Defining the regulators of neural crest lineage commitment

The neural crest is a multipotent stem cell-like population, unique to vertebrates, that contributes to a wide variety of derivatives including craniofacial cartilage and bone, sensory and autonomic ganglia of the peripheral nervous system and pigment cells. Neural crest progenitors arise at the neural plate border and first reside within the dorsal aspect of the central nervous system. Neural crest cells then delaminate from the neural tube and migrate extensively throughout the embryo. Their migratory pathways are thought to influence their subsequent cell fate choice. A complex gene regulatory network guides the progressive stages of neural crest development. Over the past decades, research in the field has provided substantial information about the early events governing neural crest determination and delamination. However, little is known about regulatory changes governing their subsequent choice of migratory pathway, or how this influences commitment versus plasticity of cell fate. This proposal aims to use new tools to study this important question in an in vivo context. In the suggested project, the applicant will investigate how the regulatory landscape of neural crest cells changes when they travel along distinct migratory pathways. First, the applicant will perform RNA-seq of distinct neural crest subpopulations associated with different migratory pathways and time points that are indicative of specific cell fate choices (e.g. becoming melanocytes versus neurons). This discovery based information promises to uncover new candidate regulatory genes that can then be functionally tested using loss of function approaches. Ultimately, the applicant will test plasticity of neural crest cells along distinct migratory pathways by challenging their fate via manipulation of the neural crest gene regulatory network. The following specific aims will be performed: Specific Aim 1: Transcriptional profiling of neural crest cells along different migratory pathways; Specific Aim 2: Validation and functional analysis of the migratory neural crest transcriptome; Specific Aim 3: Testing the plasticity of the neural crest by regulatory circuit reprogramming; Taken together, this information will allow us to "read" and modify encrypted regulatory information on a genome wide scale and resolve existing discrepancies regarding neural crest cell plasticity. Furthermore, understanding the mechanisms of neural crest migration and cell fate choice during normal embryonic development will also help to understand the cause of neural crest derived birth defects and cancers. Thus, genes and genetic sub-circuits discovered in this study could be useful targets for therapeutic interventions in the future.

The neural crest is a multipotent stem cell-like population, unique to vertebrates, that contributes to a wide variety of derivatives including craniofacial cartilage and bone, sensory and autonomic ganglia of the peripheral nervous system and pigment cells. Neural crest progenitors arise at the neural plate border and first reside within the dorsal aspect of the central nervous system. Neural crest cells then delaminate from the neural tube and migrate extensively throughout the embryo. Their migratory pathways are thought to influence their subsequent cell fate choice. Hence, different subpopulations of neural crest cells have a specific differentiation potential, which is also reflected in their unique gene expression signatures. Using the chicken embryo as a model system, we took advantage of the fact that tissue specific enhancers regulate the expression of many neural crest specific genes like Sox10 or FoxD3. The expression of Sox10 is mediated by two regulatory elements: Sox10E2, which initiates expression in cranial neural crest, and Sox10E1, which is active in the vagal and trunk neural crest. Both also mediate Sox10 expression in the otic placode. I have dissected and analyzed the Sox10E1 enhancer element to identify upstream regulatory inputs that drive its activity. I have identified two critical binding sites for Sox transcription factors with differential impact on neural crest and otic vesicle expression. I have further used the Sox10 E1 enhancer to specifically label and isolate neural crest cells migrating on the ventral pathway in the trunk, which will form the ganglia of the peripheral nervous system. The isolated cells were used for whole genome analysis, which provided me with a dataset of all active genes in this particular neural crest sub population. A total of 474 genes are significantly enriched in this cell population, which was verified by in situ hybridization of selected transcripts. Further analysis was concentrated on components of the Notch signaling pathway, because their presence is a unique feature of the trunk ventral migrating population and not found in any of the datasets we have obtained from other neural crest subpopulations. Interestingly, the present Notch components as well as many other candidate genes from the screen have been associated with neuroblastoma, a childhood tumor that derives from the trunk neural crest. Additional research is needed to investigate the role of Notch signaling during the process of trunk neural crest migration and to understand how these factors are involved in disease transformation. Ultimately, the results have the potential to yield new diagnostic markers and therapeutic intervention points for the treatment of neural crest derived diseases.