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The SOX family of transcription factors has been implicated in cell fate specification during embryogenesis. One member of this family, Sox9, has been shown to regulate both chondrogenesis and sex determination in the mouse embryo. Heterozygous mutations in Sox9 result in Campomelic Dysplasia (CD), a lethal human disorder characterized by autosomal XY sex reversal, severe skeletal malformations and several craniofacial defects. Sox9 is also expressed in neural crest progenitors but very little is known about the function of Sox9 in the neural crest. We have cloned the Xenopus homolog of the Sox9 gene. It is expressed maternally and accumulates shortly after gastrulation at the lateral edges of the neural plate, in the neural crest-forming region. As development proceeds, Sox9 expression persists in migrating cranial crest cells as they populate the pharyngeal arches. Depletion of Sox9 protein in developing embryos, using morpholino antisense oligos, causes a dramatic loss of neural crest progenitors and an expansion of the neural plate. Later during embryogenesis, morpholino-treated embryos have a specific loss or reduction of neural crest-derived skeletal elements, mimicking one aspect of the craniofacial defects observed in CD patients. We propose that Sox9 is an essential component of the regulatory pathway that leads to cranial neural crest formation.
Fig. 2. Developmental expression of Sox9 by whole-mount in situ hybridization. (A) Sox9 expression at the gastrula stage is found in a superficial ring around the blastopore (white arrows); lateral view. (B) Sox9 RNA is detected at the lateral edges of the neural plate (arrows) in a stage 12 embryo. Dorsal view, anterior towards the top. (C,D) Sox9 expression at stage 14 is in the neural crest (red arrow and arrowhead) and in the presumptive otic placode (yellow arrow). (E,F) Slug expression is shown for comparison. (C,E) are dorsal views, anterior towards the top; (D,F) are lateral views, anterior towards the right. (G) Transverse section of a stage 14 embryo. Sox9 expression is restricted to the medial (red arrowheads) and lateral (red arrows) neural crest. (H) Stage 16 embryo, dorsal view, anterior towards right. As the neural tube closes, Sox9 remains strongly expressed in both components of the neural crest (red arrow/arrowhead) and in the otic placode (yellow arrow) (I,J) Stage 23 embryos, (I) lateral view and (J) dorsal view. Genital ridge (green arrows), optic vesicle (blue arrows), otic placode (yellow arrow). (K,L) Stage 25 embryo. (K) In this frontal view, Sox9 is found in the nasal pits (purple arrows) and the prospective pineal gland (orange arrow). (L) Lateral view, anterior towards right. Sox9 is detected in the four streams of cranial neural crest (red arrows), otic placode (yellow arrows), genital ridge (green arrows) and the developing eye (blue arrows in K,L). (M-O) Transverse sections of a stage 25 embryo. (M) In this section, at the level of the forebrain, Sox9 is detected in the developing eye (blue arrow), dorsally in migrating neural crest cells (red arrow) and in discrete domains in the brain. (N) More posteriorly, Sox9 is strongly expressed in the developing otic placode (yellow arrow). (O) In the trunk region Sox9 is restricted to the genital ridge (green arrow) and the notochord (black arrow). (P) At stage 32, Sox9 expression is in the pharyngeal arches (red arrows), the otic vesicle (yellow arrow) and in restricted regions of the brain.
Fig. 5. Sox9-AS prevents Slug expression in a dose-dependent manner. Embryos were injected in one blastomere at the two-cell stage with different concentration of Sox9-AS and analyzed for Slug expression by whole-mount in situ hybridization at stage 17. (A-C) Representative embryos illustrating unperturbed Slug expression (A) and partial (B) or complete (C) loss of Slug on the injected side. Dorsal view, anterior is at the top. RNA encoding the lineage tracer β-galactosidase was co-injected with Sox9-AS to identify the injected side (red staining); the left side in all panels. (D) Quantification of Slug in situ hybridization results. (E) Early onset of Slug expression (arrow) at the late gastrula stage (stage 12) is blocked in embryos injected with 10 ng of Sox9-AS. Dorsal view, anterior is at the top, injected side (red staining) is to the left.
Fig. 6. Sox9 depletion leads to a loss of neural crest progenitors and an expansion of neural tissues. (A) Embryos were injected in one blastomere at the two-cell stage with 10 ng of Sox9-AS or Co-AS and analyzed by whole-mount in situ hybridization at stage 17-19 (Snail, Pax3, Nrp1 and Sox2) or stage 23 (Twist). Dorsal view, anterior is at the top. RNA encoding the lineage tracer β-galactosidase was co-injected to identify the injected side (red staining), the right side in all panels. Upon injection of Sox9-AS, expression of the neural crest markers Twist, Snail and Pax3 are greatly reduced, while expression of the pan-neural marker Nrp1 and Sox2 is expanded. Expression of the same markers in Co-AS-injected embryos is presented for comparison. (B) Tissue sections of representative Sox9-AS-injected embryos stained for Slug or Sox2 expression. Injected side is on the right. n, notochord; s, somite.
Fig. 7. The phenotype of Sox9-depleted embryos can be rescued by restoring Sox9 expression. (A) Rescue experiments were performed by injection of an animal dorsal blastomere at the eight-cell stage. (B) Representative case of Slug whole-mount in situ hybridization of stage 17 embryos injected with 100 pg of Sox9 plasmid (Sox9) or 5 ng of Sox9-AS (Sox9-AS), or a combination of both (Sox9-AS+Sox9) or a combination of Sox9-AS and 100 pg of a control GFP plasmid (Sox9-AS+GFP). RNA encoding the lineage tracer β-galactosidase was co-injected to identify the injected side (red staining), right side in all cases. Dorsal view, anterior is at the top. (C) Quantification of the in situ hybridization results. n, number of cases analyzed.
