XB-ART-42851Proc Natl Acad Sci U S A January 4, 2011; 108 (1): 155-60.
Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.
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.
PubMed ID: 21169220
PMC ID: PMC3017139
Article link: Proc Natl Acad Sci U S A
Genes referenced: pax3 tfap2a
Morpholinos referenced: ctnnb1 MO1 fgf8 MO4 hes4 MO1 hes4 MO2 msx1 MO1 myc MO3 pax3 MO1 sox8 MO1 tfap2a MO1 zic1 MO1
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|Fig. 1. AP2a controls NB formation. (A) NB specifier patterns from the late gastrula stage (stage 12) to late neurula stage (stage 20). ISH results for ap2a, hairy2, msx1, pax3, zic1, and snail2. (a–f) Gastrulas, side views. Arrowheads indicate the dorsal blastopore lip; arrows indicate the NB. (g–r) Anterior views of mid (stage 17; g–l) and late (stage 22; m-r) neurulae. (B) AP2a controls NB formation. NB expression of hairy2, msx1, and pax3 depends on AP2a, whereas AP2a down-regulates zic1 expression. AP2a morphants were analyzed at stage 10.5–17 for hairy2, msx1, pax3, and zic1 ISH [stage 17, anterior views; stage 10.5, blastopore (dots) views]. (C) Up-regulation, but not initiation, of ap2a at the NB depends on the other NB specifiers. ap2a ISH in Hairy2, Pax3, Msx1, or Zic1 morphants (stage 12, dorsal views, blastopore on top; stage 17, anterior views). (D) MO specificity is validated by rescue analysis: AP2 (a and b, ISH for pax3), Hairy2, and Pax3 (c–f, ISH for ap2). The star indicates the injected side.|
|Fig. 2. AP2a induces NB genes in neuralized ectoderm explants and is essential for NC induction by FGF and Wnt. (A) Although explants neuralized by Noggin express zic1, AP2 activates coexpression of pax3 and msx1. RT-PCR for NB markers pax3, msx1, and zic1 after injection of Noggin (lane 4), ap2a (lane 5), or ap2a combined with Noggin (lane 6). Controls are shown in lanes 1–3 (SI Materials and Methods). (B) FGF controls ap2a at the NB in a β-catenin–dependent manner. RT-PCR analysis of ap2a in neuralized explants (lanes 4–7) and treated with bFGF (lane 5), CHX (lane 6), or CHX followed by bFGF (lane 7). Controls are shown in lanes 1–3 (SI Materials and Methods). (C) Expansion of ap2a by FGF is abolished by β-catenin depletion. Injections of a constitutively active FGFR4 (caFGFR4), β-catenin MO, or both at stage 17, anterior views. (D) AP2a restores snail2 expression in both FGF and β-catenin morphants. Injections of ap2a, FGF8MO, and βcatMO alone and combined. (E) AP2a mediates NB induction by FGF and Wnt. AP2a morphants lack snail2, hairy2, and msx1 expression even with FGF or Wnt GOF. Injections of caFGFR4, AP2aMO, and activated β-catenin (Δβcat) alone or combined at stage 17, anterior views. The star indicates the injected side.|
|Fig. 4. AP2a induces NC downstream of Pax3 and Zic1. (A) AP2a restores snail2 and foxd3 expression in Pax3 or Zic1 morphants. Injections of Pax3MO, Zic1MO, and ap2a alone or in combination. In contrast, Pax3 and Zic1 do not maintain/increase the NC domain in AP2a morphants. Injections of Pax3, Zic1, or AP2aMO alone or in combination at stage 17, anterior views. (B) Embryos were injected ventrally with pax3 or zic1 mRNAs, AP2aMO, or all three. Ectopic snail2, but not foxd3, induction by Pax3/Zic1 is lost in AP2a morphants. Side (a, d, and f) and ventral (b, c, and e) views are shown; stars and arrows indicate injected areas. (C) The Pax3/Zic1 combination does not induce snail2 and foxd3 in AP2a-depleted explants. RT-PCR analysis of snail2 and foxd3 in animal caps injected with pax3 (lane 4), zic1 (lane 5), or AP2aMO (lane 6) or coinjected with pax3 and zic1 (lane 7) or with all three (lane 8). Controls are shown in lanes 1–3 (SI Materials and Methods).|
|Fig. 5. AP2a specifies NC independent of its NB specifier role. (A) Early APaGR induction (stage 8–17) enlarges both the NB and the NC, whereas late induction (stage 14–17) expands snail2 (b) without affecting NB markers (d, f, h, and j). Anterior views, ISH snail2, hairy2, msx1, zic1, and sox2. (B) Snail2 is an immediate-early target of AP2a. RT-PCR for snail2 in neuralized explants: Noggin (lane 1) and Noggin/AP2aGR (lanes 2–5) (Fig. S5). AP2aGR was induced (Dex+) from stage 11–13 in DMSO- or CHX-treated explants. (C) AP2aGR activation at mid-neurulation (stage 14) increases sox10 expression in the neural tube and NC (stage 22, anterior views, with a star indicating the injected side), whereas an inducible dominant interfering AP2 mutant prevents NC emigration on the injected side (stage 22 embryos, sox9 and sox10 ISH). (D) Late (stage 22) ap2a expression is regulated by other NC specifiers. c-Myc morphants exhibit decreased ap2a expression, whereas Sox8 depletion affects NC migration but not ap2a levels (a and c, uninjected side; b and d, injected side). (E) Model of AP2a dual function during NC development in vertebrates. Interactions among the neural plate, ectoderm, and paraxial mesoderm induce NB (lane 1). At a low dose, Wnt increases ap2a expression, which initiates NB formation (notably by direct activation of pax3) and triggers the NB specifiers gene cassette (lane 2). Once a robust NB pattern is established, AP2a also acts as a downstream NC specifier, with direct activation of some NC specifiers (snail2, solid arrow) but not others (foxd3, interrupted arrow) and regulation by other NC specifiers (i.e., cMyc) (lane 3).|