Pax3 and Zic1 drive induction and differentiation of multipotent, migratory, and functional neural crest in Xenopus embryos

Abstract

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.

The 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, 7–11). However, it remains unknown which of these factors are sufficient for switching on definitive NC lineage commitment. These factors should be sufficient to drive an autonomous and complete NC-like developmental program, including migration, multipotency, and differentiation. To tackle the issue, we have focused on the early neural border regulators (ap2, hairy2, msx1, pax3, and zic1) acting during gastrulation. To address which combination is sufficient to elicit NC development, we took advantage of the ability of pluripotent prospective ectoderm cells of the Xenopus blastula (also named the animal cap) to develop autonomously in culture. These early ectodermal cells respond to embryonic patterning cues and thus, provide an effective system for studying pluripotent cells. A major challenge in stem cell biology and especially, craniofacial biology will be to build new tissues in vitro for repair of injuries. As such, understanding which of the myriad factors involved in NC specification are sufficient to drive the process is a major goal. We find here that the transcription factors Pax3 and Zic1 are the best combination to provide all of the necessary information to trigger multipotent NC specification in the absence of additional inducers. We challenged both migration and differentiation of the induced cells in ectopic locations in vivo, long-term culture in vitro, and orthotopic backgrafting in vivo.

Results

Neural border specifiers are essential transcription factors that initiate NC specification in vivo. However, by itself, none of these factors induces efficient NC development from early pluripotent prospective ectoderm. Using the main neural border regulators, which act early upstream of the NC gene regulatory network (e.g., Pax3, Zic1, Msx1, Hairy2, and AP2), we tested the various possible pairs to identify which combinations would be sufficient for driving the next key steps of NC development [e.g., NC induction (snail2 expression) and cell delamination in animal cap ectoderm explants] (Fig. 1 and SI Experimental Procedures). We observed that Pax3 activation promoted modest snail2 expression and cell delamination. This activity was strongly enhanced by the coactivation of Msx1 or Zic1, whereas other pairs of factors failed to induce snail2 and delamination (Fig. 2A). Moreover, the Pax3/Zic1 combination promoted the highest snail2 induction, efficient explant attachment, and a massive cell delamination from the explants. In addition, in vivo, Pax3/Zic1 also activated NC induction in the prospective ventral ectoderm in X. laevis and the extra embryonic ectoderm in chicken embryos (Fig. S1). We have, therefore, decided to further characterize the NC-inducing activity of Pax3/Zic1.

Fig. 1.

General experimental design. X. laevis embryos were injected at the two-cell stage into both blastomeres with either WT or inducible pax3 and zic1 [e.g., dexamethasone activable glucocorticoid receptor (GR) fusions]. Embryos were grown until blastula stage 9 when blastocoele roof ectoderm was cut (about a 20-cell-wide square). Explants were further grown in 1/3 MMR or 3/4 NAM without growth factor supplements until gastrula or neurula stage equivalent 10–18 (12, 15). Control sibling embryos served as a reference to evaluate developmental stages. Dexamethasone was added at late blastula–early gastrula stage 10 unless otherwise mentioned to activate Pax3GR and Zic1GR in the explants. At neurula stage 18, the explants were processed (for RT-PCR, Western blot, or luciferase assay), put on fibronectin-coated plates for videomicroscopy, or backgrafted into the cranial NC territory of a stage 18 uninjected host sibling after ablating either a part or all of the host NC. Grafted embryos were analyzed either at migration stages 22–25 or tadpole stage 41 for differentiation. Targeted ectopic injections are described below.

Fig. 2.

