A Zebrafish Embryo Model to Screen Potential Therapeutic Compounds in Sapindaceae Poisoning.

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG) are protoxins produced by Sapindaceae plants, particularly Acer pseudoplatanus, and are responsible for causing atypical myopathy (AM) in equids. These protoxins metabolise into toxic compounds, such as methylenecyclopropylacetyl-CoA (MCPA-CoA), which alters energy metabolism and induces severe rhabdomyolysis. Currently, no specific treatment exists for this poisoning, in vitro models fail to reproduce HGA's toxic effects on equine primary myoblasts, and mammalian models are impractical for large-scale drug screening. This study aimed to develop a zebrafish embryo model for screening therapeutic compounds against AM. Zebrafish embryos were exposed to various concentrations of HGA, MCPrG, and methylenecyclopropylacetate (MCPA) for 72 h. MCPrG did not induce toxicity, while HGA and MCPA showed median lethal concentration (LC50) values of 1.7 µM and 1 µM after 72 h, respectively. The highest levels of the conjugated metabolite MCPA-carnitine were detected 24 h after HGA exposure, and the acylcarnitines profile was highly increased 48 h post-exposure. Isovaleryl-/2- methylbutyrylcarnitine levels notably rose after 24 h, suggesting potential exposition biomarkers. Glycine and carnitine effectively reduced mortality, whereas riboflavin showed no protective effect. These findings suggest that the zebrafish embryo represents a valuable model for identifying therapeutic compounds for Sapindaceae poisoning.

Identification of an evolutionarily conserved domain in Neurod1 favouring enteroendocrine versus goblet cell fate.

ARP/ASCL transcription factors are key determinants of cell fate specification in a wide variety of tissues, coordinating the acquisition of generic cell fates and of specific subtype identities. How these factors, recognizing highly similar DNA motifs, display specific activities, is not yet fully understood. To address this issue, we overexpressed different ARP/ASCL factors in zebrafish ascl1a-/- mutant embryos to determine which ones are able to rescue the intestinal secretory lineage. We found that Ascl1a/b, Atoh1a/b and Neurod1 factors are all able to trigger the first step of the secretory regulatory cascade but distinct secretory cells are induced by these factors. Indeed, Neurod1 rescues the enteroendocrine lineage while Ascl1a/b and Atoh1a/b rescue the goblet cells. Gain-of-function experiments with Ascl1a/Neurod1 chimeric proteins revealed that the functional divergence is encoded by a 19-aa ultra-conserved element (UCE), present in all Neurod members but absent in the other ARP/ASCL proteins. Importantly, inserting the UCE into the Ascl1a protein reverses the rescuing capacity of this Ascl1a chimeric protein that cannot rescue the goblet cells anymore but can efficiently rescue the enteroendocrine cells. This novel domain acts indeed as a goblet cell fate repressor that inhibits gfi1aa expression, known to be important for goblet cell differentiation. Deleting the UCE domain of the endogenous Neurod1 protein leads to an increase in the number of goblet cells concomitant with a reduction of the enteroendocrine cells, phenotype also observed in the neurod1 null mutant. This highlights the crucial function of the UCE domain for NeuroD1 activity in the intestine. As Gfi1 acts as a binary cell fate switch in several tissues where Neurod1 is also expressed, we can envision a similar role of the UCE in other tissues, allowing Neurod1 to repress Gfi1 to influence the balance between cell fates.

Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics.

Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) inverse-agonist BMS493 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes in zebrafish, including hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of zebrafish and murine RAR ChIP-seq data highlighted the conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependent manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.

Pancreatic and intestinal endocrine cells in zebrafish share common transcriptomic signatures and regulatory programmes.

