Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Research Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network Marcos Simo˜es-Costa,1 Joanne Tan-Cabugao,1 Igor Antoshechkin,1 Tatjana Sauka-Spengler,2 and Marianne E. Bronner1,3 1Division of Biology, California Institute of Technology, Pasadena, California 91125, USA; 2The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom The neural crest is an embryonic stem cell population that gives rise to a multitude of derivatives. In particular, the cranial neural crest (CNC) is unique in its ability to contribute to both facial skeleton and peripheral ganglia. To gain further insight into the molecular underpinnings that distinguish the CNC from other embryonic tissues, we have utilized a CNC- specific enhancer as a tool to isolate a pure, region-specific NC subpopulation for transcriptional profiling. The resulting data set reveals previously unknown transcription factors and signaling pathways that may influence the CNC’s ability to migrate and/or differentiate into unique derivatives. To elaborate on the CNC gene regulatory network, we evaluated the effects of knocking down known neural plate border genes and early neural crest specifier genes on selected neural crest- enriched transcripts. The results suggest that ETS1 and SOX9 may act as pan-neural crest regulators of the migratory CNC. Taken together, our analysis provides unprecedented characterization of the migratory CNC transcriptome and identifies new links in the gene regulatory network responsible for development of this critical cell population. [Supplemental material is available for this article.] The neural crest is a multipotent cell population that contributes Although all neural crest cells share common molecular and to a wide variety of derivatives, including sensory and autonomic behavioral traits, they can be subdivided operationally according ganglia of the peripheral nervous system, cartilage and bone of the to their axial level of origin, as cranial, vagal, trunk, and sacral (Le face, and pigmentation of the skin (Le Douarin and Kalcheim Douarin and Kalcheim 1999). These subpopulations differ in mi- 1999). Acquisition of the neural crest played a critical role in ver- gratory pathways and types of derivatives formed; e.g., cranial tebrate evolution, facilitating evolution of the jaw and active neural crest (CNC) has the ability to give rise to cartilage and predation that fostered diversification of vertebrates (Gans and bone, whereas trunk neural crest does not, even after trans- Northcutt 1983). Due to the extensive contributions of the plantation in the head (Nakamura and Ayer-le Lievre 1982; Le neural crest to vertebrate embryos (Hall 2000), defects in its de- Douarin et al. 2004). Recent evidence points to distinct regu- velopment contribute to a large percentage of congenital birth latory programs controlling expression of neural crest specifier defects (Trainor 2010). genes SOX10 and FOXD3 in these two subpopulations (Betancur Neural crest cells are induced during gastrulation (Basch et al. et al. 2010b; Simo˜es-Costa et al. 2012), raising the intriguing 2006), but first become apparent as ‘‘premigratory’’ cells in the possibility that a molecular framework may underlie such dorsal neural tube by expression of a suite of neural crest specifier differences. genes like FOXD3, SNAI2, and SOX9 (Sauka-Spengler and Bronner- Here, we take advantage of a cranial-specific neural crest en- Fraser 2008). They subsequently undergo an epithelial to mesen- hancer as a tool to isolate pure populations of migrating CNC cells chymal transition (EMT) to leave the neural tube, migrate long from an in vivo context, allowing us to obtain a genome-wide distances, and differentiate into diverse cell types. To understand representation of the active transcriptome of this important cell the progression of their development, we have proposed a gene population. Because migrating CNCs intermingle with other regulatory network (GRN) that underlies this complex process mesenchymal cell types, obtaining pure populations has been (Meulemans and Bronner-Fraser 2004), comprised of nested sub- particularly difficult. Moreover, no available reagents distinguish networks, each controlling distinct events from specification to CNCs from subpopulations at other axial levels. Recently, how- emigration, and ultimately to migration and differentiation ever, we have isolated NC enhancers that mediate gene expression (Betancur et al. 2010a). However, this network is far from com- at unique stages and axial levels (Betancur et al. 2010a), including plete: A relatively small number of transcriptional regulators have Sox10E2, which mediates reporter expression selectively in all been identified/characterized, and little is known about effector migrating CNC. This tool enables us to isolate these cells and ob- genes present in migrating neural crest cells (Simo˜es-Costa and tain genome-wide information regarding their active tran- Bronner 2013). Thus, elaboration of the neural crest GRN is cru- scriptome using RNA-seq. Here, we utilize this approach to char- cial to clarify how distinct differentiation programs are activated acterize the suite of genes enriched in migratory CNCs. The results in multipotential precursors to drive commitment toward specific provide unprecedented characterization of their transcriptome fates. Ó 2014 Simo˜es-Costa et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue 3Corresponding author publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After E-mail [email protected] six months, it is available under a Creative Commons License (Attribution- Article published online before print. Article, supplemental material, and pub- NonCommercial 3.0 Unported), as described at http://creativecommons.org/ lication date are at http://www.genome.org/cgi/doi/10.1101/gr.161182.113. licenses/by-nc/3.0/. 24:281–290 Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/14; www.genome.org Genome Research 281 www.genome.org Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Simo˜ es-Costa et al. and allow identification of new links in the GRN responsible for addition, 257 genes were depleted in the migrating CNC pop- development of this critical cell population. ulation relative to the whole embryo (Supplemental Table 2). As proof of principle regarding data set quality, we looked for Results known transcription factors, including bona fide neural crest markers such as TFAP2A, PAX7, SOX8 and SOX10, FOXD3, among Isolation of pure cranial neural crest population others. As expected, all were present in our data set (Fig. 3C). SOX10 is one of the genes with highest enrichment relative to Expression of the specifier gene SOX10 initiates as NC cells com- GFPÀ cells (Fig. 3D). ETS1, a key regulator of CNC formation mence emigration from the neural tube at all axial levels (Kim et al. (The´veneau et al. 2007; Betancur et al. 2010b; Simo˜es-Costa et al. 2003; McKeown et al. 2005). Previously, we identified avian NC 2012), is significantly enriched in the data set although it is enhancers that drive reporter expression in a manner that re- strongly expressed in other embryonic tissues such as endothelial capitulates endogenous SOX10 expression (Fig. 1A; Betancur et al. cells. 2010b). In particular, Sox10E2 (Fig. 1B,C,C9) mediates expression Figure 3D shows genes with high enhancement or depletion in the CNC but not in the trunk subpopulations. in the migratory CNC. In addition to transcription factors, genes To confirm specificity, we compared enhancer activity with known to play important roles in CNC formation are also present. endogenous SOX10 expression. Antibody staining in embryos For example, Collagens 2 and 9, crucial for chondrocyte de- electroporated with Sox10E2:eGFP shows that the enhancer is only velopment, are enriched (Fig. 3D), as are nuclear receptors such as active in SOX10+ neural crest (Fig. 1D–F). Although the majority of RXRG and other transcriptional regulators such as ALX1. Genes ectodermal cells are efficiently coelectroporated with Sox10E2 en- visibly down-regulated or absent from the CNC include a number hancer-driven (eGFP) and ubiquitous promoter-driven (mRFP) of HOX genes, which is expected since the CNC lacks most HOX constructs, as highlighted by H2B-RFP expression (Fig. 1G), only genes, GATA factors 4,5,6 as well as TBX5, 20 and 15 (Fig. 3C; SOX10+ cells exhibit eGFP expression (Fig. 1H–J). To examine en- Supplemental Table 2). Importantly, ZIC1, important for trunk NC hancer activity during neural crest migration, we performed time- specification and a trunk-specific input in FOXD3 (Simo˜es-Costa lapse imaging. Supplemental Movie 1 highlights the remarkable et al. 2012), is also absent from the CNC (Fig. 3C). movement of CNC cells as they migrate throughout the embryonic head (Fig. 1K–M). Thus, Sox10E2 enhancer is a useful tool for in vivo labeling and identifying migrating CNCs. Biological pathway analysis of cranial neural crest In order to obtain a transcriptional profile of the CNC, transcriptome Sox10E2:eGFP was electroporated into gastrula-stage chick em- To gain an overview of the molecular processes operational in the bryos subsequently incubated to stages of active CNC migration CNC transcriptome,