Building regulatory networks in neural crest induction: integrative approaches in vivo and in stem cells – CRESTNET

These approaches include microarray analysis, next generation sequencing and in situ hybridization to define the molecular fingerprint of neural crest progenitors; microarray analysis and in vivo validation of pax3 direct targets in neural crest induction; and pax3 regulatory region analysis to identify pax3 direct regulators.In addition, we use culture of human embryonic stem cells and xenopus tissues to decipher the mechanisms of neural crest induction in vivo and in vitro.

The results from this first 6-months are :
- to characterize the genome-wide developmental expression profile of NC progenitors from neural border stage to premigratory NC using microarrays.
- to start understanding the Pax3-centered regulatory network at the neural border.
- To determine if Pax3, in combination with Zic1, is sufficient for development of several differentiated and functional neural crest derivatives in vitro
- To elucidate whether induced neural crest is able to generate several lineages in vitro.

Our next objectives are:
- to provide a larger NC profiling using NGS
- to provide a Pax3-centered regulatory network at the neural border : Within the neural border/NC transcriptome landscape, we will provide the description of Pax3 direct and indirect targets, and a analysis of Pax3 regulation in vivo. - - to use our model of NC development to derive a protocol for programming hiPSCs into neural crest, and possibly multipotent neural crest, in vitro.

on going

Reprogramming stem cells into a precise lineage and driving them to differentiate appropriately is one of the major challenges opened by adult cell reprogramming into pluripotent cells (iPS). These cells are equivalent to ES cells and respond to the molecular signals acting in the embryo. However, there is much to study in this emerging field, to derive complex lineages in a defined and controlled manner.

Our research focuses on the earliest steps of the neural crest program. The neural crest is a multipotent and migratory cell population, which emerges from the dorsal neural tube of vertebrate embryos by an epithelial-to-mesenchymal transition, populates most of our tissues and organs and differentiates notably into peripheral neurons and glia, melanocytes, craniofacial skeleton. While the cellular and molecular events in migration and differentiation have been explored for many years, it is only in the last few years that research was conducted to understand the upstream regulations happening during gastrulation and leading to induction and specification of the neural crest lineage from the neural tube and adjacent ectoderm. Our laboratory has produced experimental basis for constructing an initial model for neural crest emergence from the neural plate, relying of the key role of the transcription factor Pax3. We have also identified partners and regulators for Pax3. Using epistasis analysis in vivo, we have thus actively participated in building the current putative neural crest gene regulatory network.

One major challenge is now to convert this network of epistatic interactions into a network of direct regulations. We have developed a series of complementary tools and approaches to investigate pax3 targets and regulators in neural crest earliest development, on a genome-wide scale, backed up by in vivo experimental validation procedures. These approaches include microarray analysis, next generation sequencing and in situ hybridization to define the molecular fingerprint of neural crest progenitors; microarray analysis and in vivo validation of pax3 direct targets in neural crest induction; and pax3 regulatory region analysis to identify pax3 direct regulators. This first objective will result in building a new network of direct regulations centered on pax3 and linked to neural crest induction with special emphasis on multipotency and epithelial to mesenchymal transition regulators.

A second challenge is to translate the knowledge accumulated in animal models to human stem cell biology. We have developed strategies using both amphibian ectoderm stem cells and human iPS, to understand how neural crest lineage and neural crest-derived fates may be induced and modulated in vitro, focusing on the roles of embryonic inducers Wnts and Pax3. We will seek specific defined conditions for neural crest but not neural induction, as well as conditions for obtaining multipotent progenitors and further allow their determination and differentiation into various lineages. This second objective will provide the experimental basis for controlling neural crest induction in vitro, and favor neural crest fate diversification at the expense of neural fates.

The four teams forming the CrestNet consortium have complementary expertise in neural crest biology, stem cell biology, genomics, and bioinformatics. We have already set up the basis for this program together, by producing many preliminary data for the two objectives. We will thus fully synergize to implement this program, which will result, first, in providing the completed regulatory network and fill missing links between neural plate regionalization and neural crest induction in vivo; and second, will allow the direct translation of these findings into protocols to induce multipotent neural crest from pluripotent stem cells.