Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells

Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human–mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1+ vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo

Study of the molecular mechanisms controlling neural crest cells proliferation in xenopus

La crête neurale (CN) est une structure transitoire apparaissant en bordure de la plaque neurale chez les embryons de vertébrés. Au cours du développement embryonnaire, les cellules de la CN prolifèrent, subissent une transition épithélio-mésenchymateuse, migrent et se différencient en de nombreux types cellulaires tels que des neurones et cellules gliales du système nerveux périphérique, des mélanocytes, des cellules musculaires lisses ou des élements du squelette cranio-facial. Afin de mieux comprendre les mécanismes moléculaires contrôlant la prolifération et la spécification des cellules de la CN, nous avons étudié le rôle de deux facteurs de transcription, Hairy2 et Stat3, via des expériences de perte et gain de fonction chez l’embryon de xénope. Le gène Hairy2 code pour un facteur de transcription bHLH-O répresseur. Il est exprimé précocement au niveau de la bordure de la plaque neurale incluant la CN présomptive. Nous avons montré que Hairy2 est requis pour la prolifération des cellules de la CN en aval de signaux FGFs et qu’il maintient les cellules dans un état indifférencié en réprimant l’expression précoce des gènes spécifiques de la CN. Hairy2 réprime aussi la transcription du gène Id3 codant pour un facteur HLH essentiel à la prolifération des cellules de la CN. Id3 affecte également Hairy2. Nous avons observé que la protéine Id3 interagit physiquement avec Hairy2 et bloque son activité, démontrant que les interactions entre Hairy2 et Id3 jouent un rôle important dans la prolifération et la spécification des cellules de la CN. \Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Self-regulation of Stat3 activity coordinates cell-cycle progression and neural crest specification

A complex set of extracellular signals is required for neural crest (NC) specification. However, how these signals function to coordinate cell-cycle progression and differentiation remains poorly understood. Here, we report in Xenopus a role for the transcription factor signal transducers and activators of transcription-3 (Stat3) in this process downstream of FGF signalling. Depletion of Stat3 inhibits NC gene expression and cell proliferation, whereas overexpression expands the NC domain and promotes cell proliferation. Stat3 is phosphorylated and activated in ectodermal cells by FGFs through binding with FGFR4. Stat3 activation is also modulated by Hairy2 and Id3 proteins that, respectively, facilitate and disrupt Stat3-FGFR4 complex formation. Furthermore, distinct levels of Stat3 activity control Hairy2 and Id3 transcription, leading to Stat3 self-regulation. Finally, high Stat3 activity maintains cells in an undifferentiated state, whereas low activity promotes cell proliferation and NC differentiation. Together, our data suggest that Stat3, downstream of FGFs and under the positive and negative feedback regulation of Hairy2 and Id3, plays an essential role in the coordination of cell-cycle progression and differentiation during NC specification

Hairy2 functions through both DNA-binding and non DNA-binding mechanisms at the neural plate border in Xenopus.

The Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest progenitor survival and maintains cells in a mitotic undifferentiated pre-neural crest state. However, its mode of action remains largely unknown. Here we show that a Hairy2 DNA-binding mutant is unable to promote cell survival and to upregulate the expression of early neural border genes but is capable to increase cell proliferation and to expand NC in late embryos. We found that Hairy2 transiently activates in a DNA-binding independent manner the expression of the Notch ligand Delta1 and that Delta1 is required for Hairy2 to promote cell proliferation and to expand NC. Finally, we provide evidence that Hairy2 induces Delta1 through the transcription factor Stat3. Together, these results suggest that Hairy2 has a dual mode of action and may function at the neural plate border through both a DNA-binding and a non-DNA-binding Stat3-Delta1 mediated mechanism.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The Na+/PO4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos.

The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Evi1 plays an essential role in distal tubule and duct formation in the Xenopus pronephros.

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Evi1 has conserved restricted expression profile in vertebrate embryos and regulates pronephros development in Xenopus.

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Evolutionarily Conserved Role of Nucleostemin: Controlling Proliferation of Stem/Progenitor Cells during Early Vertebrate Development

Nucleostemin (NS) is a putative GTPase expressed preferentially in the nucleoli of neuronal and embryonic stem cells and several cancer cell lines. Transfection and knockdown studies indicated that NS controls the proliferation of these cells by interacting with the p53 tumor suppressor protein and regulating its activity. To assess the physiological role of NS in vivo, we generated a mutant mouse line with a specific gene trap event that inactivates the NS allele. The corresponding NS(−/−) embryos died around embryonic day 4. Analyses of NS mutant blastocysts indicated that NS is not required to maintain pluripotency, nucleolar integrity, or survival of the embryonic stem cells. However, the homozygous mutant blastocysts failed to enter S phase even in the absence of functional p53. Haploid insufficiency of NS in mouse embryonic fibroblasts leads to decreased cell proliferation. NS also functions in early amphibian development to control cell proliferation of neural progenitor cells. Our results show that NS has a unique ability, derived from an ancestral function, to control the proliferation rate of stem/progenitor cells in vivo independently of p53

Retinoic acid signalling is required for specification of pronephric cell fate.

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe