Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes.
; Lavergne, Arnaud ; et al
in BMC Biology (2017), in revision
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 ▲]Detailed reference viewed: 45 (4 ULg)
Transcriptional landscape of the major pancreatic cells reveals conserved expression patterns amongst distant vertebrate species
; Lavergne, Arnaud ; Padamata, Keerthana et al
Poster (2016, July)Detailed reference viewed: 38 (11 ULg)
ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis.
Dupont, Laura ; ; et al
Conference (2016, June)Detailed reference viewed: 23 (5 ULg)
Pancreatic Beta Cell Regeneration: Duct Cells Act as Progenitors in Adult Zebrafish
Bergemann, David ; ; 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 ▲]Detailed reference viewed: 108 (9 ULg)
Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.
; Bergemann, David ; Tarifeño Saldivia, Estefania 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 ▲]Detailed reference viewed: 103 (10 ULg)
Use of the CRISPR/Cas9 system to generate simultaneously mutant and GFP transgenic zebrafish for the Foxo1a gene
Bernard, Alice ; Peers, Bernard ; Voz, Marianne
Poster (2015, September)Detailed reference viewed: 77 (7 ULg)
ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis.
; ; Bekhouche, Mourad 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 ▲]Detailed reference viewed: 44 (22 ULg)
ADAMTS-3 deficiency is embryonic lethal in mouse and zebrafish.
Janssen, Lauriane ; ; Dupont, Laura et al
Conference (2013, November)Detailed reference viewed: 48 (20 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: 76 (22 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: 234 (14 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: 33 (8 ULg)
Enteroendocrine cells ontogenesis in zebrafish
Stern, David ; Voz, Marianne
Poster (2012, July 04)
Endocrine cells of the digestive tract include pancreatic cells clustered in the islets of Langerhans and the enteroendocrine cells, scattered throughout the digestive epithelium. Notch pathway plays a ... [more ▼]
Endocrine cells of the digestive tract include pancreatic cells clustered in the islets of Langerhans and the enteroendocrine cells, scattered throughout the digestive epithelium. Notch pathway plays a crucial role in endocrine cell fate determination and mediates cell fate decisions. The goal of this project is to decipher the molecular cascade triggered by Notch signaling that controls the endocrine cell differentiation in the digestive system. The targets of Notch signaling are usually members of the bHLH family and more precisely of the Achaete scute-like (Ascl) family or of the atonal related proteins (ARP) family. In this study, we searched for all ARP and Ascl factors expressed in the endocrine lineage of the pancreas and in the gastrointestinal tract in zebrafish. [less ▲]Detailed reference viewed: 19 (1 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: 47 (10 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: 35 (8 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: 57 (10 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: 69 (20 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: 20 (4 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: 25 (4 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: 60 (23 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: 98 (10 ULg)