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See detailTranscriptome analysis of pancreatic cells across distant species highlights novel important regulator genes.
tarifeno-Saldiva, estefania; Lavergne, Arnaud ULg; Bernard, Alice et al

in BMC Biology (2017), 15

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 ... [more ▼]

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 mean to pinpoint genes under strong evolutionary constrains due to their crucial function and which have 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 pathways 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. [less ▲]

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See detailHabenular Neurogenesis in Zebrafish Is Regulated by a Hedgehog, Pax6 Proneural Gene Cascade
Halluin, Caroline; Madelaine, Romain; Naye, Francois et al

in PLoS ONE (2016), 11(7), 0158210

The habenulae are highly conserved nuclei in the dorsal diencephalon that connect the forebrain to the midbrain and hindbrain. These nuclei have been implicated in a broad variety of behaviours in humans ... [more ▼]

The habenulae are highly conserved nuclei in the dorsal diencephalon that connect the forebrain to the midbrain and hindbrain. These nuclei have been implicated in a broad variety of behaviours in humans, primates, rodents and zebrafish. Despite this, the molecular mechanisms that control the genesis and differentiation of neural progenitors in the habenulae remain relatively unknown. We have previously shown that, in zebrafish, the timing of habenular neurogenesis is left-right asymmetric and that in the absence of Nodal signalling this asymmetry is lost. Here, we show that habenular neurogenesis requires the homeobox transcription factor Pax6a and the redundant action of two proneural bHLH factors, Neurog1 and Neurod4. We present evidence that Hedgehog signalling is required for the expression of pax6a, which is in turn necessary for the expression of neurog1 and neurod4. Finally, we demonstrate by pharmacological inhibition that Hedgehog signalling is required continuously during habenular neurogenesis and by cell transplantation experiments that pathway activation is required cell autonomously. Our data sheds light on the mechanism underlying habenular development that may provide insights into how Nodal signalling imposes asymmetry on the timing of habenular neurogenesis. [less ▲]

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See detailADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis.
Dupont, Laura ULg; Janssen, Lauriane; Bekhouche, Mourad et al

Conference (2016, June)

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See detailPancreatic Beta Cell Regeneration: Duct Cells Act as Progenitors in Adult Zebrafish
Bergemann, David ULg; Ghaye, Aurélie; Tarifeño, Estefania et al

Poster (2016, March 18)

Diabetes is characterized by the loss of insulin producing beta cells. Although different therapeutic strategies do exist, they lack precise and dynamic control of glycemia as carried out by endogenous ... [more ▼]

Diabetes is characterized by the loss of insulin producing beta cells. Although different therapeutic strategies do exist, they lack precise and dynamic control of glycemia as carried out by endogenous beta cells. One promising alternative is to replenish the pancreas with bona fide functional beta cells by triggering regeneration mechanisms. Previous studies have shown beta cell neogenesis but still remain controversial about their origin as they used different models. However, among the different hypotheses, it is tempting to assume that pancreatic ducts contain progenitor/precursor cells in adults. The latter is supported by the fact that the embryonic duct epithelium gives rise to the endocrine lineage, and that in healthy and diabetic human adults, insulin positive cells could be found next to or in pancreatic ducts. Despite these observations, mammals show very limited regenerative capabilities, making it difficult to investigate those mechanisms. In contrast, zebrafish are extensively used for regeneration studies. The ability of adult zebrafish to regenerate its beta cells and restore normoglycemia after massive beta cell ablation has already been shown. Our work focuses on the understanding of the underlying mechanisms leading to this retained potential. Here we show that adult pancreatic duct cells act as progenitors, giving rise to beta cells, in physiological and induced diabetic condition in vivo. To get insight into this process, we conducted RNA-seq experiments on zebrafish pancreatic duct cells. By this mean we could identify new ductal markers and noticed that adult duct cells also show strong expression of embryonic pancreatic progenitor markers. In our ongoing comparative analyses we are deciphering the key genes and pathways needed to set in motion the regenerative machinery. The differences between zebrafish and mammal duct cells that will thereby be underlined might then be transposed to mammalian model s to restore regenerative processes. [less ▲]

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See detailPhenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity
Verstraelen,, Sandra; Peers, Bernard ULg; Maho, w et al

in Journal of Applied Toxicology (2016)

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See detailCopy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene
ishibashi, Minaka; Manning, Elisabeth; Shoubridge, Cheryl et al

in Human Genetics (2015), 134

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See detailProgenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.
Ghaye, Aurelie P.; Bergemann, David ULg; Tarifeño Saldivia, Estefania ULg et al

in BMC biology (2015), 13

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 ... [more ▼]

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. [less ▲]

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See detailComparative transcriptomic analysis of the distinct pancreatic cell types
Peers, Bernard ULg

Conference (2015, June 28)

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See detailBiologie du développement (partim. animal)
Peers, Bernard ULg

Learning material (2015)

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See detailADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis.
Janssen, Lauriane; Dupont, Laura; Bekhouche, Mourad ULg et al

in Angiogenesis (2015)

The only documented activity of a subclass of ADAMTS proteases comprising ADAMTS2, 3 and 14 is the cleavage of the aminopropeptide of fibrillar procollagens. A limited number of in vitro studies suggested ... [more ▼]

The only documented activity of a subclass of ADAMTS proteases comprising ADAMTS2, 3 and 14 is the cleavage of the aminopropeptide of fibrillar procollagens. A limited number of in vitro studies suggested that ADAMTS3 is mainly responsible for procollagen II processing in cartilage. Here, we created an ADAMTS3 knockout mouse (Adamts3-/-) model to determine in vivo the actual functions of ADAMTS3. Heterozygous Adamts3+/- mice were viable and fertile, but their intercrosses demonstrated lethality of Adamts3-/- embryos after 15 days of gestation. Procollagens I, II and III processing was unaffected in these embryos. However, a massive lymphedema caused by the lack of lymphatics development, an abnormal blood vessel structure in the placenta and a progressive liver destruction were observed. These phenotypes are most probably linked to dysregulation of the VEGF-C pathways. This study is the first demonstration that an aminoprocollagen peptidase is crucial for developmental processes independently of its primary role in collagen biology and has physiological functions potentially involved in several human diseases related to angiogenesis and lymphangiogenesis. [less ▲]

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See detail“Transcriptome analysis by RNAseq of the distinct pancreatic cell types
Peers, Bernard ULg

Conference (2014, March 30)

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See detailADAMTS-3 deficiency is embryonic lethal in mouse and zebrafish.
Janssen, Lauriane ULg; Dubail, Johanne; Dupont, Laura ULg et al

Conference (2013, November)

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See detailAscl1b and Neurod1, instead of Neurog3, control pancreatic endocrine cell fate in zebrafish
Flasse, Lydie; Pirson, Justine; Stern, David ULg 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 ▲]

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See detailCharacterization of the zebrafish beta-cell transcriptome by RNA-seq
Manfroid, Isabelle ULg; Tarifeno, Estefania; Voz, Marianne ULg 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 ▲]

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See detailThe bHLH transcription factor Ascl1a is essential for the specification of the intestinal secretory cells and mediates Notch signaling in the zebrafish intestine.
Flasse, Lydie C.; Stern, David ULg; Pirson, Justine ULg 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 ▲]

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See detailSyntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish.
Lambaerts, Kathleen; Van Dyck, Stijn; Mortier, Eva 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 ▲]

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See detailCharacterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.
Arkhipova, Valeriya; Wendik, Bjorn; Devos, Nathalie 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 ▲]

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