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

First published online 11 August 2004
doi: 10.1242/dev.01329

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 Perez-Alcala, S. Articles by Barbas, J. A. Search for Related Content PubMed PubMed Citation Articles by Perez-Alcala, S. Articles by Barbas, J. A.

LSox5 regulates RhoB expression in the neural tube and promotes generation of the neural crest

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

View larger version (110K): [in a new window]   Fig. 1. Expression patterns of LSox5 in the cephalic region of chick embryos. Dorsal views of stage 7 (A), 8 (D), 9 (F) and 10 (G) embryos show the expression of LSox5 mRNA in the neural folds/neural tube, following a pattern compatible with premigratory and migratory cephalic neural crest. Transverse sections of stage7 (B,C) and stage 10 (H,I) embryos show LSox5 expression all along the dorsoventral axis of the neural tube at prosencephalic levels (B,H), and a restriction to the most dorsal region at more caudal levels (C,I). (E) A dorsal view of a stage 8 embryo showing the expression of Slug in territories competent to form neural crest, and its absence from the non-crest producing prosencephalic regions. (J-L) A transverse section of a stage 10 embryo at the hindbrain level labelled with both anti-LSox5 (green) and anti-Slug (red) antibodies. Only a subpopulation of the cells within the neural tube (nt) express both Slug and LSox5, whereas all the migratory cells express both genes. LSox5 expression increases along the cell migratory tracts, while Slug expression diminishes in some migratory cells. (M) A transverse section of a stage 10 embryo at the level of the rostral hindbrain shows that as migration proceeds, LSox5 immunoreactive cells (green) acquire the HNK1 epitope (red). (N) Parasagittal section of a stage 25 embryo showing the proximal segment of the oculomotor nerve. LSox5 expression (green) is high in the precursors of the Schwann cells, where it coincides with the P0 marker. (O) Section through the ophthalmic lobule of the trigeminal ganglion of an E5.5 (stage 28) embryo double stained for Islet (red) and LSox5 (green). The small nuclei, morphologically associated with satellite glia, express LSox5, while those of the neuroblasts express Islet.

View larger version (106K): [in a new window]   Fig. 2. LSox5 misexpression in the cephalic neural tube augments the neural crest population. (A) Dorsal view of an embryo 20 hours after co-electroporation with pCX-LSox5 and PCX-EGFP on the right-hand side of the cephalic neural tube. The EGFP expression is seen on the treated side and in the emerging streams of migratory neural crest cells (white arrowheads). (B,C) Transverse sections of an electroporated embryo showing the coincident expression of EGFP and ectopic LSox5 all along the dorsoventral axis of the right hand side of the neural tube. (D,E) Lateral views of an electroporated embryo immunostained for HNK1 where the increase in HNK1-positive migratory cells in the electroporated (ep) side can be observed, particularly in the circumpharyngeal stream (black arrowhead). (F,G) A transverse section at the level of the caudal hindbrain shows the supernumerary HNK1-positive migratory cells (red) in the electroporated side of the embryo. (H) A magnification of G shows the appearance of ectopic HNK1-positive cells within the treated side of the neural tube (arrowheads). (I,J) At the hindbrain level, in a double immunostained transverse section, the overexpression of Pax7 (red) in cells expressing EGFP (green) can be seen in both neuroepithelial and migratory cells on the LSox5 transfected side. (K,L) An overlay of RhoB hybridisation and double immunostaining for Pax7 (green) and laminin 1 (red) shows that severe damage of the basement membrane coincides with the expanded region of neural crest delamination on the electroporated side of the neural tube. (M,N) Transverse sections of electroporated embryos hybridised for LSox5 and Slug (M), and N-cadherin (N). The territory competent for neural crest generation extends ventrally on the electroporated side, as judged by the expanded expression of Slug (M) and the coincident inhibition of N-cadherin expression (compare the length of the brackets on both sides of the neural tube). (O) At more ventral regions of the neural tube, scattered cells could be detected that ectopically expressed RhoB on the treated side of the neural tube, breaking the basal lamina and invading into the lateral mesenchyme (open arrowhead). ov, otic vesicle.

