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First published online 1 October 2003
doi: 10.1242/dev.00808

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Neural crest development is regulated by the transcription factor Sox9

Developmental Neurobiology, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK

View larger version (122K): [in a new window]   Fig. 1. Sox9 is expressed in prospective neural crest cells. Expression of the indicated genes in sections of the posterior and anterior open neural plate (PNP and ANP, respectively), and neural tube of HH stage 10 and stage 12 chick embryos. In posterior neural plate (B), Sox9 expression is restricted to the dorsal tips of the neural fold region, overlapping with FoxD3 (E), and precedes expression of Sox8 (A), Sox10 (C) and Slug (D). Anterior to this, expression of Sox10 (H) and Slug (I) are also found in a similar domain to Sox9 (G) and FoxD3 (J), but Sox8 (F) is still not detected in this domain. After neural tube closure, Sox8 (K), Sox9 (L), Sox10 (M), Slug (N) and FoxD3 (O) are expressed in the dorsal midline, where premigratory neural crest is located. Stage 10 images are adjacent sections from the open neural plate region of a HH stage 10 embryo. Stage 12 sections are from the prospective forelimb level of HH stage 12 embryos. (P) Schematic diagram of HH stage 10 chick embryo showing anterior and posterior levels of sections for the images in Q-T. (Q-T) Confocal images of Sox9 expression in HH stage 10 chick embryo from posterior to anterior levels. (Q) Sox9 is not detected in the most posterior regions. Anterior to this, Sox9 is upregulated in the dorsal tips of the closing neural folds (R,S). Sox9 expression is detected in the premigratory crest region at the more anterior region of closed neural tube (T). Expression of Sox9 is also seen at high level in the notochord (R-T).

View larger version (122K): [in a new window]   Fig. 2. Sox9 induces HNK-1 expression and neural crest differentiation. (A-I) Neural tubes electroporated with Sox9, 6 (A-C), 12 (D-F) and 24 (G-I) hours post transfection (hpt) analysed for HNK-1 expression. High level expression of the transfected construct was detected within 6 hpt (A-C) and robust ectopic induction of HNK-1 was detected by 12 hpt (D-F) and continued at 24 hpt (G-I) following Sox9 electroporation (EP). Confocal images indicate that the effect of Sox9 on HNK-1 expression is cell autonomous (C,F,I and data not shown). (G-I) An increase in the number of neural crest cells delaminating dorsally was evident and several transfected cells were observed delaminating from the neural tube in the intermediate and ventral region of the neural tube at 24 hpt (white arrows). (J) Dorsal view of the trunk neural tube electroporated with Sox9 at 40 hpt and assayed for HNK-1 expression by whole-mount immunofluorescence (K). Sox9-expressing cells are observed in the neural tube and in the delaminating neural crest cells (J, white brackets). An increase in the amount of delaminating HNK-1-expressing neural crest cells is detected on the transfected side of the embryo (K, white brackets). (L) Sox9-expressing cells are observed delaminating from the pial surface of the dorsal and intermediate region (white arrowhead) of the neural tube, corresponding to regions where laminin production is lost. (M) Sox9-expressing cells with ectopic HNK-1 expression are observed delaminating from the ventral neural tube (white arrowhead). (N-U) Induction of neural crest cells by Sox9 in neural plate explants. Vectors encoding either Sox9 and GFP or GFP alone were electroporated into the open neural plate region of the HH stage 10 chick embryos, [i] regions dissected and cultured for 48 hours in vitro before examining HNK-1 expression. (N-P) Explants of [i] regions transfected with GFP alone do not induce HNK-1 expression (O,P) and the DAPI image indicates cells remain confined to the explant (Q). By contrast, ectopic HNK-1 expression is detected in [i] explants transfected with Sox9 and many cells emigrate from the explants (R-U). A, anterior; P, posterior.

View larger version (100K): [in a new window]   Fig. 4. Sox9 induces neural crest markers but not RhoB. All panels show embryos electroporated with Sox9 and analysed at the indicated times after transfection; images are oriented with the transfected side of the neural tube to the right. Slug (A-C) and Cad6B (D-F) are induced transiently by Sox9; upregulation can be detected at 6 hpt (A,D) but expression levels have returned to basal by 12 hpt and 24 hpt (B,C,E,F). (G-I) Ectopic Cad7 expression is observed at all time points and ectopic expression of Cad7 is observed in cells migrating away from the neural tube (I, red arrow). (J-L) Ectopic FoxD3 expression is also detected in transfected neural tube but not until 24 hpt (L). Sox10 expression is normal at 6 hpt (M) but ectopic induction can be detected at 12 hpt (N) and continues at 24 hpt (O). By contrast, RhoB is not induced in any time point examined (P-R) and reduction of endogenous expression is observed at 6 hpt (P), 12 hpt (Q) and 24 hpt (R).

