Identification and characterization of pancreatic progenitors and endocrine precursors during embryogenesis in zebrafish

Le diabète survient lorsque le nombre ou la fonction des cellules β, productrices d’insuline est affecté. Le nombre de personnes atteintes par cette maladie croît de manière impressionnante d’année en année. Bien qu’il puisse être contrôlé par des injections régulières d’insuline, ce traitement est contraignant, coûteux et ne permet pas d’éliminer toute une série d’effets secondaires chez le patient diabétique. Un des challenges à l’heure actuelle est de développer des stratégies qui permettraient de remplacer ces cellules. La régénération in vivo constitue une approche thérapeutique attrayante. Cependant, cette régénération est peu efficace chez les mammifères et un défi majeur consisterait à la stimuler. Contrairement aux mammifères, le poisson-zèbre (Danio rerio), est un modèle de choix pour étudier la régénération puisqu’il possède le remarquable pouvoir de régénérer les cellules β rapidement et efficacement après leur ablation ciblée. Néanmoins, il est important d’identifier et de caractériser les cellules pancréatiques qui donnent naissance aux cellules β afin de connaître l'ensemble des facteurs et voies de signalisation contrôlant leur formation. Le but de mon doctorat s’est inscrit dans cette démarche d’identification et de caractérisation des cellules progénitrices. Pour ce faire, nous avons généré deux lignées transgéniques qui nous ont permis de suivre le destin des cellules exprimant le facteur de transcription Nkx6.1 et Ascl1b. Par des expériences de traçage de lignée, nous avons montré que les cellules Nkx6.1+ marquent des progéniteurs pancréatiques qui donnent naissance à toutes les lignées pancréatiques alors que les cellules Ascl1b marquent des précurseurs endocrines qui ne donnent naissance qu’à la lignée endocrine. Nous avons aussi montré qu’au début du développement pancréatique, les deux facteurs sont exprimés dans les mêmes cellules pancréatiques puis se séparent rapidement. Cette ségrégation n’est pas la conséquence d’une répression mutuelle entre Ascl1b et Nkx6.1 mais est due à un effet opposé de la voie de signalisation Notch qui maintient l’expression de nkx6.1 et réprime l’expression d’ascl1b

Surgical Treatment of Chest Wall Tumors

peer reviewedThe observation of a primary chest wall desmoid tumor discovered incidentally in a young patient is an opportunity to review the nosology, diagnosis and treatment of this uncommon pathology. Surgical intervention should aim at resecting completely the lesion with sufficient margins. Subsequent reconstruction of the bony thorax uses synthetic materials and muscle or myocutaneous flaps

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

Pancreatic Beta Cell Regeneration: Duct Cells act as Progenitors in Adult Zebrafish

Diabetes is characterized by the loss of insulin-producing beta cells. One promising therapeutic approach is to replenish the pancreas with bona fide functional beta cells by triggering regeneration mechanisms. Previous studies have reported beta cell neogenesis but still remain controversial about their origin and are hampered by very limited regenerative capabilities of mammals. This is contrasting with the astonishing ability of zebrafish to spon-taneously regenerate its beta cells and restore normoglycemia after massive beta cell ablation1. Among the different pancreatic cell types, duct cells have been proposed as a promising source for beta cell replacement in regard to their progenitor capabilities during development. Our project focuses on the role of pancreatic duct cells and the molecular mechanisms involved during beta cell regeneration in adult Zebrafish. To that end we used and de-veloped tools to analyze pancreatic duct cells in normal and regenerating conditions and conducted RNA-sequencing experiments.Pancreatic Regeneration in Zebrafis

RUNX3, EGR1 and SOX9B form a regulatory cascade required to modulate BMP-signaling during cranial cartilage development in zebrafish

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the fsta gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.status: publishe

Pancreatic Beta Cell Regeneration: Duct Cells Act as Progenitors in Adult Zebrafish

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.Identification of the molecular mechanisms underlying pancreatic beta cell regeneration in zebrafis

RUNX3, EGR1 and SOX9B Form a Regulatory Cascade Required to Modulate BMP-Signaling during Cranial Cartilage Development in Zebrafish

<div><p>The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the <em>fsta</em> gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of <em>runx2b</em>, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that e<em>gr1</em> depletion leads to increased expression of <em>fsta</em> and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.</p> </div

Bmp signaling is down-regulated in <i>egr1</i> morphants.

<p>Pharyngeal cartilage precursor cells were visualized by immunohistochemistry using anti-GFP antibodies (green) in <i>fli-</i>GFP embryos. Activity of the BMP signaling pathway was assessed using antibodies against phospho-Smad1/5/8 (red) in 32 hpf embryos. Ventral view of pharyngeal arches, scale bar 40 µm. (A–F) Pharyngeal cartilage precursor cells were visualized by immunohistochemistry using anti-GFP antibodies (green) in <i>fli1-</i>GFP embryos. Activity of the BMP signaling pathway was assessed using antibodies against phospho-Smad1/5/8 (red) in 32 hpf embryos. Ventral view of pharyngeal arches, scale bar 40 µm. (A,B,C) 4 ng MOcon injected embryos, (D, E, F) 4 ng MOegr1 spl injected embryos. <i>fli1-</i>GFP embryos express the GFP transgene in cartilage precursors and endothelial cells in control (A) and in e<i>gr1</i> morphants (D). In contrast, phospho-Smad1/5/8 is is clearly down regulated in e<i>gr1</i> morphants (E) compared to control embryos (B). (C,F) Overlay images of the two anti-body signals clearly show that phospho-Smad1/5/8 is present in GFP-epressing cartilage precursor cells in control embryos (C), while no colocalization is observed in e<i>gr1</i> morphants (F). (a1) first arch, (a2) second arch, (a3) third arch, (a4) fourth arch, (bv) blood vessel.</p

Runx3, Egr1 and Sox9b form a regulatory cascade required to modulate Bmp-signaling during cranial cartilage development in zebrafish.

<p>Signaling model in wild-type embryos (A) and in embryos lacking of endodermal regulatory cascade (B). (A) In wild-type embryos, pharyngeal endoderm expresses a regulatory cascade composed of three transcription factors, Runx3, Egr1 and Sox9b, which down-regulates <i>fsta</i> expression that codes for a Bmp antagonist. This down-regulation of <i>fsta</i> enables Bmp ligands to bind to their heterodimeric receptor (BmpRI and BmpRII) and induce <i>runx2b</i> expression in cranial neural crest cells (cNCC). (B) Embryos lacking of any member of Runx3-Egr1-Sox9b cascade have an over-expression of <i>fsta</i>, which its coding protein is secreted from the endoderm. Antagonist Fsta binds to Bmp ligands and inhibit them to bind to their receptor, having for consequence no Bmp-signaling towards the cNCC and no <i>runx2b</i> expression.</p