BACKGROUND: Endocrine cells of the zebrafish digestive system play an important role in regulating metabolism and include pancreatic endocrine cells (PECs) clustered in the islets of Langerhans and the enteroendocrine cells (EECs) scattered in the intestinal epithelium. Despite EECs and PECs are being located in distinct organs, their differentiation involves shared molecular mechanisms and transcription factors. However, their degree of relatedness remains unexplored. In this study, we investigated comprehensively the similarity of EECs and PECs by defining their transcriptomic landscape and comparing the regulatory programmes controlled by Pax6b, a key player in both EEC and PEC differentiations. RESULTS: RNA sequencing was performed on EECs and PECs isolated from wild-type and pax6b mutant zebrafish. Data mining of wild-type zebrafish EEC data confirmed the expression of orthologues for most known mammalian EEC hormones, but also revealed the expression of three additional neuropeptide hormones (Proenkephalin-a, Calcitonin-a and Adcyap1a) not previously reported to be expressed by EECs in any species. Comparison of transcriptomes from EECs, PECs and other zebrafish tissues highlights a very close similarity between EECs and PECs, with more than 70% of genes being expressed in both endocrine cell types. Comparison of Pax6b-regulated genes in EECs and PECs revealed a significant overlap. pax6b loss-of-function does not affect the total number of EECs and PECs but instead disrupts the balance between endocrine cell subtypes, leading to an increase of ghrelin- and motilin-like-expressing cells in both the intestine and pancreas at the expense of other endocrine cells such as beta and delta cells in the pancreas and pyyb-expressing cells in the intestine. Finally, we show that the homeodomain of Pax6b is dispensable for its action in both EECs and PECs. CONCLUSION: We have analysed the transcriptomic landscape of wild-type and pax6b mutant zebrafish EECs and PECs. Our study highlights the close relatedness of EECs and PECs at the transcriptomic and regulatory levels, supporting the hypothesis of a common phylogenetic origin and underscoring the potential implication of EECs in metabolic diseases such as type 2 diabetes.

Nifurpirinol: A more potent and reliable substrate compared to metronidazole for nitroreductase-mediated cell ablations.

The zebrafish is a popular animal model with well-known regenerative capabilities. To study regeneration in this fish, the nitroreductase/metronidazole-mediated system is widely used for targeted ablation of various cell types. Nevertheless, we highlight here some variability in ablation efficiencies with the metronidazole prodrug that led us to search for a more efficient and reliable compound. Herein, we present nifurpirinol, another nitroaromatic antibiotic, as a more potent prodrug compared to metronidazole to trigger cell-ablation in nitroreductase expressing transgenic models. We show that nifurpirinol induces robust and reliable ablations at concentrations 2,000 fold lower than metronidazole and three times below its own toxic concentration. We confirmed the efficiency of nifurpirinol in triggering massive ablation of three different cell types: the pancreatic beta cells, osteoblasts, and dopaminergic neurons. Our results identify nifurpirinol as a very potent prodrug for the nitroreductase-mediated ablation system and suggest that its use could be extended to many other cell types, especially if difficult to ablate, or when combined pharmacological treatments are desired.

Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes.

BACKGROUND: Defining the transcriptome and the genetic pathways of pancreatic cells is of great interest for elucidating the molecular attributes of pancreas disorders such as diabetes and cancer. As the function of the different pancreatic cell types has been maintained during vertebrate evolution, the comparison of their transcriptomes across distant vertebrate species is a means to pinpoint genes under strong evolutionary constraints due to their crucial function, which have therefore preserved their selective expression in these pancreatic cell types. RESULTS: In this study, RNA-sequencing was performed on pancreatic alpha, beta, and delta endocrine cells as well as the acinar and ductal exocrine cells isolated from adult zebrafish transgenic lines. Comparison of these transcriptomes identified many novel markers, including transcription factors and signaling pathway components, specific for each cell type. By performing interspecies comparisons, we identified hundreds of genes with conserved enriched expression in endocrine and exocrine cells among human, mouse, and zebrafish. This list includes many genes known as crucial for pancreatic cell formation or function, but also pinpoints many factors whose pancreatic function is still unknown. A large set of endocrine-enriched genes can already be detected at early developmental stages as revealed by the transcriptomic profiling of embryonic endocrine cells, indicating a potential role in cell differentiation. The actual involvement of conserved endocrine genes in pancreatic cell differentiation was demonstrated in zebrafish for myt1b, whose invalidation leads to a reduction of alpha cells, and for cdx4, selectively expressed in endocrine delta cells and crucial for their specification. Intriguingly, comparison of the endocrine alpha and beta cell subtypes from human, mouse, and zebrafish reveals a much lower conservation of the transcriptomic signatures for these two endocrine cell subtypes compared to the signatures of pan-endocrine and exocrine cells. These data suggest that the identity of the alpha and beta cells relies on a few key factors, corroborating numerous examples of inter-conversion between these two endocrine cell subtypes. CONCLUSION: This study highlights both evolutionary conserved and species-specific features that will help to unveil universal and fundamental regulatory pathways as well as pathways specific to human and laboratory animal models such as mouse and zebrafish.

Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

BACKGROUND: In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. RESULTS: Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. CONCLUSIONS: We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish. Our findings support the hypothesis that nkx6.1+ pancreatic progenitors contribute to beta cell regeneration. Further characterization of these cells will open up new perspectives for anti-diabetic therapies.

Ascl1b and Neurod1, instead of Neurog3, control pancreatic endocrine cell fate in zebrafish.

BACKGROUND: NEUROG3 is a key regulator of pancreatic endocrine cell differentiation in mouse, essential for the generation of all mature hormone producing cells. It is repressed by Notch signaling that prevents pancreatic cell differentiation by maintaining precursors in an undifferentiated state. RESULTS: We show that, in zebrafish, neurog3 is not expressed in the pancreas and null neurog3 mutant embryos do not display any apparent endocrine defects. The control of endocrine cell fate is instead fulfilled by two basic helix-loop-helix factors, Ascl1b and Neurod1, that are both repressed by Notch signaling. ascl1b is transiently expressed in the mid-trunk endoderm just after gastrulation and is required for the generation of the first pancreatic endocrine precursor cells. Neurod1 is expressed afterwards in the pancreatic anlagen and pursues the endocrine cell differentiation program initiated by Ascl1b. Their complementary role in endocrine differentiation of the dorsal bud is demonstrated by the loss of all hormone-secreting cells following their simultaneous inactivation. This defect is due to a blockage of the initiation of endocrine cell differentiation. CONCLUSIONS: This study demonstrates that NEUROG3 is not the unique pancreatic endocrine cell fate determinant in vertebrates. A general survey of endocrine cell fate determinants in the whole digestive system among vertebrates indicates that they all belong to the ARP/ASCL family but not necessarily to the Neurog3 subfamily. The identity of the ARP/ASCL factor involved depends not only on the organ but also on the species. One could, therefore, consider differentiating stem cells into insulin-producing cells without the involvement of NEUROG3 but via another ARP/ASCL factor.

The bHLH transcription factor Ascl1a is essential for the specification of the intestinal secretory cells and mediates Notch signaling in the zebrafish intestine.

Notch signaling has a fundamental role in stem cell maintenance and in cell fate choice in the intestine of different species. Canonically, Notch signaling represses the expression of transcription factors of the achaete-scute like (ASCL) or atonal related protein (ARP) families. Identifying the ARP/ASCL genes expressed in the gastrointestinal tract is essential to build the regulatory cascade controlling the differentiation of gastrointestinal progenitors into the different intestinal cell types. The expression of the ARP/ASCL factors was analyzed in zebrafish to identify, among all the ARP/ASCL factors found in the zebrafish genome, those expressed in the gastrointestinal tract. ascl1a was found to be the earliest factor detected in the intestine. Loss-of-function analyses using the pia/ascl1a mutant, revealed that ascl1a is crucial for the differentiation of all secretory cells. Furthermore, we identify a battery of transcription factors expressed during secretory cell differentiation and downstream of ascl1a. Finally, we show that the repression of secretory cell fate by Notch signaling is mediated by the inhibition of ascl1a expression. In conclusion, this work identifies Ascl1a as a key regulator of the secretory cell lineage in the zebrafish intestine, playing the same role as Atoh1 in the mouse intestine. This highlights the diversity in the ARP/ASCL family members acting as cell fate determinants downstream from Notch signaling.

Pax4 is not essential for beta-cell differentiation in zebrafish embryos but modulates alpha-cell generation by repressing arx gene expression.

