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.

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.

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.)

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).

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.)

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.)