Team members: David Sassoon, Giovanna Marazzi, Ludovic Arandel, Vanessa Besson, Nathalie Didier, Ara Parlakian


The Vangogh-like-2 protein is involved in non-canonical Wnt-signaling and is non-functional in Looptail mutant mice (Vangl2Lp). Vangl2Lp mice display histological uterine abnormalities at birth suggesting that loss of Vangl2 affects FRT development. Vangl2Lp mutant mice display a septate vagina and loss of oviduct coiling similar to the phenotype in Wnt7a mutants. Vangl2 protein localizes to epithelial (epi) membranes leading to the hypothesis that the epi is a key site of Vangl activity. E-cadherin staining revealed pseudostratified epi, and Scribble is mislocalized to the apical domain, implying that it governs cell polarity. Cytoskeletal actin staining confirmed that Vangl2Lp epi has altered cell polarity. To date, we do not detect an alteration in localization of either Cdc42 or RhoA. Similar localization of Vangl2 was seen in adult epithelium but appeared to be restricted to regions of cell-cell contact and glandular membranes. Xenografted Vangl2Lp uteri reveal expanded pseudostratified epi with abnormal actin polarization, E-cadherin and Scribble localization, also a variety of histological abnormalities including epithelium which is detached from underlying mesenchyme and hyperpolar-appearing cells. Taken together, these studies provide evidence that Vangl2 functions in maintaining cell polarity in uterine tissues, and the loss of cell polarity caused by the Vangl2Lp mutation has lasting consequences on female reproductive tract development.



Identification of a Pax7-dependent pre-myogenic cell population in postnatal muscle

Alice Pannerec, Ph.D. Student

Kathryn Mitchel (Postdoctoral Alumnus)


     Non-satellite cells possess myogenic potential and can participate in muscle regeneration, however, their precise position, origin and relationship to satellite cells remain unclear. During postnatal growth, PW1, a gene co-expressed with Pax7 in satellite cells, is also expressed in a sub-population of muscle resident interstitial cells, termed PICs (PW1 interstitial cells). In primary cultures, we readily obtain PW1+/Pax7+/MyoD+ cells (myoblasts) but fail to observe PW1+/Pax7- cells (PICs). We isolated an enriched population of PICs by FACS, separating cells based on their expression of stem cell markers, and immediately determining their PW1/Pax7 expression profile. We obtain a population of Sca+/CD34+ cells that is highly enriched for PICs (PW1+ Pax7-; >80%), distinct from the Sca1-/CD34+ satellite cells (PW1+ Pax7+; >70%). In culture, PICs predominantly acquire a smooth muscle identity but do show a low spontaneous skeletal myogenic conversion. When GFP+ PICs are co-cultured with wildtype satellite cells, a large proportion of the myotubes are GFP+, revealing that PICs readily contribute towards myogenesis in vitro. In the Pax7 mutant, we note a marked increase in the PIC population in vivo. In culture, purified Pax7 mutant satellite cells show pronounced myogenic capacity. In contrast, Pax7 mutant PICs cannot convert to the myogenic lineage, whether cultured alone or in the presence of satellite cells, suggesting that Pax7 is required to recruit PICs to the myogenic lineage.  When low numbers of labelled satellite cells and PICs are injected into regenerating muscle, distinct cell fates are observed: whilst satellite cells readily terminally differentiate, PICs mainly acquire an interstitial position, with some contribution to myofibre regeneration. This behaviour suggests that PICs represent a more ‘stem-like’ population.

 

PW1: a p53 response gene that underlies muscle atrophy

Ara Parlakian, Postdoc

PW1, initially obtained from a strategy to identify factors involved in muscle stem cell commitment, is expressed in specific skeletal muscle compartments (myofibers, satellite and interstitial cells). During postnatal growth, PW1 expression becomes restricted to interstitial cells with myogenic potential as well as satellite cells suggesting a role for PW1 in muscle stem cells. Recent data from our laboratory showed that PW1 acts through a p53 dependent pathway to regulate both number and behavior of muscle stem cells including their response to tumor load mediated muscle wasting. We sought to investigate the potential role of PW1 in muscle fibrosis and atrophy. We initially examined a laminA/C mutant muscle from LmnaH222P knock-in mice, which are fibrotic and dystrophic . We observe ectopic myonuclear expression of PW1 specifically in atrophic fibers that also show myonuclear expression of activated p53. The induction of PW1 correlates with an increase in the number of Pax7 positive satellite cells. We further note that denervation of muscle leads to myonuclear PW1 expression. To directly and functionally test whether myonuclear PW1 expression plays a central role in muscle atrophy, we have generated 2 mouse models in which PW1 is either conditionally induced or inhibited. Analysis of these mice is under way.