Comparative transcriptomic analysis of the distinct pancreatic cell types
Conference (2015, June 28)Detailed reference viewed: 9 (0 ULg)
“Transcriptome analysis by RNAseq of the distinct pancreatic cell types
Conference (2014, March 30)Detailed reference viewed: 18 (0 ULg)
ADAMTS-3 deficiency is embryonic lethal in mouse and zebrafish.
Janssen, Lauriane ; ; Dupont, Laura et al
Conference (2013, November)Detailed reference viewed: 38 (19 ULg)
Ascl1b and Neurod1, instead of Neurog3, control pancreatic endocrine cell fate in zebrafish
; ; Stern, David et al
in BMC Biology (2013), 11
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 ... [more ▼]
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 herein 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 a couple of bHLH 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. [less ▲]Detailed reference viewed: 51 (19 ULg)
Characterization of the zebrafish beta-cell transcriptome by RNA-seq
Manfroid, Isabelle ; ; Voz, Marianne et al
Poster (2013, May 27)
The loss of pancreatic insulin-producing cells (beta-cells) is a hallmark of diabetes and more knowledge is needed to find new treatments. Thus, it is crucial to identify novel regulatory genes ... [more ▼]
The loss of pancreatic insulin-producing cells (beta-cells) is a hallmark of diabetes and more knowledge is needed to find new treatments. Thus, it is crucial to identify novel regulatory genes specifically expressed in this pancreatic cell subtype. In the present study, the main pancreatic islet was dissected from transgenic Tg(insulin:GFP) adult zebrafish and beta-cells were selectively recovered by FACS with 98% of purity. Illumina RNA-seq was used to sequence the transcriptome. 20 millions of sequenced reads (paired-end) were obtained, aligned on the zebrafish genome and assembled into transcripts (Tophat/Cufflinks softwares). The zebrafish beta-cells transcriptome includes all known regulatory genes involved in beta-cell differentiation such as pdx1, mnx1, pax6b, neuroD, isl1, insm1, as well as Hopx and Hdac9 genes, both recently identified in human beta-cells. In contrast, the alpha-cell specific transcription factor arx and the acinar marker ptf1a were not detected, confirming the high purity of our beta-cell preparation. Interestingly, many miRNAs were detected, such as dre-mir-375 and dre-mir-7, as well as several lncRNA recently described at embryonic stages. We are currently applying the same approach to the Tg(somatostatin:GFP) and Tg(glucagon:GFP) transgenic lines in to characterize the transcriptome of delta- and alpha-cells. The comparison of these different data will allow us to identify coding and non-coding genes specifically expressed in the different endocrine subtype cells, paving the way for further functional studies. [less ▲]Detailed reference viewed: 179 (6 ULg)
The bHLH transcription factor Ascl1a is essential for the specification of the intestinal secretory cells and mediates Notch signaling in the zebrafish intestine.
; Stern, David ; Pirson, Justine et al
in Developmental Biology (2013), 376(2), 187-97
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 21 (8 ULg)
Essential roles of zebrafish bmp2a, fgf10 and fgf24 in the specification of the ventral pancreas
Naye, François ; Voz, Marianne ; Detry, Nathalie et al
in Molecular Biology of the Cell (2012)Detailed reference viewed: 46 (10 ULg)
Syntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish.
; ; et al
in Journal of Cell Science (2012), 125(Pt 5), 1129-40
Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance ... [more ▼]
Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin-syndecan-PIP2-Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development. [less ▲]Detailed reference viewed: 14 (3 ULg)
Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.
; ; et al
in Developmental Biology (2012), 365(1), 290-302
Differentiation of insulin producing beta-cells is a genetically well defined process that involves functions of various conserved transcription factors. Still, the transcriptional mechanisms underlying ... [more ▼]
Differentiation of insulin producing beta-cells is a genetically well defined process that involves functions of various conserved transcription factors. Still, the transcriptional mechanisms underlying specification and determination of beta-cell fate are poorly defined. Here we provide the description of a beta-cell progenitor specific enhancer as a model to study initial steps of beta-cell differentiation. We show that evolutionary non-conserved upstream sequences of the zebrafish hb9 gene are required and sufficient for regulating expression in beta-cells prior to the onset of insulin expression. This enhancer contains binding sites for paired-box transcription factors and two E-boxes that in EMSA studies show interaction with Pax6b and NeuroD, respectively. We show that Pax6b is a potent activator of endodermal hb9 expression and that this activation depends on the beta-cell enhancer. Using genetic approaches we show that pax6b is crucial for maintenance but not induction of pancreatic hb9 transcription. As loss of Pax6b or Hb9 independently results in the loss of insulin expression, the data reveal a novel cross-talk between the two essential regulators of early beta-cell differentiation. While we find that the known pancreatic E-box binding proteins NeuroD and Ngn3 are not required for hb9 expression we also show that removal of both E-boxes selectively eliminates pancreatic specific reporter expression. The data provide evidence for an Ngn3 independent pathway of beta-cell specification that requires function of currently not specified E-box binding factors. [less ▲]Detailed reference viewed: 21 (3 ULg)
A feedback loop between the liver-enriched transcription factor network and mir-122 controls hepatocyte differentiation.