View larger version (80K): [in a new window]   Fig. 3. Neural crest cell induction by Sox9 is not dependent on BMP or Wnt signals. Panels show embryos electroporated with Sox9 (A-L) or Wnt3a (M-R) and analysed at the indicated times after transfection; images are oriented with the transfected side of the neural tube to the right. Induction of BMP4 (A-C), BMP7 (D-F) and Wnt1 (G-I) was not detected at any time points examined. Expression of BMP4 (A-C), BMP7 (D-F) and Wnt1 (G-I) was reduced in the dorsal neural tube, which might be a consequence of a change in fate of these cells from neural progenitor to neural crest. (J-L) By contrast, Wnt3a is induced throughout the dorsal/ventral region of the neural tube at 6 hpt (J) and 12 hpt (K) but returns to basal levels by 24 hpt (L). (M-O) Ectopic expression of Wnt3a does not induce HNK-1 expression. (P) Wnt3a transfection increases BrdU incorporation in the ventral neural tube (white bracket). Moreover, CyclinD1 (Q) expression is induced ventrally and expression of the neuronal marker TuJ1 (R) is reduced. The increased proliferation results in deformities on the transfected side of the embryo (blue arrowhead).

View larger version (96K): [in a new window]   Fig. 5. Ectopic Sox9 expression suppresses neurogenesis. Immunohistochemical detection of neural progenitor markers (A-I) and interneuron markers (J-R) on transverse sections of neural tubes 24 hours (A-I) and 48 hours (J-R) after electroporation with Sox9. Ectopic Sox9 suppresses Pax7 (A-C), Pax6 (D-F) and Nkx6 (G-I) expression in the neural progenitor cells in a cell-autonomous manner. Moreover, neuronal differentiation is inhibited by Sox9 expression. Induction of Lbx1-expressing interneurons in the dorsal neural tube is inhibited by Sox9 expression (J-L), as are the induction of Pax2-expressing interneurons (M-O) and HB9/MNR2-expressing motor neurons (P-R). The effect of Sox9 is cell autonomous, with untransfected cells differentiating into neuronal subtypes in a position-appropriate manner.

View larger version (69K): [in a new window]   Fig. 6. Forced expression of Sox9 influences the fate of neural crest cells in the periphery. (A-F) Transverse sections of the neural tube electroporated with GFP alone (A-C) and Sox9 IRES GFP (D-F) analysed 48 hpt. (A,B) GFP+ neural crest cells migrate into core and peripheral regions of dorsal root ganglion (DRG) and sympathetic ganglion (SG), and some cells express the neuronal marker Islet1/2 (blue arrow). (C) GFP+ cells are also observed along the peripheral nerve (pn) identified by ß-tubulin (TuJ1) expression. (D,E) In a Sox9-electroporated embryo, GFP+ cells are not observed coexpressing Isl1/2 in either the DRG (D) or SG (E) and cells are predominantly located in the periphery of the ganglia (D,E). (F) Sox9-transfected cells are also observed along the TuJ1+ peripheral nerve. (G,H) Embryos electroporated with GFP alone (G) or Sox9 (H) analysed 24 hpt. Cells transfected with only GFP migrate exclusively along the medial-lateral migration route leading to the DRG (G). By contrast, cells expressing Sox9 migrate along both medial-lateral and dorsal-lateral migration routes (H) (white arrow). (I) Co-expression of Sox9 and the gene encoding the Schwann cell marker protein zero (P0) is observed in neural crest cells (yellow arrow, inset) after 72 hpt. (J-L) Quantitative analysis of the effects of Sox9 expression on neural crest cells fate (n=9, ±s.e.m.). Following electroporation of Sox9-IRES-GFP, the number of cells in dorsal-lateral migratory route increased compared with the GFP control (J), whereas the number of cells co-producing Islet1/2 was reduced (K), Moreover a small increase in the number of cells co-producing P0 was evident in Sox9-IRES-GFP-transfected embryos (L). (M) The experimental approach. Following electroporation of GFP alone or Sox9-IRES-GFP, neural plate explants [i] are dissected and transplanted into the region between the neural tube and somite at stage 12-14 of the chick embryos at forelimb level and incubated for 72 hours before processing. (N-Q) Cells from [i] regions transfected with GFP alone do not migrate away from the position of transplantation. Many of these cells express Pax7 (N) and Islet1/2 (Q) but few if any GFP+ cells were observed expressing HNK-1 (O) or P0 (P). Blue arrows in (N-Q) indicate the high magnification of GFP+ cells shown in the insets. (R-U) By contrast, Sox9-expressing cells show extensive migration from sites of transplantation. These cells do not express Pax7 (R) or Islet1/2 (U) but many express HNK-1 (S) and P0 (T). White arrows in (R-U) indicate the high magnification of the Sox9+ cells shown in the insets. nc, notochord; da, dorsal aorta.