Pax3/Zic1 is the best combination to induce NC specification in vitro with a time schedule reflecting the steps of induction in vivo. (A) Comparison of neural border specifiers’ combinations in NC induction and emigration from ectoderm. Neural border specifiers, which expand the NC domain in vivo, were tested for initiating NC specification (indicated by snail2 induction) and delamination in “animal cap” prospective ectoderm explants. Delamination was analyzed after plating the explants onto a fibronectin substratum. Ten different combinations of the main five neural border specifiers (pax3, zic1, msx1, ap2, and hairy2) were tested. Results were scored as follows: percent of explants showing delamination (an average of 15 explants per condition was analyzed); relative levels of snail2 induction (from 24 explants per condition) compared with the maximal induction observed using quantitative RT-PCR: −, value ≤ 25%; +, 25% < value ≤ 50%; ++, 50% < value ≤ 75%; +++, value ≥ 75%. Single injections were analyzed for pax3 and hairy2, because they had not been described for snail2 induction in ectoderm explants before. att, attachement; NA, not analyzed. (B) RT-PCR analysis was done after induction and lysis at various time points during gastrulation and neurulation for the following NC specifiers: snail2, foxd3, sox8, 9, and 10, myc, and snail1. When induction was done at stage 10 and lysis at increasing developmental time points during gastrulation and neurulation (stages 11, 12, 15, and 18), appearance of NC specifiers followed the sequential appearance described in vivo. Similarly, when induction was done at various times during gastrulation (stages 10, 10.5, and 11.5) and lysis was done at stage 18, responsiveness drastically decreased, indicating the same stage limit in ectoderm competence as described in vivo. Lane 1, uninjected whole embryo; lane 2, − reverse transcriptase (RT) control; lane 3, uninjected ectoderm; induction/lysis, stage of dexamethasone addition/stage of analysis.

NC specification involves a clear temporal sequence of gene activation starting at late gastrula–early neurula stage (stages 11.5–12) (12) for the earliest NC specifiers (snail1, sox8, sox9, and myc) followed by genes activated at early–midneurula stage (stages 12.5–13; snail2 and foxd3) and finally, genes activated during later neurulation (stage 14+; sox10) (13, 14). We, therefore, first explored whether Pax3/Zic1 recapitulated the normal progression of gene activation using inducible constructs activated at early gastrulation (stage 10). Inducible Pax3 and Zic1 display similar NC inducing activity as the WT forms (Fig. S2) (1, 11, 15, 16). Indeed, a group of early genes (snail1, sox8, and myc) was activated during early neurulation (between stages 12 and 15); a group of intermediate genes, including foxd3 and sox9, was expressed at midneurula stage 15. Finally, snail2 and sox10 were induced at a later neurula stage (Fig. 2B). Thus, the sequential gene activation observed in vivo was clearly reproduced in vitro, albeit with slight delay.

Because early ectoderm is competent to form NC only until late gastrula/early neurula stages in vivo, we then tested the developmental window of ectodermal competence to respond to Pax3/Zic1 in vitro (17). To this end, we activated Pax3 and Zic1 at gastrula through neurula stages for assay at the late neurula stage (Fig. 2B and Fig. S3A). Similar to the normal competence for ectoderm, we observed efficient NC specifiers’ induction only when the activation was done before neurulation (stage 11.5). We also asked whether Pax3/Zic1 might alter the specification of an already committed tissue. When Pax3 and Zic1 were activated in already committed mesoderm (stage 10.5 dorsal, dorsal–lateral, or ventral mesoderm), the mesodermal marker myod was maintained, and activation of the NC marker snail2 was not observed (Fig. S3 B and C).

We conclude that the Pax3/Zic1 combination faithfully activates the schedule of NC specification in pluripotent prospective ectoderm. This result raised the question of whether the tissue continued NC-like development, including epithelial-to-mesenchymal transition (EMT), migration, and terminal differentiation into multiple types of derivatives.

