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First published online 30 August 2006
doi: 10.1242/dev.02558

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Functional analysis of Sox8 during neural crest development in Xenopus

Michael O'Donnell^*,, Chang-Soo Hong^*, Xiao Huang, Raymond J. Delnicki and Jean-Pierre Saint-Jeannet^¶

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.

View larger version (91K): [in a new window]   Fig. 1. Sox8 expression in neural crest progenitors and their derivatives. (A) Sox8 is first detected at the gastrula stage in a ventrolateral domain around the blastopore. At stage 11.5 (B-D) Sox8 expression around the blastopore persists and additional expression is detected lateral to the neural plate (arrows). Vegetal (A), lateral (B,C; anterior towards right) and dorsal (D; anterior towards the top) views. Comparison of Sox8 (E) Sox9 (F) and Sox10 (G) expression in sibling stage 14/15 embryos illustrates that all three genes are expressed in the presumptive neural crest. Dorsal views, anterior towards the top. (H) Section of a stage 15 embryo illustrates the expression of Sox8 in both the lateral (arrows) and the medial neural crest (arrowheads). At stage 17 (I,J), Sox8 persists in the neural crest region and is also expressed anterior to the neural plate in the prospective cement gland (arrow). As the neural tube closes (K,L), Sox8 is detected in the migrating neural crest cells in the cranial region (arrows), and the premigratory cells in the trunk neural crest (arrowheads). Dorsal view, anterior towards top (I,K); lateral view, anterior towards the left (L); cranial view (J). (M) Comparison of Sox8, Sox9 and Sox10 expression at the tailbud stages. Dorsal views of stage 25 embryos, anterior towards the right. Sox8, Sox9 and Sox10 are co-expressed in the migrating cranial neural crest. Posteriorly, although Sox8 and Sox10 are both expressed in trunk neural crest cells, Sox9 is downregulated in this cell population. At stage 35 (left panels, lateral views), Sox8 is detected in the cranial neural crest similar to Sox9; however, at this stage, Sox10 starts to be downregulated in the branchial arches. Sox8, Sox9 and Sox10 are co-expressed in the otic vesicle at this stage (arrows). Ventral views (right panels) showing colocalization of Sox8 and Sox9 in the pancreatic rudiments (arrowheads) where Sox10 is not detected.

View larger version (47K): [in a new window]   Fig. 2. Comparison of the onset expression of Sox8 with other neural plate border-specific genes. (A) Summary of the onset of expression of Sox8 and seven other neural plate border-specific genes in Xenopus. The developmental stages are according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1956). (B) Developmental expression of Pax3, Snail, Myc, Slug and Sox10 from stage 11 to stage 14 by whole-mount in situ hybridization. Dorsal views, anterior towards the top.

View larger version (51K): [in a new window]   Fig. 3. Sox8-depleted embryos fail to express Sox10 at the neural plate border. (A) Increasing amounts of Sox8mo (10 ng, 100 ng and 1000 ng) blocks translation directed by Sox8 mRNA. The same morpholino (500 ng) fails to block Sox9 and Sox10 translation. (B) Embryos injected in one blastomere at the two-cell stage with 30 ng of Sox8mo exhibit reduced Sox10 expression at stage 17, while Slug, Sox9 and Sox2 expression appears largely unaffected at this stage. (C) Quantification of the in situ hybridization results. The numbers in parenthesis indicate the number of embryos analyzed. (D) Sox8mo (500 ng) does not interfere with translation of a mutated Sox8 mRNA (mSox8). (E) Rescue experiments were performed by injection of an animal dorsal blastomere at the eight-cell stage. Bilateral Sox10 expression is rescued in Sox8mo-injected embryo by co-injection of mSox8 mRNA (Sox8mo+mSox8). Single injection of mSox8 expanded the Sox10 expression domain. Injection of a 5 bp mis-matched Sox8 morpholino (Sox8mis) had no effect on Sox10 expression. (F) Quantification of the in situ hybridization results. The numbers in parentheses indicate the number of embryos analyzed. (B,E) Dorsal view, anterior is towards the top. RNA encoding the lineage tracer ß-galactosidase was co-injected to identify the injected side (red staining) (left side in B and right side in E).

View larger version (25K): [in a new window]   Fig. 4. Sox8 regulates the onset of expression of Slug and Sox9. (A) Embryos injected in one blastomere at the two-cell stage with 30 ng of Sox8mo exhibited reduced Sox9 and Slug expression at stage 12.5 and stage 14. Lateral view in all panels, anterior is towards the right (control side) or to the left (injected side). (B) Quantification of the in situ hybridization results. The numbers in parentheses indicate the number of embryos analyzed.

