino oligonucleotides. Very early steps of the gland formation seemed unaffected based on morphology and expression of early placodal marker pitx. Prop1 knock-down reduced the expression of pit1, prl (prolactin) and gh (growth hormone), as expected if the function of Prop1 is conserved throughout vertebrates. Less expectedly, lim3 was down regulated. This gene is expressed from early stages of vertebrate pituitary development but is not known to be Prop1-dependent. In situ hybridizations on prop1 morphants using probes for the pan pituitary gene pitx3 and for the hormone gene markers prl, gh and tshß, revealed abnormal shape, growth and cellular organization of the developed adenohypophysis. Strikingly, the effects of prop1 knock-down on adenohypophysis morphology and gene expression were gradually reversed during late development, despite persistent splice-blocking of transcripts. Therefore, prop1 function appears to be conserved between mammals and fish, at least for the mediation of hormonal cell type differentiation via pit1, but the existence of other fish-specific pathways downstream of prop1 are suggested by our observations.

Characterization of the AP-1 µ1A and µ1B adaptins in zebrafish (Danio rerio).

Protein transport between the trans-Golgi network and endosomes is mediated by transport vesicles formed by the adaptor-protein complex AP-1, consisting of the adaptins γ1, ß1, µ1, σ1. Mammalia express µ1A ubiquitously and isoform µ1B in polarized epithelia. Mouse γ1 or µ1A 'knock out's revealed that AP-1 is indispensable for embryonic development. We isolated µ1A and µ1B from Danio rerio. Analysis of µ1A and µ1B expression revealed tissue-specific expression for either one during embryogenesis and in adult tissues in contrast to their expression in mammalia. µ1B transcript was detected in organs of endodermal derivation and "knock-down" experiments gave rise to embryos defective in formation of intestine, liver, and pronephric ducts. Development ceased at 7-8 dpf. µ1B is not expressed in murine liver, indicating loss of µ1B expression and establishment of alternative sorting mechanisms during mammalian development.