NC cells undergo an EMT that allows dispersal from their birthplace in the dorsal neural tube and migration to their final positions. If Pax3/Zic1 drives NC specification, one would expect that these cells would display the molecular signature of cells undergoing an EMT. In particular, a cadherin switch occurs before EMT: e-cadherin is expressed in cohesive epithelial/neuroepithelial cells, whereas n-cadherin is expressed in migrating NC in quail, mouse, and frog (18, 19). On Pax3 or Zic1 induction, e-cadherin expression was decreased, whereas Pax3 activated n-cadherin expression. Pax3/Zic1 coexpression potentiated these two effects (Fig. 3 A–D). This clear-cut cadherin switch was, thus, consistent with EMT initiation in the induced ectoderm in vitro in register with the time when NC undergoes EMT in vivo. To validate a functional EMT in vitro, we compared the ability of NC cells and GFP-labeled Pax3/Zic1-induced ectoderm to undergo EMT and migrate on fibronectin (18). When uninduced animal caps attached and spread on fibronectin, these control cells did not emigrate from the explant. In contrast, both NC and Pax3/Zic1-induced ectoderm efficiently attached, spread, and underwent EMT (Fig. 3 B–G). Individual cells detached from the explant, produced numerous protrusions, and actively migrated, with a slightly slower overall velocity for Pax3/Zic1-induced ectoderm compared with NC cells (Fig. 3H and Movies S1, S2, and S3). Altogether, these in vitro results showed that Pax3/Zic1 activation is sufficient to induce EMT and migration in early prospective ectoderm.

Fig. 3.

Pax3/Zic1-induced ectoderm displays cadherin switch and migratory activity in vitro and migrates in vivo. (A) n- and e-cadherin expression was analyzed by RT-PCR on explants induced at gastrula stage 10 and lysed at neurula stage 18. Low e-cadherin and high n-cadherin mark the Pax3/Zic1-induced ectoderm. Lane 1, uninjected whole embryo; lane 2, −RT control; lanes 3 and 7, uninjected ectoderm; −Dex, ethanol-treated Pax3GR/Zic1GR-injected ectoderm; +Dex, dexamethasone-treated Pax3GR/Zic1GR-injected ectoderm. (B–H) Using histone2b-GFP mRNA coinjections, we plated either Pax3GR/Zic1GR/GFP-injected ectoderm (B and E; uninduced controls; C and F; induced explants) or GFP-labeled NC (D and G) at stage 18 on fibronectin-coated plates. NC and Pax3/Zic1-induced (+Dex) cells attached, spread, and exhibited EMT. E-cadherin was prominent at cell junctions in uninduced explants, whereas actin staining showed numerous protrusions in both NC and induced explants. Videomicroscopy (H) indicated that individual cells actively migrated outside of the Pax3/Zic1-induced ectoderm, albeit slightly slower than control NC cells (t test: P < 0.0001; error bars: SEM). Uninduced cells (−Dex) did not migrate. (Scale bars: 100 μm.) (I–K) When grafted into the cranial NC territory, the control (−Dex) explants integrated the ectoderm and remained at the graft site (I and I′; 0% migration, n = 24, white arrow), whereas the induced (+Dex) ectoderm actively migrated along the normal NC migration paths a few hours postgrafting (J and J′; 77%, n = 70, red arrows) when host NC was both present and fully ablated. Pax3GR-only injected grafts exhibited some but less-efficient migration (K). (Scale bars: 500 μm.)

Because Pax3/Zic1 cells display the molecular signature of premigratory NC and EMT and undergo migration in vitro, we asked if they were capable of migration in vivo. We grafted such cells, unilaterally, into an unlabeled host after the time when endogenous NC induction had been completed (Fig. 1). Thus, we ensured that the potential NC induction only depended on the in vitro activation of Pax3 and Zic1. Control explants healed into the host ectoderm and did not migrate (Fig. 3I). Pax3/Zic1-induced ectoderm efficiently migrated in host embryos and followed normal NC migration paths around the eye and along the mandibular, hyoid, and branchial arches (Fig. 3J). Ectoderm forced to express Pax3 only also migrated, albeit far less efficiently (only 30% of the grafted explants migrated) (Fig. 3K). In conclusion, when backgrafted in vivo, after the time when NC can be induced, the Pax3/Zic1-induced ectodermal cells migrate along the normal NC streams of migration, indicating a fine recognition of the spatial cues that guide NC migration in the embryo.