View larger version (65K): [in a new window]   Fig. 5. SoxE factors function redundantly at the neural plate border. (A) Sox8, Sox9 or Sox10 can equally rescue Slug expression levels at stage 14 and expand Sox10 expression domain in Sox8-depleted embryos. In these experiments, Sox10 expression was evaluated using a probe against Sox10 3' UTR. (B) Western blot analysis. Detection of SoxE-GR proteins in extracts from injected embryos collected at stage 17 after injection at the two-cell stage. The fusion proteins are expressed at similar levels, as revealed with -GR antibody. -, uninjected control embryo; -tubulin is presented as a loading control.

View larger version (24K): [in a new window]   Fig. 6. Sox8 and Sox9 regulate Sox10 expression in animal explants. (A) Real-time RT-PCR of animal explants isolated from embryos injected with Sox8GR, Sox9GR or Sox10GR. The histograms indicate the relative expression levels of Sox8 (yellow), Sox9 (blue) and Sox10 (green) in animal explants collected 4 hours after dexamethasone treatment (+Dex). (B) In animal explants, Sox8GR, Sox9GR and Sox10GR strongly induce Sox10 expression after 4 hours of dexamethasone treatment (+Dex). The induction of Sox10 by Sox8GR, Sox9GR or Sox10GR occurs independently of protein synthesis (+CHX), indicating that not only can Sox10 regulate its own expression but that Sox8 and Sox9 can directly activate Sox10. (C) Hierarchy of SoxE genes activation in the developing neural crest.

View larger version (80K): [in a new window]   Fig. 7. Sox8-depletion leads to a broad range of defects in neural crest derivatives. (A) Embryos were co-injected in one blastomere on the dorsal side at the eight-cell stage with 20-30ng of Sox8mo and RNA encoding the lineage tracer ß-galactosidase. Well-defined pharyngeal arches are absent on the injected side when compared with the control side (arrow). Longitudinal section of the embryo shown on the left panel; the asterisks indicate individual pharyngeal arches on the control side. (B) Flat-mount Alcian Blue-stained skeletal preparations from control and Sox8mo-injected embryos at stage 45. Injected side is on the right. Sox8mo-injected embryos present different levels of skeletal defects. (C) A reduced number of Trp2-positive cells is observed in embryos that received 30 ng of Sox8mo in one blastomere at the two-cell stage. The control side shows a normal pattern of Trp2-expressing cells (arrows) at stage 33. Embryo is viewed from the lateral side, anterior towards the right (left panel) or anterior towards the left (right panel). (D) Histological analysis of Sox8mo- and Sox8mis-injected embryos at stage 42 and stage 47. In each panel, an arrow indicates the position of the dorsal root ganglia. Sox8-depleted embryos (Sox8mo) have no or reduced dorsal root ganglia on the injected side when compared with sibling to Sox8mis-injected embryos. For stage 42 embryos, only the injected side is shown. In sections of stage 47 embryos, the injected side is indicated by an asterisk.

View larger version (79K): [in a new window]   Fig. 8. Sox8mo-injected embryos have defects in neural crest migration. (A) Phosphohistone H3 immunostaining (-pH3) shows no difference in the pattern of cell proliferation in stage 17 embryos that received unilateral injection of Sox8mo (left side; FITC label) when compared with the uninjected side. (B) TUNEL staining shows no difference in the pattern of cell death in stage 17 embryos that received unilateral injection of Sox8mo (left side; FITC label) when compared with the uninjected side. In A,B, embryos are viewed from the dorsal side, anterior towards the top. (C) The migration of cranial neural crest cells into the pharyngeal arches visualized by Sox9 and Sox10 expression is severely perturbed in Sox8-depleted embryos (brackets). These cells appear to accumulate lateral to the hindbrain. RNA encoding the lineage tracer ß-galactosidase was co-injected to identify the injected side (red staining). Embryos are viewed from the lateral side, anterior towards the left (left panels) or anterior towards the right (right panels). (D) Tissue section of Sox8mo-injected embryos showing accumulation of Sox10-positive cells (small bracket; outlined in red) lateral to the hindbrain on the injected side (arrow); on the control side, Sox10-positive cells have initiated their migration (large bracket; outlined in red). The black outline indicates the position of the hindbrain (hb). (E) TUNEL staining of a stage 25 embryo that received unilateral injection of Sox8mo (left panel; dorsal view, anterior to top). The injected side (arrow) is characterized by reduced pharyngeal arches. Higher power views of the cranial regions on the injected side show no significant increase in TUNEL-positive cells (right panel; lateral view, anterior towards left, dorsal towards top) when compared with the uninjected side (middle panel; lateral view, anterior towards the right, dorsal towards the top).