QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Spokony, R. F. Articles by Saint-Jeannet, J.-P. Search for Related Content PubMed PubMed Citation Articles by Spokony, R. F. Articles by Saint-Jeannet, J.-P.

The transcription factor Sox9 is required for cranial neural crest development in Xenopus

Rebecca F. Spokony^*, Yoichiro Aoki^*, Natasha Saint-Germain, Emily Magner-Fink and Jean-Pierre Saint-Jeannet

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA
^* These authors contributed equally to this work

View larger version (87K): [in a new window]   Fig. 1. Sequence, structure and expression of Xenopus Sox9. (A) Deduced amino acid sequence from Xenopus, human, mouse, chicken, alligator and zebrafish Sox9 were aligned using Mac Vector CustalW Alignment. Identical and similar amino acids are in black and gray boxes, respectively. Conserved regions in human and chicken Sox9 sequences selected to design degenerate primers are indicated in green. The HMG box is underlined in blue. The peptide corresponding to the C terminus of human Sox9 used to generate rabbit antibodies against Sox9 (Bridgewater et al., 1998) is underlined in red. (B) Northern hybridization analysis. Developmental expression of Sox9 mRNA. Stages are according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Ethidium bromide-stained rRNAs are shown as loading control.

View larger version (58K): [in a new window]   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.

View larger version (56K): [in a new window]   Fig. 3. Sox9-AS prevents neural fold formation. Embryos were injected in one blastomere at the two-cell stage with 10 ng of Sox9-AS (A-C) or 10 ng of Co-AS (D-F) and analyzed at stage 17. RNA encoding the lineage tracer ß-galactosidase was co-injected to identify the injected side (blue), shown on the right in all panels. (B,C) Transverse section of the embryo presented in A shows that the neural fold is missing on the injected side (blue staining) when compared with the uninjected side (arrowhead). (E,F) Transverse sections of the embryo presented in D shows that the neural fold is unaffected on the injected side (blue staining). n, notochord; s, somite. Insets in B,E depict whole embryo ß-galactosidase staining (blue) of the specimens presented in A,D, respectively. (G) Scanning electron micrograph of a Sox9-AS-injected embryo at the early neurula stage. Note the absence of the neural fold in the injected side when compared with the uninjected control side (arrowheads).

View larger version (27K): [in a new window]   Fig. 4. Analysis of Sox9-AS specificity. (A) A Sox9 specific antibody recognizes only the major product of Sox9 in vitro translation reactions. (B) Western blot of Sox9 in vitro translated products. Sox9-AS blocked translation of Sox9 mRNA containing the 5'UTR target sequence (Sox9+5'UTR), but did not affect translation of Sox9 mRNA lacking the target sequence (Sox9). A nonspecific control oligo (Co-AS) had no effect on the translation of Sox9 mRNA derived from either construct. (C) Sox9 protein can be visualized by indirect immunofluorescence (green) in the nucleus (arrows) of the cells derived from animal explants injected with 1 ng of Sox9 mRNA at the two-cell stage. DAPI (blue) staining is shown for nucleus identification. (D) In vivo depletion of Sox9 protein. Detection of Sox9 protein in extracts from stage 17 embryos co-injected at the two-cell stage with 1 ng Sox9 mRNA (Sox9+5'UTR) and 10 ng of Co-AS or Sox9-AS. Tubulin is presented as a loading control.

View larger version (45K): [in a new window]   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 ß-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.

View larger version (90K): [in a new window]   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 ß-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.

View larger version (48K): [in a new window]   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 ß-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.

View larger version (127K): [in a new window]   Fig. 8. Sox9-depleted embryos develop abnormal pharyngeal arches and altered pattern of skeletal elements. (A) Ventral view of a tailbud stage embryo injected with 5 ng of Sox9-AS and RNA encoding the lineage tracer ß-galactosidase in one blastomere at the eight-cell stage. The bracket indicates the pharyngeal arches on the uninjected side. On the injected side (arrow, red ß-galactosidase staining), the pharyngeal arches are missing. (B) Longitudinal section of an embryo similar to the one presented in A. Note the absence of well-defined pharyngeal arches on the injected side (arrow) when compared with the control side (bracket). The asterisks indicate individual pharyngeal arch. cg, cement gland. Migration (C) and contribution to cranial skeletal elements (D) of individual neural crest segments. Modified from Sadaghiani and Thiebaud (Sadaghiani and Thiebaud, 1987). Meckel’s (Me), quadrate (Qu), ethmoid (Et), cerathoyal (Ce), basihyal (Ba) and branchial/gills (Br) cartilages. Embryos were co-injected in one blastomere on the dorsal side at the eight-cell stage with 3 ng of Co-AS (E,F) or 3 ng of Sox9-AS (G,H) and RNA encoding the lineage tracer ß-galactosidase. At stage 45, tadpoles were fixed and stained for ß-galactosidase to identify the injected side (red staining), right side in all cases. Dorsal (E,G) and ventral (F,H) views indicate an overall reduction of the cranial structures in Sox9-AS-injected embryos (G,H). (I-K) Flat-mounts of Alcian Blue stained skeletal structures from Stage 45 tadpoles. Injected side is on the left. (I) Normal pattern of skeletal elements in a Co-AS-injected embryo. (J,K) Sox9-AS-injected embryos presenting different levels of skeletal defects including loss of Meckel’s cartilage and reduction of ceratohyal and branchial cartilages. Color-coded arrows indicate the origin of each skeletal element according to D. The mesoderm-derived basihyal (black arrows) and infrarostral (black arrowheads) cartilages are unaffected in Sox9-AS-injected embryo (J,K).