View larger version (90K): [in a new window]   Fig. 3. Rapid activation of RhoB after ectopic LSox5 expression in the cephalic neural tube. Expression of EGFP (A); LSox5 (B,E,F) or RhoB (C,D,F) in embryos fixed 6-7 hours after electroporation with pCX-LSox5. A few transformed cells ectopically expressing LSox5 can be detected scattered within the neural tube with the anti-LSox5 serum (A,B). In situ hybridisation for RhoB at a mesencephalic level shows that the ectopic activation of this gene in cells at the electroporated side is analogous to the expression of LSox5 (C). The cells that ectopically express RhoB correspond to those with higher levels of ectopic LSox5 expression in their nuclei (D-F).

View larger version (118K): [in a new window]   Fig. 4. Ectopic LSox5 expression in the cephalic neural tube upregulates the expression of specific neural crest markers. Embryos were electroporated with pCX-LSox5 on the right-hand side of the neural tube, left to develop for 12 (A-D) or 20-24 hours (E-M), and then subjected to in situ hybridisation to visualise the expression of LSox5 and/or FoxD3 or Sox10. (A,C) Dorsal view of the midbrain and anterior hindbrain of two embryos showing an increase in FoxD3- and Sox10-expressing cells on the transfected side. Transverse sections of these embryos (B,D) show the appearance of ectopic premigratory cells expressing these markers. Dorsal (E) or lateral views (F,G) of two treated embryos show the dramatic increase in FoxD3 and Sox10 expression on the transfected side (ep) 20-24 hours after electroporation. The effect is particularly remarkable in transverse sections at the level of the circumpharyngeal crest (J,K). (H,I) Diagrams representing the area covered by FoxD3- or Sox10-expressing cells in 40 µm serial sections through the cephalic region of the same embryos. These data enable us to estimate the number of cells expressing these markers along the anteroposterior axis (in arbitrary units). A larger area of expression is associated with the transfected side (red bars) in most sections. The red lines correspond to the transverse section in B (A), D (C), J (E,H) and K (F,G,I). (L-M) Transverse sections at a mesencephalic level showing transformed neuroepithelial cells that seem to leave the neural tube beyond the dorsal competence domain (arrowheads) and do not express FoxD3 or Sox10. ov, otic vesicle.

View larger version (97K): [in a new window]   Fig. 5. Deviation of neural crest derivatives to the glial lineage. Parasagittal sections of the head region of embryos 48 hours after electroporation of LSox5 showing the formation of the trigeminal (A,B), facial (C,D), ciliary (E,F), and superior (G,H) ganglia. Serial sections immunostained for LSox5 (green in A,C,E,G) or EGFP (green in B,D,F,H) show the distribution of transformed neural crest cells that maintain LSox5 overexpression. The exposure time was reduced in A,C,E,G to visualise only LSox5-overexpressing cells with signals above the normal level in glial cells. These cells are preferentially located at the proximal entrance of the corresponding cranial nerve (yellow arrowheads) or at the distal exit point (white arrowheads). Transformed cells are excluded from the proximal region of the trigeminal ganglion (broken line in A,B), where neural crest derived neurons should differentiate, and there is a consistent failure to colocalise with the neuronal marker (Islet, in red).

View larger version (30K): [in a new window]   Fig. 6. SSox5 mimics the effects of LSox5 in misexpression assays. (A) Comparison of the LSox5-II and SSox5 polypeptides. HMG, HMG binding domain; cc, potential coiled-coil domains. (B,C) Transverse sections at rostral hindbrain levels hybridised with RhoB or FoxD3 12 hours after electroporation with pCX-SSox5. Both the ectopic expression of RhoB and the upregulation of FoxD3 observed here are very similar to those produced after LSox5 misexpression (Figs 3, 4, 5).