Claire Chazaud

We investigated the role of Netrin-1 (NTN1) in human naïve pluripotency using complementary loss- and gain-of-function approaches. In primate embryos and human embryonic stem cells (hESCs), Netrin-1 expression is associated with the naïve pluripotent state. Disruption of NTN1 had no detectable effect on hESCs maintained on murine embryonic fibroblasts. However, under sub-optimal culture conditions, NTN1-knockout cells exhibited compromised naïve pluripotency, which was rescued with feeder cells overexpressing Netrin-1. Netrin-1 overexpression in hESCs accelerated acquisition of the naïve state and markedly increased resistance to differentiation. These effects were accompanied by extensive epigenetic remodeling, including H3K27ac and H2K27me3. Proteomic and phospho-proteomic analyses further revealed rapid Netrin-1-dependent alterations in pathways controlling cell adhesion, signaling, and chromatin regulation. Together, these findings extend the role of Netrin-1 beyond its established functions and identify it as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naïve pluripotency.

The epiblast is the source of all mammalian embryonic tissues and of pluripotent embryonic stem cells. It differentiates alongside the primitive endoderm in a “salt and pepper” pattern from inner cell mass (ICM) progenitors during the preimplantation stages through the activity of NANOG, GATA6 and the FGF pathway. When and how epiblast lineage specification is initiated is still unclear. Here, we show that the coordinated expression of pluripotency markers defines epiblast identity. Conversely, ICM progenitor cells display random cell-to-cell variability in expression of various pluripotency markers, remarkably dissimilar from the epiblast signature and independently from NANOG, GATA6 and FGF activities. Coordination of pluripotency markers expression fails inNanogandGata6double KO (DKO) embryos. Collectively, our data suggest that NANOG triggers epiblast specification by ensuring the coordinated expression of pluripotency markers in a subset of cells, implying a stochastic mechanism. These features are likely conserved, as suggested by analysis of human embryos.

Fagraea berteroana is a tree used in traditional medicine in various islands of the South Pacific. Here, we studied its hair growth-inducing properties as suggested by one of its Marquesan ethno-uses in haircare. The ethyl acetate extract of the fruits of F. berteroana (FEAE) and four resulting fractions (FEAE-F0, FEAE-F1, FEAE-F2, and FEAE-F3) were tested on hair follicle dermal papilla cells to determine their cell proliferative activity. Furthermore, RT-qPCR analysis enabled gene modulation analysis, while immunostaining of the β-catenin protein was used to follow protein regulation. We found that the plant extracts induced a controlled, dose-dependent cell proliferation. FEAE-F0 simultaneously down-regulated Bone Morphogenetic Protein 2 (BMP2) mRNA expression and upregulated Cyclin-D1 (CCND1) gene expression, which suggests an involvement in the regulation of the Wnt and Transforming Growth Factor beta (TGFβ) pathways that control the hair cycle. FEAE-F0 exhibited a 1.34-fold increase of nuclear β-catenin protein. This is indicative of an active hair growth state. Thus, we conclude that FEAE-F0 could be an innovative candidate in hair care, which opens interesting leads to promote the Marquesan cosmetopoeia.

Hair loss is becoming increasingly prevalent as dietary and living habits change. The search for natural products to limit hair loss has led to tapping into traditional cosmetic knowledge. We studied three plants of the Polynesian cosmetopoeia, Bidens pilosa, Calophyllum inophyllum and Fagraea berteroana, to determine their ability to promote hair growth. Their chemical content was characterized by liquid chromatography coupled to mass spectrometry (LC-MS). Their proliferative activity on dermal papilla cells (DPCs) was assessed via MTT assay and molecular targets were evaluated by RT-qPCR analysis of seven factors involved in the modulation of the hair cycle, CCND1, LEF1, DKK1, WNT5A PPARD, TGFΒ1, PPARD and RSPO2. Our results show that our extracts significantly increased proliferation of dermal papilla cells. Furthermore, LC-MS/MS analysis revealed a diversity of molecules, flavonoids, iridoids and organic acids, some known for hair-inducing properties. Finally, specific extracts and fractions of all three plants either upregulated CCND1, LEF1 and PPARD involved in stimulating hair follicle proliferation and/or lowered the gene expression levels of hair growth inhibiting factors, DKK1 and TGFB1. Our findings suggest that extracts from B. pilosa, C. inophyllum and F. berteroana are interesting candidates to stimulate hair growth.

A couple of days after fertilization of a mouse oocyte by a sperm, two sequential cell differentiation events segregate pluripotent cells that can be identified by the presence of specific markers. Early mammalian embryos are relatively easy to recover as they are not yet implanted in the uterus matrix. Several decades of experimentation have enabled to find appropriate media to culture them, and therefore provide an excellent way to test different experimental setups such as the use of signaling inhibitors. We provide here a commonly used protocol to culture preimplantation embryos as well as a method to detect pluripotent cells in blastocysts.

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation occurs in ICM precursor cells, in differentiated primitive endoderm (PrE) cells as well as in the mature, formative state epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative-feedback loops of the FGF pathway, are differently regulated. Whereas DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into the PrE while protecting the Epi identity from autocrine signalling.