The molecular signatures and cell migratory behaviors suggested that Pax3/Zic1 cells were equivalent to the NC cells developing in vivo. However, the ultimate test for NC cell commitment is their ability to differentiate into diverse cell types. We, therefore, asked if the Pax3/Zic1-induced ectoderm could undergo autonomous NC-type terminal differentiation in vitro. Strikingly, Pax3/Zic1 expressing ectodermal explants frequently developed some pigmented and stellate cells reminiscent of NC-derived melanocytes (in 44% of the explants), whereas the controls formed only ciliated ectoderm like uninjected animal caps (Fig. 4 A and B and Fig. S4A). Occasionally, a few melanocytes differentiated within Pax3 alone-induced explants (14% of the explants) (Fig. S4A). Importantly, Pax3/Zic1-induced ectoderm expressed mitf, a key molecular marker for terminal melanocyte differentiation (Fig. 4 and Fig. S4) (20, 21).

Fig. 4.

Pax3/Zic1-induced ectoderm differentiates into multiple NC derivatives in vitro. (A–C) We have grown the control (A) and induced (B) explants in 3/4 NAM or 1/3 MMR without any supplements (except for gentamicin) for several days (6–8 d at 15 °C), from late neurula stage 18 to swimming tadpole stage 41 (differentiation stage). Melanocytes differentiated into the induced (B) but not the control (A) explants. (C) RT-PCR analysis showed that markers for various NC derivatives were expressed when both WT and inducible pax3 and zic1 were coinjected. Lanes 1 and 7, uninjected whole embryo; lanes 2 and 8, −RT control; lanes 3 and 9, uninjected explant; −Dex, ethanol-treated Pax3GR/Zic1GR-injected explants; +Dex, dexamethasone-treated Pax3GR/Zic1GR-injected explants. (D–I) Using histone2b-GFP mRNA coinjections, we plated control ectoderm (D and G), Pax3GR/Zic1GR-induced ectoderm (E and H), or GFP-labeled NC (F and I) on fibronectin-coated plates after the initial induction in 3/4 NAM in vitro. The cells were grown either in 3/4 NAM for 3–4 d or switched to Neurobasal/B27 medium after 1 d on fibronectin. In 3/4 NAM, only NC formed neurites (F; red, antineurofilament immunostaining). When Neurobasal/B27 medium was added, both NC and Pax3GR/Zic1GR-induced cells formed neurites (H and I), whereas the uninduced cells did not (G).

Because a major function of NC cells in vivo is to differentiate into the chondrocytes that build the skeletal elements of the face and jaw, we asked if Pax3/Zic1 expressing ectoderm ultimately formed chondrocytes. We found that the explants expressed sox9 and runx2, which indicate chondrocyte and bone commitment, respectively (Fig. 4C) (22, 23). In addition, the NC differentiates into neurons and glia of the peripheral nervous system. We found that the Pax3/Zic1 explants expressed the neuronal markers c-ret and phox2b, which mark autonomous neurons of the sympathetic and enteric nervous systems as well as the glial markers sox10 and foxd3 (Fig. 4C) (24–30). Similar results were observed with both WT and activable pax3 and zic1 injections (Fig. 4C). In contrast, when Zic1 alone was activated, only neural tissue was formed, indicated by robust ncam activation, but no other differentiation marker tested was found (31). The few Pax3 alone-induced explants that formed pigment were picked up for RT-PCR: they also expressed ncam, sox9 (Fig. 4C), sox10, and mitf (Fig. S4B), which is in line with Pax3 being a direct mitf activator during melanocyte differentiation (32).

To test if the induced cells could form morphologically differentiated neurons, we used a defined medium enriched to allow neuronal growth and survival after the initial induction (Fig. 4 D–I). Control NC formed numerous neurofilament-positive neurites in both saline and supplemented medium (Fig. 4 F and I). Pax3/Zic1-induced ectoderm cells efficiently formed neuritis only in the supplemented medium (Fig. 4 E and H), whereas control cells did not form neurites (Fig. 4 D and G). Hence, the ectoderm induced by Pax3 and Zic1 has the potential to differentiate into neurons. Because Msx1 coinjected with Pax3 was the second best combination to activate early NC development from animal caps (Fig. 2A), we have then tested if Msx1 would promote neuronal differentiation in Pax3- or Pax3/Zic1-induced ectoderm. However, coexpression of Msx1 did not improve neuron formation in minimal culture conditions (Fig. S5).

We then tested the differentiation of cells induced ectopically in vivo using injections targeted to either the prospective ventral ectoderm or the prospective endoderm of 16-cell stage Xenopus embryos (33). The induced cells formed a pigmented tissue mass either ventrally or within the endoderm (Fig. 5 A–C and Fig. S6); they also expressed ectopic sox9 (Fig. 5 M–O), sox10 (Fig. 5 J–L), neural tubulin (Fig. 5 D–F), tyrosine hydroxylase (TH) (Fig. 5 G–I), and low levels of runx2 (Fig. S6 P–R). These ectopic stainings indicated the formation of multiple NC-like derivatives, such as melanocytes, TH-positive peripheral neurons, and cartilage, in ectopic locations, including the prospective endoderm in vivo.

Fig. 5.

Pax3 and Zic1 ectopic coactivation induces NC-like differentiation in prospective ventral ectoderm and endoderm. (A–C) Pax3GR/Zic1GR/β-gal mRNAs coinjections were targeted to the prospective ventral epidermis (B Inset; blastomere V1.1) or the prospective endoderm (C Inset; vegetal V2.2/3d blastomere) in 16-cell stage blastulas. Dexamethasone activation was performed at the 32-cell stage. Stage 41-injected tadpoles exhibit ectopic melanocytes in the ventral ectoderm (B) and endoderm (C), respectively (white arrows) compared with control siblings (A). (D–O) After β-gal staining to localize the injected area (red), these tadpoles were stained for various NC markers (WISH; purple-blue staining) and sectioned. In tadpoles injected into both the prospective ventral epidermis and the prospective endoderm, we observed ectopic staining for neural tubulin (E and F), TH (H and I), sox10 (K and L), and sox9 (N and O) compared with control uninjected embryos (D, G, J, and M). (Scale bars: A–C, 1 mm; D–O, 100 μm.)

Finally, because the Pax3/Zic1 cells induced in vitro and backgrafted in vivo specifically followed endogenous NC migration paths, we have challenged the ability of the induced ectoderm to form differentiated tissues in host embryos. To favor differentiation from the graft, the whole cranial NC was ablated on one side of the unlabeled host before grafting. NC removal resulted in a dramatic failure of craniofacial development that could be rescued by backgrafting premigrating GFP-labeled NC cells taken at neurulation stages 17–18 (Fig. S7). The control graft (i.e., injected Pax3/Zic1/GFP but uninduced) formed skin epithelium along with WT blastocoele roof ectoderm (Fig. 6A and Fig. S8). In contrast, after Pax3/Zic1 activation in vitro, we noted long distance migration of the grafted Pax3/Zic1/GFP-labeled cells either dorsally or to the craniofacial areas of the host, and in the best cases, we noted restoration of the general head morphology on the grafted side at swimming tadpole stage (Fig. 6 C and F). Moreover, the GFP-positive cells differentiated into pigmented and stellate melanocytes under the ectoderm and were also found in deeper locations around the eye, along the optic nerve, and in the head mesenchyme (Fig. 6B and Fig. S8). They formed GFP-positive fibroblasts in the branchial arches mesenchyme, well-shaped sox9+/GFP+ Meckel’s cartilage, and other cartilage elements on the grafted side (Fig. 6 D–G), whereas contralateral cartilages were formed by sox9+/GFP− host cells (Fig. 6H). Occasionally, at the rhombencephalon level, GFP-labeled cells were found adjacent to craniofacial muscles and ganglia on the grafted side, but we could not clearly identify those cells as muscle or glial precursors.

Fig. 6.

