Pax7 is regulated by cMyb during early neural crest development through a novel enhancer

Abstract

SUMMARYThe neural crest (NC) is a migratory population of cells unique to vertebrates that generates many diverse derivatives. NC cells arise during gastrulation at the neural plate border (NPB), which is later elevated as the neural folds (NFs) form and fuse in the dorsal region of the closed neural tube, from where NC cells emigrate. In chick embryos, Pax7 is an early marker, and necessary component of NC development. Unlike other early NPB markers, which are co-expressed in lateral ectoderm, medial neural plate or posterior-lateral mesoderm, Pax7 early expression seems more restricted to the NPB. However, the molecular mechanisms controlling early Pax7 expression remain poorly understood. Here, we identify a novel enhancer of Pax7 in avian embryos that replicates the expression of Pax7 associated with early NC development. Expression from this enhancer is found in early NPB, NFs and early emigrating NC, but unlike Pax7, which is also expressed in mesodermal derivatives, this enhancer is not active in somites. Further analysis demonstrates that cMyb is able to interact with this enhancer and modulates reporter and endogenous early Pax7 expression; thus, cMyb is identified as a novel regulator of Pax7 in early NC development. KEY WORDS: Neural plate border, Neural crest, PAX7, cMyb, Enhancer, ChickPax7 is regulated by cMyb during early neural crest development through a novel enhancerStephanie Vadasz, Jonathan Marquez, Maria Tulloch, Natalia A. Shylo and Martín I. García-Castro* DEVELOPMENT 3692 regulatory elements and TFs that initiate the expression of Pax7 at the NPB have not been reported.Here, we identify a novel Pax7 cis-regulatory element that directs expression in the avian NPB, NFs and early migrating NC cells, similar to the endogenous Pax7 expression associated with early NC. This element does not provide expression in later NC or in mesoderm, where endogenous Pax7 is seen. Through mutagenesis and overexpression studies, we unveil cMyb as a regulator of this enhancer and of endogenous early Pax7 expression. Our study is the first to demonstrate the ability of cMyb to regulate a border specifier directly during NC development and thus proposes a fundamental role for cMyb during the early phases of the NC-GRN. MATERIALS AND METHODS Sequence conservation and TF-binding analysisSequence conservation was determined using VISTA Browser (Visel et al., 2007), over a 50 bp window with 70% conserved identity. Putative TFbinding sites were identified using the JASPAR core vertebrate database (Bryne et al., 2008). Plasmid constructsEnhancer constructs were PCR amplified (DNeasy Kit, Qiagen) from genomic chick or human DNA (a gift from J. Noonan, New Haven, CT, USA), and cloned into ptkEGFP (a gift from H. Kondoh, Osaka, Japan) or ptkmCherry (a gift from D. Meulemans, Boulder, CO, USA) using KpnI and XhoI. pRFP was generated by removing the miRNA cassette from pRFPRNAi (ARK Genomics). Deletion constructs were generated by first amplifying each end with primers containing 15 bp of internal overlapping sequence fol...

Regulatory Logic Underlying Diversification of the Neural Crest

“…In addition, transcription factors Myb, Myc, and Prdm1 are also required for specification [42–44]. Myb directly interacts with Pax3/7 as well as Sox10 in chick, and Prdm1 directly interacts with Tfap2a and Foxd3 in fish [43–46]. …”

Specification and formation of the neural crest: Perspectives on lineage segregation

“…cMyb is known to regulate Pax7 expression in chick at neural plate border stages (Vadasz et al, ), and was also found to regulate the expression of the neural crest specifier snai2 (Karafiat et al, ). Gene expression analysis following cMyb knockdown in chick revealed downregulation of Pax7 and Twist1 , among others, and an upregulation of Zic1 (Betancur, Simões‐Costa, Sauka‐Spengler, & Bronner, ), placing cMyb as a regulator of neural plate border and neural crest specifier genes.…”

“…The direct regulation of Pax7 expression is a major question. In chick, this has been shown to occur through cMyb binding to a Pax7 enhancer (Vadasz, Marquez, Tulloch, Shylo, & Garcia-Castro, 2013), and studies in Xenopus indicate that Pax3 directly activates its own expression and that of pax7 (Maczkowiak et al, 2010;Plouhinec et al, 2014).…”

Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density

“…In this assay, Hamburger-Hamilton stage 11 embryos are electroporated with DNA reporter constructs in which the putative enhancer is cloned upstream of a minimal promoter driving EGFP expression (see Materials and Methods ). This assay has been previously used to validate enhancers for multiple signaling pathways [ 20 , 54 , 55 , 56 , 57 , 58 , 59 , 60 ]. Endogenous Sonic Hedgehog (SHH) produced by the notochord and floorplate drives expression of Hh-dependent enhancers in the ventral half of the neural tube [ 53 ].…”