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Overexpression of Snail family members highlights their ability to promote chick neural crest formation

Instituto Cajal, CSIC, Doctor Arce, 37 28002 Madrid, Spain

View larger version (67K): [in a new window]   Fig. 1. Slug overexpression increases neural crest production in the chick hindbrain. (A) Stage 13 control embryo showing the normal Slug expression in the premigratory and migratory neural crest. (B-D) Embryos electroporated with plasmids containing chick Slug and GFP cDNAs, injected with DiI at stage 9 and analysed 15 hours later (stage 13-14). GFP expression is observed in the right hand side of the neural tube and in cells migrating from this side (B). DiI labelling is observed within the neural tube and in all crest cells that have emigrated from the neural tube after electroporation (C). (D) HNK-1 staining in the same embryo. Both DiI labelling and HNK-1 immunohistochemistry confirm the increase in neural crest production in the electroporated side. White arrows in C indicate the r4 and r6 crest streams where an increase in the migratory cell population can be observed when compared to the control side. Black arrowhead shows DiI-labelled cells adjacent to rhombomere 5 (see also Fig. 2H). A representative section taken at the level of r4 is shown in E to better assess the relative increase in crest production. (F-H) A section taken from a different embryo at the level of r6 showing (F) exogenous GFP-Slug expression, (G) DiI labelling and (H) the merged image. ov, otic vesicle; r, rhombomere. In all experiments, the control side is to the left.

View larger version (88K): [in a new window]   Fig. 2. Slug expression directly or indirectly induces the expression of rhoB and Pax3 in the chick hindbrain. (A, B) Embryo electroporated with chick Slug at stage 9 and analysed 15 hours later (stage 13) by in situ hybridisation with chick Slug (A) and subsequently subjected to HNK-1 immunohistochemistry (B). The electroporated cells on the right hand side of the neural tube express high levels of Slug and more Slug-expressing cells can be observed migrating from the post-otic hindbrain. It is interesting to note that although many Slug-expressing migratory cells are also HNK-1 positive (white arrowheads in A and B), some migrating cells do not show HNK-1 reactivity (black arrowheads in A and B). The dotted line shows the level of the section in Fig. 3A. (C,D) Different embryos subjected to the same electroporation process and hybridised with rhoB or Pax3 at stage 13. Note the ectopic rhoB expression in the premigratory crest of rhombomeres 4 and 5 and a greater area covered by the rhoB-expressing cells emigrating from r4 and r6. This can be better observed in the sections (G,H) taken from this embryo at the levels indicated by the dotted lines in (C). White arrows in H indicate cells migrating out from r5. (D) An increase in the levels of Pax3 expression can be seen all along the AP axis of the hindbrain. (E,F) Another stage 15 embryo illustrating rhoB (E) and HNK-1 expression (F) after Slug electroporation. Note that there is an increase in crest cells migration from the right hand side, which expresses the two markers. Abbreviations as in Fig. 1.

View larger version (66K): [in a new window]   Fig. 3. Slug overexpression in the chick hindbrain increases the production of neural crest cells from the dorsal neural tube. (A) Embryo showing ectopic Slug expression at the right side of the hindbrain all along its dorsoventral axis. This section was taken from the embryo shown in Fig. 2B at the level indicated by the dotted line. Observe that Slug-expressing cells do not migrate from intermediate or ventral levels of the neural tube. A higher number of migratory cells can be observed in the electroporated side, some of which do not express HNK-1. This can be better observed in the magnification shown in B, where the existence of different migratory populations is readily apparent. Black arrowheads indicate Slug-positive/HNK-1-negative cells; white arrowheads exemplify the double-labelled population and black arrows indicate examples of Slug-negative/HNK-1-positive cells (see text). These populations are also detected both in the control side of the electroporated embryos (A) and in control embryos such as that shown in C-E, hybridised with Slug and subsequently stained with HNK-1. A section at the level of r6 is shown in E.

View larger version (130K): [in a new window]   Fig. 4. Slug overexpression in the chick hindbrain induces ectopic epithelial-mesenchymal transition in the dorsal part of the neural tube. (A) A section from an embryo hybridised with rhoB (blue) and neomycin (red; to detect electroporated cells, see text). There is a clear increase in premigratory crest cells in the dorsal neural tube and a higher amount of rhoB-expressing migratory cells (B). (C,D) Composit images of high power views of the boxed regions in A, corresponding to control and electroporated sides, respectively. Observe the Slug-induced ectopic EMT concomitant with the breakdown of the basement membrane indicated by the white arrow in D. The site of breakdown is confirmed by immunostaining with the basement membrane marker laminin and indicated by stars in E and F.

View larger version (75K): [in a new window]   Fig. 5. Slug overexpression increases the migratory neural crest population in the cranial region of the chick. Control (A) and electroporated sides (B) of the same whole-mounted embryo hybridised with rhoB 24 hours after electroporation with Slug (stage 15). In B, observe the higher number of rhoB positive cells in the crest cells migrating from the forebrain, midbrain and hindbrain regions (arrowheads). (C,D) Another Slug electroporated embryo double labelled for rhoB and HNK-1 at stage 17. (E) A section taken from this embryo at the different levels indicated by the dotted lines in C and D allows the visualization of different crest populations that are increased in the electroporated side (top in this picture). (F,G) High power views of the boxed areas in E. Black arrowheads, rhoB-positive/HNK-1-positive cells; white arrowheads, rhoB positive cells; black arrows, HNK-1-positive cells.

View larger version (131K): [in a new window]   Fig. 6. Slug overexpression increases the premigratory neural crest population in the trunk. (A) Trunk level of an embryo processed for rhoB and HNK-1 expression 30 hours after being electroporated with Slug at stage 12. No differences can be detected in the migratory neural crest of control and experimental sides although numerous electroporated GFP-expressing cells had migrated from the tube and can be seen in a similar pattern to that observed for HNK-1 (B). (D,E) A section of this embryo, confirming that the migratory population is similar in both sides. However, note an increase in the rhoB-expressing area in the right half of the spinal cord (E, brackets). This is better observed in a similar embryo only hybridised with rhoB (F). (C) Flat mount of the posterior hindbrain of an embryo electroporated at stage 9. The increase in rhoB-expressing crest cells both in the premigratory and in the migratory population can be better assessed in the section taken at the level of the dotted line (G).

View larger version (120K): [in a new window]   Fig. 7. Ectopic expression of chick Snail increases neural crest production in the chick hindbrain. Electroporations were carried out as described in the legend to Fig. 1. (A) GFP expression and (B) DiI labelling to visualise the neural crest cells that have emigrated from the hindbrain. C illustrates a similar embryo showing rhoB expression (blue). This embryo was also hybridised with a neomycin probe to detect the electroporated area (pale red). (D) A high power view showing the increase in premigratory and migratory neural crest (arrowheads). (E,F) Sections taken from the same embryo at the levels indicated by the dotted lines in C.

View larger version (103K): [in a new window]   Fig. 8. Ectopic expression of mouse Snail also increases neural crest production in the chick hindbrain. Electroporations were carried out as described in the legend to Fig. 1. (A,B) GFP expression and DiI labelling identifying neural crest emigrating from the hindbrain. In C a similar embryo shows ectopic rhoB expression (blue). (D) A high power view of premigratory and migratory neural crest. Arrowheads indicate regions of ectopic expression. Note the amount of rhoB-positive cells at the level of r5.