Pax3/Zic1-induced ectoderm differentiates into multiple cranial NC derivatives in vivo. Embryos were grafted orthotopically as previously described (Figs. 1 and 3) and grown until stage 41. Although control GFP+ cells (i.e., ethanol-treated Pax3GR/Zic1GR/H2bGFP-injected cells) integrated the skin (A), induced GFP+ cells (i.e., dexamethasone-treated Pax3GR/Zic1GR/H2bGFP-injected cells) formed melanocytes (B Inset; note adjacent black melanosome and GFP+ nucleus) and migrated into deeper (thus out of focus) locations dorsally, around the eye, and into the branchial arches (B, stage 45; C, stage 35). Scheme of a stage-41 tadpole head in transverse section (D), indicating the location of the three NC-type derivatives found in operated embryos (shown in B and E–H). Transverse head sections processed with sox9 in situ hybridization and anti-GFP immunostaining analysis show GFP+ fibroblasts in the maxillary mesenchyme (D, 2 and E) and GFP+ Meckel’s cartilage (D, 3, F, and G) on the grafted side but only sox9+/GFP− cartilage on the contralateral control side (H). (Scale bars: A–C, 500 μm; E–H, 100 μm.)

In conclusion, the cells committed to a NC fate by Pax3 and Zic1 activation, integrated several NC destinations in the craniofacial area in vivo, and underwent terminal differentiation into multiple bona fide NC-type derivatives.

Discussion

The mechanisms of NC induction and development have been extensively studied. Wnt, BMP, and FGF secreted by the superficial ectoderm and paraxial/intermediate mesoderm activate a network of genes at the neural plate border, which then specifies the NC (3, 34–36). It was shown that AP2, Pax3, Msx1, and Zic1 are key transcription factors that initiate expression of the earliest NC markers after neural border formation (3, 5, 7, 8). In particular, Pax3 and Zic1 cooperate with Wnt signals, whereas AP2 cooperates with BMP antagonists to initiate snail2 expression in the prospective ectoderm (3, 5, 7). Despite these numerous studies, the key question of exactly which cell autonomous factors may be sufficient to commit ectoderm to a multipotent NC fate and drive its complete development until differentiation remained elusive. Because individual neural border regulators fail to activate de novo NC development from early prospective ectoderm, we looked for a combinatorial activity of pairs of neural border specifiers, which would be sufficient to drive full NC development from uncommitted ectoderm. It was shown previously that coinjection of the two neural border specifiers Pax3 and Zic1 activates ectopic expression of early premigratory NC markers in frog ventral ectoderm in vivo (5, 7). Positive cross-regulation activates Pax3 or Zic1 expression when Zic1 or Pax3 are activated, respectively, in Wnt-treated animal caps (5). Moreover, the balance of activity of these two factors is critical for a choice between NC, placode, and hatching gland fates (11). Whether other combinations of factors were playing a similar role remained unknown. Altogether, our results indicate that activating the two factors Pax3 and Zic1 efficiently commits early cells—whether they are prospective ectoderm or endoderm—to multipotent and functional NC. This coactivation triggers NC specifiers’ expression in the normal sequence and schedule followed by EMT, migration, and definitive NC-type differentiation both in vitro and in vivo. Although the WT NC autonomously forms neurons in minimal conditions, the Pax3/Zic1-induced cells formed neurites only in supplemented medium. In addition, the induced cells undergo a random migration in vitro, which was described for isolated NC cells devoid of a chemoattractant source, but directional migration in vivo, indicating appropriate recognition of guidance cues (18, 37). Our data, thus, provide an experimental paradigm to elucidate the fine-tuning of the NC gene regulatory network as well as develop assays for differentiating NC derivatives from stem cells in vitro.

During ectoderm patterning in vivo, balanced levels of Pax3 and Zic1 are essential to control the choice between NC, placode, and other cell fates at the neural border (7, 11). When Pax3/Zic1 levels are appropriate, a full NC development program is, thus, triggered by these two transcription factors, similar to activation of the skeletal muscle program by MyoD and cardiac development by a combination of Nkx2.5, GATA4, and myocardin (38). Within the general context of understanding adult cell reprogramming and controlling pluripotent cell differentiation into chosen lineages (39), the NC commitment triggered by Pax3 and Zic1 provides a framework to study NC and craniofacial development from stem cells. It also suggests that coregulation of these two factors is an essential node in the NC gene regulatory network that may have allowed NC formation during vertebrate evolution.

Experimental Procedures

Xenopus Embryo Manipulation and Explant Culture.

