Massimo Nichane

Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/progenitor cells during early vertebrate development.

ABSTRACT 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.

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-Id3 interactions play an essential role in Xenopus neural crest progenitor specification.

Loss of function studies have shown that the Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest formation and maintains cells in a mitotic undifferentiated state. However, its position in the genetic cascade regulating neural crest formation and its relationship with other neural crest regulators remain largely unknown. Here we find that Hairy2 is regulated by BMP, FGF and Wnt and that it is only required downstream of BMP and FGF for neural crest formation. We show that Hairy2 overexpression represses neural crest and upregulates neural border genes at early stages while it expands a subset of them in later embryos. We show that Hairy2 downregulates Id3, another essential HLH neural crest regulator, through attenuation of BMP signaling. Knockdown and rescue experiments indicate that Id3 protein, which physically interacts with Hairy2, negatively regulates Hairy2 activity. However, Id3 is required to allow Hairy2 to promote neural crest formation. Together, our results provide evidence that Hairy2 acts downstream of FGF and BMP signals at the neural border to maintain cells in an undifferentiated state, and that Hairy2-Id3 interactions play an essential role in neural crest progenitor 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.

14-P013 Implication of XHairy1/2 transcription factors in retinal stem cell maintenance

lysed Wnt signalling activity (target gene expression, transgenic reporter line) following Hedgehog pathway pharmacological interference, or vice versa, and (iii) investigated retinal cell proliferation and determination phenotypes following simultaneous inhibition or activation of the two pathways. Altogether, our data suggest that Wnt and Hedgehog morphogens form opposite gradients within retinal stem cell niche and that these signalling pathways antagonize with each other to control retinal stem cell proliferation and multipotence. Interestingly, our retinal gradient model is reminiscent to the neural tube patterning model.

04-P014 Stat3 self-regulates its activity downstream of fibroblast growth factors to coordinate cell cycle progression and specification of neural crest

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 inXenopus a role forStat3 inthis process downstream of FGF signaling. Depletion of Stat3 inhibited NC gene expression and proliferation while its overexpression expanded the NC domain and promoted cell proliferation. Stat3 was phosphorylated and activated in ectodermal cells by FGFs through FGFR4 binding. Stat3 activation was also modulated at the protein level by the transcription factors Hairy2 and Id3, Hairy2 facilitating and Id3 disrupting Stat3–FGFR4 complex formation. Furthermore, Hairy2 and Id3 expression were found to be under opposite transcriptional control by distinct levels of Stat3. Finally, the level of Stat3 activity was found also to critically regulate NC cell fate and proliferation, low levels inducing cell proliferation and differentiation and high levels promoting the maintenance of cells in an undifferentiated state.Together, our data indicate 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.