FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, we have analyzed the role of the transcription factors Msx1 and Pax3. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, we show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction.

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9),whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However,neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations:while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.

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...

The neural crest is a transient and multipotent cell population arising at the edge of the neural plate in vertebrates. Recent findings highlight that neural crest patterning is initiated during gastrulation, i.e. earlier than classically described, in a progenitor domain named the neural border. This chapter reviews the dynamic and complex molecular interactions underlying neural border formation and neural crest emergence.

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

Defining which key factors control commitment of an embryonic lineage among a myriad of candidates is a longstanding challenge in developmental biology and an essential prerequisite for developing stem cell-based therapies. Commitment implies that the induced cells not only express early lineage markers but further undergo an autonomous differentiation into the lineage. The embryonic neural crest generates a highly diverse array of derivatives, including melanocytes, neurons, glia, cartilage, mesenchyme, and bone. A complex gene regulatory network has recently classified genes involved in the many steps of neural crest induction, specification, migration, and differentiation. However, which factor or combination of factors is sufficient to trigger full commitment of this multipotent lineage remains unknown. Here, we show that, in contrast to other potential combinations of candidate factors, coactivating transcription factors Pax3 and Zic1 not only initiate neural crest specification from various early embryonic lineages in Xenopus and chicken embryos but also trigger full neural crest determination. These two factors are sufficient to drive migration and differentiation of several neural crest derivatives in minimal culture conditions in vitro or ectopic locations in vivo. After transplantation, the induced cells migrate to and integrate into normal neural crest craniofacial target territories, indicating an efficient spatial recognition in vivo. Thus, Pax3 and Zic1 cooperate and execute a transcriptional switch sufficient to activate full multipotent neural crest development and differentiation.neural crest developmental program | ectoderm to neural crest transcriptional switch T he neural crest, a transient embryonic cell population, develops into an amazing array of derivatives, including peripheral nervous system, pigment cells, cartilage, mesenchyme, and bone (1). During neural development, definitive neural crest (NC) induction is preceded by formation of a neural border territory between the neural plate and the nonneural ectoderm. This region is initiated by transcription factor TFAP2-α (AP2a, transcription factor activating enhancer binding protein 2 alpha) and reinforced by Hairy2, Msx1, and AP2a itself along with secreted bone morphogenetic protein (BMP) antagonists. In addition, Pax3/Pax7, Gbx2, and Zic1 are also essential for neural border specification (2-8). In turn, these transcription factors cooperate to activate the NC specifiers snail2 (snai2), soxE (sox8, 9, 10), and foxd3 in the ectoderm (reviewed in ref. 9). Although each neural border specifier is necessary for NC formation in vivo, none of these factors alone is sufficient to initiate NC induction in the ectoderm (3,7,8). Addition of a secreted BMP antagonist, a Wnt signal, or another transcription factor is needed to activate early NC specifiers (reviewed in ref. 10). In particular, Pax3 and Zic1 can synergize and initiate expression of early NC markers in blastula ectoderm (3,5,(7)(8)(9)(10)(11). However, it remains unknown which of t...

Loss of function studies have shown that the Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest formation and maintains cells in a mitotic undifferentiated state. However, its position in the genetic cascade regulating neural crest formation and its relationship with other neural crest regulators remain largely unknown. Here we find that Hairy2 is regulated by BMP, FGF and Wnt and that it is only required downstream of BMP and FGF for neural crest formation. We show that Hairy2 overexpression represses neural crest and upregulates neural border genes at early stages while it expands a subset of them in later embryos. We show that Hairy2 downregulates Id3, another essential HLH neural crest regulator, through attenuation of BMP signaling. Knockdown and rescue experiments indicate that Id3 protein, which physically interacts with Hairy2, negatively regulates Hairy2 activity. However, Id3 is required to allow Hairy2 to promote neural crest formation. Together, our results provide evidence that Hairy2 acts downstream of FGF and BMP signals at the neural border to maintain cells in an undifferentiated state, and that Hairy2-Id3 interactions play an essential role in neural crest progenitor specification.

The neural crest is induced at the edge between the neural plate and the nonneural ectoderm, in an area called the neural (plate) border, during gastrulation and neurulation. In recent years, many studies have explored how this domain is patterned, and how the neural crest is induced within this territory, that also participates to the prospective dorsal neural tube, the dorsalmost nonneural ectoderm, as well as placode derivatives in the anterior area. This review highlights the tissue interactions, the cell-cell signaling and the molecular mechanisms involved in this dynamic spatiotemporal patterning, resulting in the induction of the premigratory neural crest. Collectively, these studies allow building a complex neural border and early neural crest gene regulatory network, mostly composed by transcriptional regulations but also, more recently, including novel signaling interactions.