X. laevis embryos were obtained and staged using standard procedures (12, 15). H2b-GFP mRNA was coinjected as a lineage tracer for migration and grafting assays, and β-gal was used as a lineage tracer for ectopic injection assays. mRNAs were synthesized using the mMESSAGE mMACHINE kit (Ambion). The plasmids used as templates were pax3, zic1, pax3-GR, zic1-GR, β-gal, and H2bGFP (Tables S1, S2, and S3). For animal cap experiments, the two blastomeres of stage 2 embryos were injected at the animal pole. The blastocoele roof ectoderm was dissected at stage 9 and aged in 3/4 Normal Amphibian Medium (NAM) or 1/3 Mark’s Modified Ringer (MMR) until the sibling controls reached stage 18 or 41. For grafting, the cranial NC of stages 17–18 host embryos was partially or totally ablated on one side. A piece of animal cap explant grown to stage 18 was carefully rinsed and immediately put in place of the ablated NC. Operated embryos were then grown until stages 22–41.

Chicken Embryos Electroporation.

Gallus gallus embryos were incubated until late blastula/early gastrula stage (2–4 Hamburger–Hamilton stage). They were then placed in the electroporation chamber as described in ref. 40. The plasmid solution containing Xenopus Pax3 and Zic1 constructs was then electroporated. The embryos were reincubated overnight on albumin, fixed, and processed for in situ hybridization (adapted from ref. 41).

In Vitro Cell Culture and Videomicroscopy.

For in vitro migration assay, NC explants of stages 17–18 control embryos and injected animal cap ectoderm grown until stage 18 were plated on fibronectin (10 μg/mL on plastic) in six-well plates, cultured, and imaged in 3/4 NAM (18). Individual cell tracking was performed using the Manual Tracking Image J plugin (http://rsbweb.nih.gov/ij/plugins/track/track.html) (42). Statistical analysis by two-way ANOVA indicated an extremely significant difference of migration overall velocity between control NC and induced ectoderm explants (P < 0.0001). For in vitro neuronal differentiation, similar NC or explants were plated on fibronectin-coated coverslips (100 μg/mL) in six-well plates and cultured 24 h in 3/4 NAM. Then, 2/3 of the NAM was removed, and an equivalent volume of Neurobasal medium supplemented with antibiotics and B-27 supplement was added (#21103 and #17504; Gibco). Neuronal differentiation occurred within 2–3 additional d of culture for NC and 3–4 d for Pax3/Zic1-induced ectoderm.

In Situ Hybridization and Immunochemistry.

After β-gal staining, whole mount in situ hybridization on ectopically injected whole embryos was performed (41); then, embryos were embedded in Albumin/Gelatin mix for vibratome sectioning. Grafted embryos were first embedded in paraffin for sectioning and then stained by in situ hybridization using DIGoxigenin-labeled probes (http://geisha.arizona.edu). Anti-GFP immunostaining was performed after in situ hybridization. For in vitro explants staining, NC explants (taken from stages 17–18 control embryos) and injected ectoderm explants grown until stage 18 were plated on fibronectin (10 mg/mL) in six-well plates and cultured in 3/4 NAM (18). Explants were fixed in 3.7% formaldehyde/96.3% PBS (vol/vol) and stained with phalloidin, anti–E-Cadherin, or antineurofilament P200 antibodies (Tables S1, S2, and S3).

Pharmacological Treatments.

Inducible Pax3 and Zic1 (Pax3-GR and Zic1-GR) were activated by dexamethasone (16). Controls included pax3GR/zic1-GR–injected embryos grown in 0.2% ethanol and noninjected embryos grown in dexamethasone.

RT-PCR.

Semiquantitative and quantitative RT-PCR (15) included the following controls (Figs. 2B, 3A, and 4C): whole embryo (lane 1), embryo without reverse-transcriptase treatment (lane 2), and uninjected caps (lane 3). Elongation factor 1a or Ornithine Decarboxylase served as a loading control, and muscle actin controlled for mesoderm contamination (not shown). Details and primer pairs are presented in Tables S1, S2, and S3.