Efficient gene transfer by electroporation of chick embryos in ovo has allowed the development of new approaches to the analysis of gene regulation, function and expression, creating an exciting opportunity to build upon the classical manipulative advantages of the chick embryonic system. This method is applicable to other vertebrate embryos and is an important tool with which to address cell and developmental biology questions. Here we describe the technical aspects of in ovo electroporation, its different applications and future perspectives.

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3␤ (GSK-3␤) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3␤ hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3␤ stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.Noonan syndrome (NS) (MIM163950) is a relatively frequent (ϳ1/2,000 births) autosomal dominant disease primarily characterized by facial dysmorphism, heart defects, and short stature. LEOPARD syndrome (LS) (MIM151100) is a rarer but related disorder that associates, roughly, NS symptoms with deafness and cutaneous abnormalities. Both NS and LS belong to the family of "neuro-cardio-facial-cutaneous" (NCFC) syndromes, a group of developmental disorders that display different combinations of the above-mentioned symptoms with mental retardation and tumor predisposition (4, 13).At least 80% of LS and 50% of NS patients carry germ line missense mutations in PTPN11, the gene encoding SHP2. SHP2 is a widely expressed protein tyrosine phosphatase (PTP) that contains Src homology 2 (SH2) domains and promotes Rasmitogen-activated protein kinase (MAPK) activation through different molecular mechanisms (14,27,39,47). Other LS or NS forms are caused by mutations in KRAS, SOS1, RAF1, BRAF, and SHOC2 genes, which encode key factors of the Ras-MAPK pathway (6,32,34,37,38,41).Although genetic studies have provided essential advances, how PTPN11 mutations cause the diseases' symptoms remains an open question. Biochemical studies have shown that NS mutations are located at contact points between the catalytic and the SH2 domains and therefore disrupt SHP2 autoinhibitory conformation (11,19), thereby stimulating SHP2 catalytic activity (gain-of-function mutations). Conversely, LS mutations are confined within the catalytic domain and repress SHP2 activity (15,21,40).A key issue in understanding LS/NS pathogenesis is the identification of the signaling pathways that are altered by these mutations. It is now well ac...