BACKGROUND: Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies ß- and δ-cell fate at the expense of α-cell identity by repressing Arx gene expression and ectopic expression of PAX4 in α-cells is sufficient to convert them into ß-cells. Surprisingly, no Pax4 orthologous gene can be found in chicken and Xenopus tropicalis raising the question of the function of pax4 gene in lower vertebrates such as in fish. In the present study, we have analyzed the expression and the function of the orthologous pax4 gene in zebrafish. RESULTS: pax4 gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating δ- and ε-cells but was not detected in differentiating ß-cells. pax4 knock-down in zebrafish embryos caused a significant increase in α-cells number while having no apparent effect on ß- and δ-cell differentiation. This rise of α-cells is due to an up-regulation of the Arx transcription factor. Conversely, knock-down of arx caused to a complete loss of α-cells and a concomitant increase of pax4 expression but had no effect on the number of ß- and δ-cells. In addition to the mutual repression between Arx and Pax4, these two transcription factors negatively regulate the transcription of their own gene. Interestingly, disruption of pax4 RNA splicing or of arx RNA splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. Such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards α-cells. CONCLUSIONS: In zebrafish, pax4 is not required for the generation of the first ß- and δ-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the differentiation of these cell types in mouse. On the other hand, the mutual repression between Arx and Pax4 is observed in both mouse and zebrafish. These data suggests that the main original function of Pax4 during vertebrate evolution was to modulate the number of pancreatic α-cells and its role in ß-cells differentiation appeared later in vertebrate evolution.

Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration.

Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells are derived from pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the zebrafish sox9b gene is expressed in pancreatic ducts where it labels the pancreatic Notch-responsive cells previously shown to be progenitors. Inactivation of sox9b disturbs duct formation and impairs regeneration of beta cells from these ducts in larvae. sox9b expression in the midtrunk endoderm appears at the junction of the hepatic and ventral pancreatic buds and, by the end of embryogenesis, labels the hepatopancreatic ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal morphogenesis and differentiation are specifically disrupted in sox9b mutants, with the dysmorphic hepatopancreatic ducts containing misdifferentiated hepatocyte-like and pancreatic-like cells. We also show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts requires FGF and Notch activity, respectively, both pathways known to prevent excessive endocrine differentiation in these ducts. Furthermore, beta cell recovery after specific ablation is severely compromised in sox9b mutant larvae. Our data position sox9b as a key player in the generation of secondary endocrine cells deriving from pancreatic ducts in zebrafish.

Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing.

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.

Essential roles of zebrafish bmp2a, fgf10, and fgf24 in the specification of the ventral pancreas.

In vertebrates, pancreas and liver arise from bipotential progenitors located in the embryonic gut endoderm. Bone morphogenic protein (BMP) and fibroblast growth factor (FGF) signaling pathways have been shown to induce hepatic specification while repressing pancreatic fate. Here we show that BMP and FGF factors also play crucial function, at slightly later stages, in the specification of the ventral pancreas. By analyzing the pancreatic markers pdx1, ptf1a, and hlxb9la in different zebrafish models of BMP loss of function, we demonstrate that the BMP pathway is required between 20 and 24 h postfertilization to specify the ventral pancreatic bud. Knockdown experiments show that bmp2a, expressed in the lateral plate mesoderm at these stages, is essential for ventral pancreas specification. Bmp2a action is not restricted to the pancreatic domain and is also required for the proper expression of hepatic markers. By contrast, through the analysis of fgf10(-/-); fgf24(-/-) embryos, we reveal the specific role of these two FGF ligands in the induction of the ventral pancreas and in the repression of the hepatic fate. These mutants display ventral pancreas agenesis and ectopic masses of hepatocytes. Overall, these data highlight the dynamic role of BMP and FGF in the patterning of the hepatopancreatic region.

The Pax6b homeodomain is dispensable for pancreatic endocrine cell differentiation in zebrafish.

Pax6 is a well conserved transcription factor that contains two DNA-binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain, and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harboring a mutation in the pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised a question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation, as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no beta cells, a strongly reduced number of delta cells, and a significant increase of epsilon cells. Through the use of various morpholinos targeting intron-exon junctions, pax6b RNA splicing was perturbed at several sites, leading either to retention of intronic sequences or to deletion of exonic sequences in the pax6b transcript. By this strategy, we show that deletion of the Pax6b homeodomain in zebrafish embryos does not disturb pancreas development, whereas lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in alpha cell number and in glucagon expression, emphasizing the importance of the fine tuning of the Pax6b level to its biological activity.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors.

