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The neural crest (NC) is a multipotent population of migratory cells unique to the vertebrate embryo, contributing to the development of multiple organ systems. Transcription factors pax3 and zic1 are among the earliest genes activated in NC progenitors, and they are both necessary and sufficient to promote NC fate. In order to further characterize the function of these transcription factors during NC development we have used hormone inducible fusion proteins in a Xenopus animal cap assay, and DNA microarray to identify downstream targets of Pax3 and Zic1. Here we present the results of this screen and the initial validation of these targets using quantitative RT-PCR, in situ hybridization and morpholinos-mediated knockdown. Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. We also obtained several factors with no known function in Xenopus NC, which represent novel regulators of NC fate. The comprehensive characterization of Pax3 and Zic1 targets function in the NC gene regulatory network, are essential to understanding the mechanisms regulating the emergence of this important cell population.
Fig. 1. Strategy to isolate Pax3-Zic1 targets: (A) procedures used to identify Pax3-Zic1 targets in the developing NC. Xenopus embryos were injected at the 2-cell stage with mRNA encoding GR (not shown), Pax3GR and Zic1GR (250 pg each), alone or in combination. At the blastula stage (stage 9), animal cap explants (AC) were dissected and cultured for 8 h in the presence of dexamethasone. RNA were extracted from each sample, analyzed by qPCR and subsequently used to screen a GeneChip Xenopus laevis Genome 2.0 Array (Affymetrix). (B) RNA extracted from GR, Pax3GR, Zic1GR, or Pax3GR+Zic1GR injected animal cap explants, were analyzed by qPCR for the expression of various marker genes, keratin (epidermis), xhe (HE), snail2 (NC), and six1 (PE) to confirm that the expected pattern of gene expression was observed in each injection group. The relative expression levels have been normalized to ef1α. P3, Pax3GR; Z1, Zic1GR.
Fig. 2. Flow chart and criteria for the selection of Pax3-Zic1 targets.
Fig. 3. Regulation of Pax3-Zic1 targets expression by NC-inducing signals: (A) in animal caps, induction of snail2 and pax3 by co-expression of Chordin (10 pg) and Fgf8a (5 pg) (C+F) is dramatically reduced in the context of embryos injected with Wnt8MO (W8MO; 50 ng). Interference with Wnt signaling pathway did not affect sox2 expression. (B) A majority of Pax3-Zic1 targets followed a pattern of activation in response to NC-inducing signals similar to that of snail2 and pax3, while a limited number of genes did not (C and D). The data are an average of at least three independent experiments. The values were normalized to odc and presented as mean±s.e.m.; (âŽ), p<0.05 versus C+F.
Fig. 4. Developmental expression pattern of selected Pax3-Zic1 targets. The spatiotemporal expression pattern of 10 Pax3-Zic1 targets was analyzed by qPCR and whole-mount ISH. Expression levels of the target genes were analyzed by qPCR at the indicated stage (right panels). The values were normalized to odc and presented as mean±s.d. The data are an average of at least three sets of embryos. For ISH embryos were analyzed at the neurula (left panels; dorsal view, anterior to top) and tailbud (middle panels; lateral and dorsal views, anterior to the left) stages.
Fig. 5. Pax3 or Zic1 morpholino mediated knockdown blocked Pax3-Zic1 targets expression at the neurula stage: (A) Pax3MO (40 ng) or Zic1MO (40 ng) were injected in one blastomere at the 2-cell stage embryos. Embryos were collected at the neurula stage (stage 15–18) and analyzed by ISH (upper left). (B) ISH with a sox8 probe was used as a positive control for the activity of each morpholino. (C) The 10 Pax3-Zic1 targets analyzed showed reduced expression upon Pax3 or Zic1 knockdown. All panels are showing dorsal views, anterior to top. The injected side is on the right as indicted by the presence of the lineage tracer (red staining).
ak3 (adenylate kinase 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
npr3 (natriuretic peptide receptor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
rassf10 (Ras association (RalGDS/AF-6) domain family (N-terminal) member 10) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
tspan18 (tetraspanin 18)gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
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