; Manfroid, Isabelle ; et al
in Gastroenterology (2012), 142(1), 119-29
BACKGROUND & AIMS: Hepatocyte differentiation is controlled by liver-enriched transcription factors (LETFs). We investigated whether LETFs control microRNA expression during development and whether this ... [more ▼]
BACKGROUND & AIMS: Hepatocyte differentiation is controlled by liver-enriched transcription factors (LETFs). We investigated whether LETFs control microRNA expression during development and whether this control is required for hepatocyte differentiation. METHODS: Using in vivo DNA binding assays, we identified miR-122 as a direct target of the LETF hepatocyte nuclear factor (HNF) 6. The role and mechanisms of the HNF6-miR-122 gene cascade in hepatocyte differentiation were studied in vivo and in vitro by gain-of-function and loss-of-function experiments, using developing mice and zebrafish as model organisms. RESULTS: HNF6 and its paralog Onecut2 are strong transcriptional stimulators of miR-122 expression. Specific levels of miR-122 were required for proper progression of hepatocyte differentiation; miR-122 stimulated the expression of hepatocyte-specific genes and most LETFs, including HNF6. This indicates that HNF6 and miR-122 form a positive feedback loop. Stimulation of hepatocyte differentiation by miR-122 was lost in HNF6-null mice, revealing that a transcription factor can mediate microRNA function. All hepatocyte-specific genes whose expression was stimulated by miR-122 bound HNF6 in vivo, confirming their direct regulation by this factor. CONCLUSIONS: Hepatocyte differentiation is directed by a positive feedback loop that includes a transcription factor (HNF6) and a microRNA (miR-122) that are specifically expressed in liver. These findings could lead to methods to induce differentiation of hepatocytes in vitro and improve our understanding of liver cell dedifferentiation in pathologic conditions. [less ▲]Detailed reference viewed: 98 (26 ULg)
Pax4 is not essential for beta-cell differentiation in zebrafish embryos but modulates alpha-cell generation by repressing arx gene expression.
; ; et al
in BMC Developmental Biology (2012), 12
BACKGROUND: Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies beta- and delta-cell fate at the expense of ... [more ▼]
BACKGROUND: Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies beta- and delta-cell fate at the expense of alpha-cell identity by repressing Arx gene expression and ectopic expression of PAX4 in alpha-cells is sufficient to convert them into beta-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 delta- and epsilon-cells but was not detected in differentiating beta-cells. pax4 knock-down in zebrafish embryos caused a significant increase in alpha-cells number while having no apparent effect on beta- and delta-cell differentiation. This rise of alpha-cells is due to an up-regulation of the Arx transcription factor. Conversely, knock-down of arx caused to a complete loss of alpha-cells and a concomitant increase of pax4 expression but had no effect on the number of beta- and delta-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 alpha-cells. CONCLUSIONS: In zebrafish, pax4 is not required for the generation of the first beta- and delta-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 alpha-cells and its role in beta-cells differentiation appeared later in vertebrate evolution. [less ▲]Detailed reference viewed: 26 (7 ULg)
Fast Homozygosity Mapping and Identification of a Zebrafish ENU-Induced Mutation by Whole-Genome Sequencing.
Voz, Marianne ; Coppieters, Wouter ; Manfroid, Isabelle et al
in PLoS ONE (2012), 7(4), 34671
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 32 (7 ULg)
Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration
Manfroid, Isabelle ; Ghaye, Aurélie ; et al
in Developmental Biology (2012)
Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells derive from pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the ... [more ▼]
Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells derive 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 Notchresponsive 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. [less ▲]Detailed reference viewed: 59 (19 ULg)
ADAMTS-2, -3 and -14 functions by the phenotypical analysis of knock-out mice and epxression studies in zebrafish.
Dubail, Johanne ; Voz, Marianne ; Peers, Bernard et al
Poster (2011)Detailed reference viewed: 13 (3 ULg)
The Pax6b homeodomain is dispensable for pancreatic endocrine cell differentiation in zebrafish.
; ; et al
in Journal of Biological Chemistry (2010), 285(18), 13863-73
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 18 (3 ULg)
Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells.
; Manfroid, Isabelle ; Flasse, Lydie et al
in Developmental Biology (2010), 340(2), 397-407
In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in ... [more ▼]
In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation. [less ▲]Detailed reference viewed: 54 (22 ULg)
Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.
; Flasse, Lydie ; et al
in Development (2010), 137(2), 203-12
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 94 (10 ULg)
Zebrafish Sox7 and Sox18 function together to control arterial-venous identity
Pendeville-Samain, Hélène ; Winandy, Marie ; Manfroid, Isabelle et al
in Developmental Biology (2008), 317(2), 405-16
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 213 (33 ULg)