BACKGROUND: PAX6 is a transcription factor playing a crucial role in the development of the eye and in the differentiation of the pancreatic endocrine cells as well as of enteroendocrine cells. Studies on the mouse Pax6 gene have shown that sequences upstream from the P0 promoter are required for expression in the lens and the pancreas; but there remain discrepancies regarding the precise location of the pancreatic regulatory elements. RESULTS: Due to genome duplication in the evolution of ray-finned fishes, zebrafish has two pax6 genes, pax6a and pax6b. While both zebrafish pax6 genes are expressed in the developing eye and nervous system, only pax6b is expressed in the endocrine cells of the pancreas. To investigate the cause of this differential expression, we used a combination of in silico, in vivo and in vitro approaches. We show that the pax6b P0 promoter targets expression to endocrine pancreatic cells and also to enteroendocrine cells, retinal neurons and the telencephalon of transgenic zebrafish. Deletion analyses indicate that strong pancreatic expression of the pax6b gene relies on the combined action of two conserved regulatory enhancers, called regions A and C. By means of gel shift assays, we detected binding of the homeoproteins PDX1, PBX and PREP to several cis-elements of these regions. In constrast, regions A and C of the zebrafish pax6a gene are not active in the pancreas, this difference being attributable to sequence divergences within two cis-elements binding the pancreatic homeoprotein PDX1. CONCLUSION: Our data indicate a conserved role of enhancers A and C in the pancreatic expression of pax6b and emphasize the importance of the homeoproteins PBX and PREP cooperating with PDX1, in activating pax6b expression in endocrine pancreatic cells. This study also provides a striking example of how adaptative evolution of gene regulatory sequences upon gene duplication progressively leads to subfunctionalization of the paralogous gene pair.

Zebrafish Sox7 and Sox18 function together to control arterial-venous identity.

Sox7 and Sox18 are members of the F-subgroup of Sox transcription factors family and are mostly expressed in endothelial compartments. In humans, dominant mutations in Sox18 are the underlying cause of the severe hypotrichosis-lymphedema-telangiectasia disorder characterized by vascular defects. However little is known about which vasculogenic processes Sox7 and Sox18 regulate in vivo. We cloned the orthologs of Sox7 and Sox18 in zebrafish, analysed their expression pattern and performed functional analyses. Both genes are expressed in the lateral plate mesoderm during somitogenesis. At later stages, Sox18 is expressed in all axial vessels whereas Sox7 expression is mainly restricted to the dorsal aorta. Knockdown of Sox7 or Sox18 alone failed to reveal any phenotype. In contrast, blocking the two genes simultaneously led to embryos displaying dysmorphogenesis of the proximal aorta and arteriovenous shunts, all of which can account for the lack of circulation observed in the trunk and tail. Gene expression analyses performed with general endothelial markers on double morphants revealed that Sox7 and Sox18 are dispensable for the initial specification and positioning of the major trunk vessels. However, morphants display ectopic expression of the venous Flt4 marker in the dorsal aorta and a concomitant reduction of the artery-specific markers EphrinB2a and Gridlock. The striking similarities between the phenotype of Sox7/Sox18 morphants and Gridlock mutants strongly suggest that Sox7 and Sox18 control arterial-venous identity by regulating Gridlock expression.

Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development.

In amniotes, the pancreatic mesenchyme plays a crucial role in pancreatic epithelium growth, notably through the secretion of fibroblast growth factors. However, the factors involved in the formation of the pancreatic mesenchyme are still largely unknown. In this study, we characterize, in zebrafish embryos, the pancreatic lateral plate mesoderm, which is located adjacent to the ventral pancreatic bud and is essential for its specification and growth. We firstly show that the endoderm, by expressing the fgf24 gene at early stages, triggers the patterning of the pancreatic lateral plate mesoderm. Based on the expression of isl1, fgf10 and meis genes, this tissue is analogous to the murine pancreatic mesenchyme. Secondly, Fgf10 acts redundantly with Fgf24 in the pancreatic lateral plate mesoderm and they are both required to specify the ventral pancreas. Our results unveil sequential signaling between the endoderm and mesoderm that is critical for the specification and growth of the ventral pancreas, and explain why the zebrafish ventral pancreatic bud generates the whole exocrine tissue.

Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

Evi1 is specifically expressed in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation.

The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor.