Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network

Abstract

The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates.he neural crest (NC), a vertebrate embryo multipotent population, gives rise notably to peripheral nervous system, melanocytes and craniofacial structures (1). Combined Wnt, FGF, and BMP signals emanating from the paraxial mesoderm, neural plate, and nonneural ectoderm activate NC specifiers (e.g., snail2, foxd3, sox10) that are expressed in the premigratory NC and essential for further NC development (2-5). NC induction starts during gastrulation with the establishment of a broad competence domain at the neural border (NB) (6-8). The NB specifiers (e.g., pax3, msx1, zic1, hairy2) are essential for NC induction, but usually are not maintained in the NC cells after induction (5, 6, 9). They integrate FGF, Wnt, and BMP signals into a coherent induction of NC specifiers by diverse actions (6, 9-11). The hierarchical organization of this network is conserved across vertebrate evolution (12). Our understanding of this complex network remains preliminary, however; defining the roles of early actors is essential to understanding how this network might have emerged in vertebrates. Comparative analyses in amphioxus and lamprey have highlighted ap2 transcription factor (tfap2) up-regulation at the NB in vertebrates (13). AP2 transcription factors are well conserved in vertebrates and are essential for both nonneural ectoderm development and NC induction (14-16). Expression in the ectoderm is shared among chordates, whereas the up-regulation at the NB is a hallmark of vertebrates (13). AP2a depletion or mutation in mice or zebrafish results in a variety of NC defects (16)(17)(18). Recent data in a basal vertebrate (lamprey) suggest its role in NB formation, whereas results in zebrafish embryos identified a function during NC postspecification steps (8,19,2...

Signaling pathways and tissue interactions in neural plate border formation

“…16 However, both direct Wnt/b-Catenin target genes, tfap2a and gbx2, are already expressed at that stage and in the case of tfap2a this early expression also depends on BMP signaling. 30,32,38 Moreover, tfap2a induction in na€ ıve ectodermal explants required only low levels of Wnt/ b-Catenin activity, 30 suggesting that low endogenous activity is sufficient to induce tfap2a in the NPB. During gastrulation, NPB genes apparently exhibit differential sensitivity toward Wnt/b-Catenin signaling.…”

“…27,29 The transcription factor AP2-alpha (gene: tfap2a) is considered as the central player in NPB specification. 30 In blastula and early gastrula stage Xenopus embryos, tfap2a is expressed in the entire non-neural ectoderm and the prospective NPB territory and a recent study showed that this expression depends on BMP activity. 31,32 The source of BMP ligands is likely the non-neural ectoderm or the prospective NPB ectoderm itself, which expresses BMP ligands in zebrafish, frog, chick and mouse embryos.…”

“…30,39 BMP and Wnt/b-Catenin signaling functionally interact with Pax3, Zic1 and Ap2a in later phases to induce bona fide NC fate. 19,30,40,41 Likewise, Wnt/b-Catenin signaling is essential for NPB specification in epiblast explants from chick blastulae, where Wnt activity precedes and upregulates bmp4, 17 as well as for expression of NPB genes in hESCs. 11 Steventon and colleagues have analyzed Wnt/ b-Catenin activity in Xenopus embryos using the TOPFlash reporter gene assay.…”

“…In Xenopus, Wnt/b-Catenin signaling, putatively activated by Wnt8, directly regulates the expression of the earliest NPB markers tfap2a and gbx2. 30,38 AP2-alpha is considered as the key transcription factor responsible for further establishing and reinforcing NPB specification, since it is required for the upregulation of multiple other early NPB specifiers including hairy2, msx1 and pax3 at early gastrula stages. 30 Similarly and at approximately the same time in development, Gbx2 depletion resulted in failure to upregulate pax3 and msx1.…”

“…30,38 AP2-alpha is considered as the key transcription factor responsible for further establishing and reinforcing NPB specification, since it is required for the upregulation of multiple other early NPB specifiers including hairy2, msx1 and pax3 at early gastrula stages. 30 Similarly and at approximately the same time in development, Gbx2 depletion resulted in failure to upregulate pax3 and msx1. 38 So far, direct transcriptional regulation of gbx2 by AP2-alpha or vice versa has not been reported.…”