Reprogramming of avian neural crest axial identity and cell fate

Abstract

Neural crest populations along the embryonic body axis of vertebrates differ in developmental potential and fate, so that only the cranial neural crest can contribute to the craniofacial skeleton in vivo. We explored the regulatory program that imbues the cranial crest with its specialized features. Using axial-level specific enhancers to isolate and perform genome-wide profiling of the cranial versus trunk neural crest in chick embryos, we identified and characterized regulatory relationships between a set of cranial-specific transcription factors. Introducing components of this circuit into neural crest cells of the trunk alters their identity and endows these cells with the ability to give rise to chondroblasts in vivo. Our results demonstrate that gene regulatory circuits that support the formation of particular neural crest derivatives may be used to reprogram specific neural crest-derived cell types.

Figure 1. Identification of cranial-specific regulators by comparative transcriptomics

(A) Diagram depicting dorsal view of chick embryo. (B, C) Embryos electroporated with FoxD3 axial-specific enhancers NC1 and NC2, active in cranial and trunk neural crest (NC), respectively. (D) Comparative transcriptome analysis of FACS sorted cranial and trunk neural crest populations identified 216 cranially-enriched genes. (E) Summary of gene ontology analysis for the cranial-enriched genes. (F) Enrichment levels of transcription factors expressed in the cranial neural crest. HH: Hamburger and Hamilton stages.

Figure 2. Spatial and temporal expression of cranial neural crest transcription factors (TFs)

(A, B) Dorsal views of embryos after in situ hybridization for cranial neural crest-specific TFs reveals expression at early (A) or later (B) stages. (C) Double in situ hybridization reveals that cranial regulator Dmbx1 is expressed in the anterior neural plate border, whereas Msx1 is expressed along the entire neural axis. (D) Fate map of neural crest progenitors (red and green dots) at stage HH8- confirms cranial specific expression of Dmbx1 (purple). (E) Diagram summarizing timing of expression of early and late cranial regulators.

Figure 3. Cranial-specific transcriptional circuit underlying neural crest axial identity

(A–D) Whole mount dorsal views of embryos after morpholino (Mo) targeted to indicated transcription factor (TF) was transfected to the right side (green) and control morpholino (CoMo) to the left side (blue) of each embryo. (E–H) Same embryos as above after in situ hybridization for indicated downstream TF; blue arrows indicate transcript downregulation. (I) Comparing control to loss of function (LOF) neural folds by qPCR reveals differential regulation of downstream targets with significant changes indicated by * (Student’s t-test, P<0.05). (J) Diagram summarizing cranial specific gene regulatory circuit delineated by functional assays. (K) Chromatin immunoprecipitation (ChIP) demonstrates direct association of cranial specific TFs with promoters of their downstream targets. No enrichment was observed for intergenic negative control regions (NCR), or when the procedure was performed with mock-transfected embryos (see Materials and Methods for more information). Error bars on (I) and (K) represent standard deviation.

Figure 4. Reprogramming of neural crest axial identity and fate

(A) Diagram of chick embryo electroporated with the Sox10E2 enhancer, expressed only in migrating cranial (Cr) neural crest (NC). (B) Transfection of trunk neural crest cells with control RFP expression vector shows no Sox10E2 expression. (C) Reprogramming of trunk neural crest cells with cranial specific regulators Sox8, Tfap2b and Ets1 results in robust expression of the cranial Sox10E2 enhancer in the trunk region (n=15/15). (D, E) Flow cytometry analysis of dissociated embryonic trunks shows a large number of Sox10E2+ trunk neural crest cells after reprogramming. (F, G) Reprogrammed (Rep) trunk neural crest display increased expression of the chondrocytic genes Runx2 and Alx1, while trunk genes Dbx2 and Hes6 are strongly downregulated. Error bars represent standard deviation. (H, I) Histological sections of E7 embryonic heads show that wild type (WT) trunk neural crest cells (GFP+; green) fail to form cartilage (Col9a+; red) after transplantation to the cranial region (n=0/5). (J, K) Inset of (H–I), showing absence of GFP+ chondrocytes. (L, M) Reprogrammed trunk neural crest cells (expressing Sox8, Tfap2b and Ets1) from GFP donor embryos transplanted to the head form ectopic cartilage nodules. (N, O) Inset of (L–M), showing chondrocytes derived from trunk neural crest cells (GFP+ and Col9a+).