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First published online 29 September 2004
doi: 10.1242/dev.01419

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A vertebrate crossveinless 2 homologue modulates BMP activity and neural crest cell migration

Edward Coles^1,2, Jeff Christiansen^1,*, Androulla Economou¹, Marianne Bronner-Fraser² and David G. Wilkinson^1,

¹ Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (111K): [in a new window]   Fig. 1. Sequence of chick Cv-2. Amino acid sequence alignment of chicken, mouse, human and Drosophila Cv-2 with the ClustalW multiple sequence alignment using the BLOSUM30 algorithm. Identical and conserved amino acid substitutions are boxed, the former being identified by dark shading. Protein motifs identified by Pfam are coloured as follows: secretory signal in light blue; CR 1-5, cysteine-rich motif in green; VWFD, von Willebrand Factor type D domain in red; and TIL, trypsin inhibitor-like cysteine-rich domain in purple. The black bars highlight the amino acid sequence CGLCG, a motif for potential multimerisation by disulphide crosslinking. GenBank Accession Number AY731507.

View larger version (93K): [in a new window]   Fig. 2. Expression pattern of Cv-2 in the chick embryo. Whole-mount in situ hybridisations of Cv-2 (A-F) and Slug (K-N) and transverse sections of Cv-2 hybridised embryos (G-J). (A) HH stage 9– (seven-somite stage). Cv-2 expression is detected in posterior lateral mesoderm (lm) and in premigratory neural crest (nc) in the hindbrain and anterior spinal cord. (B) Stage 9 (eight somites). Cv-2 expression is upregulated in premigratory neural crest in the hindbrain and spinal cord, and in some cells in the midbrain. (C) Stage 9+ (10 somites). Expression seen at stage 9 persists but (D) by stage 10+ (13 somites) Cv-2 expression in the hindbrain and anterior spinal cord is downregulated. Low levels of Cv-2 expression occur in the hindbrain in rhombomeres 3 and 5. (E) Stage 13. Cv-2 transcripts are detected at the posterior lip of the otic vesicle (ov), the outflow tract of the heart (ot) and sclerotome surrounding the dorsal aorta between the branchial arches (sc). (F) Stage 19. Cv-2 transcripts are detected in the intersomitic region (is), in sympathetic ganglia (sg), in the outflow tract of the heart (ot) and in nephrogenic mesenchyme (nm). (G,H) Transverse sections through the neural tube of stage 9+ as indicted in C show that Cv-2 transcripts are restricted to premigratory neural crest cells (nc) in the dorsal neural tube. (I,J) Transverse sections through a stage 19 embryo, showing Cv-2 expression in (I) sympathetic ganglia (sg) adjacent to the dorsal aorta (da) and in (J) nephrogenic mesenchyme (nm) surrounding the nephric ducts. (K-N) In situ hybridisation to detect slug transcripts, a marker of premigratory and migrating neural crest from stage 9– to stage 10+. Black arrowheads identify the midbrain/hindbrain boundary, black arrows indicate the posterior limit of the hindbrain.

View larger version (42K): [in a new window]   Fig. 3. In vitro analysis of Cv-2 activity. (A) Western blot analysis and co-immunoprecipitation reveals the direct interaction of V5-tagged Cv-2 protein with recombinant human BMP4 protein. V5-tagged chordin is used as a positive control for BMP binding. (B-E) Effect of Cv-2 injection on axis formation in Xenopus at stage 14 (B) and stage 27 (C-E). Ventral injections of Cv-2 RNA induce a partial secondary axis (arrows; B,C,E). Transverse section through the trunk of a stage 27 embryo injected ventrally with Cv-2 confirms the presence of an ectopic secondary neural epithelium (arrow, D). (F,G) RT-PCR analyses of Xenopus animal cap explants. (F) Xenopus Noggin mRNA induces expression of the general neural marker NCAM and anterior neural marker BF1. Increasing amounts of Cv-2 mRNA do not induce expression of either neural marker. (G) Co-injection of 2 ng Cv-2 RNA plus 0.1 ng noggin RNA increases the induction of BF-1 when compared with injection of 0.1 ng noggin RNA alone. The mesodermal marker muscle actin is used to confirm the absence of mesoderm in the explants and elongation factor-1 alpha (EF-1) is used to assess the relative amounts of recovered mRNA.

View larger version (37K): [in a new window]   Fig. 4. Elevation of BMP4 activity by Cv-2. (A-D) Injection of Cv-2 (A), BMP4 (B) or BMP4 plus Cv-2 mRNA (C,D) with fluorescein dextran lineage tracer into a single animal pole blastomere at the 32-cell stage embryo, and Xbra expression detected at stage 10.5. Fluorescein dextran is detected in red, and Xbra in blue, with the normal expression domain around the blastopore lip indicated with a black ellipse. Ectopic BMP4 with or without Cv-2 induces ectopic expression of Xbra, either co-extensive with the normal expression around the blastopore lip (B,C) or as an isolated patch (D). (E) Summary of the effects of BMP4 and Cv-2 injections on the proportion of embryos with ectopic Xbra expression (%, y-axis). For each amount of BMP4 RNA, co-injection of Cv-2 RNA increases the induction of Xbra expression. The number of embryos analysed are indicated at the top of the chart. (F) Injection of BMP4 and/or Cv-2 mRNA at the one-cell stage embryo, followed by excision of animal caps at stage 8-9, and RT-PCR analysis of Xbra transcripts at stage 10.5. Quantitation with a phosphoimager reveals that Xbra expression is fourfold higher in BMP4 plus CV-2 injected animal caps than with BMP-4. Cv-2 alone does not induce Xbra expression.

View larger version (85K): [in a new window]   Fig. 5. Effect of elevated Cv-2 expression on trunk neural crest cell migration. (A-E) Representative images of the trunk region of stage 16 chick embryos electroporated with pCIG (A,B) or co-electroporated with pCS2Cv-2 and pCIG (C-E) expression constructs at HH stage 10+. (A) In situ hybridisation with Sox10 of a control pCIG electroporated embryo. (B) Transverse section of embryo (A) at axial level indicated; black and white arrows identify equal progression of Sox10-positive migratory neural crest cells on control and electroporated sides. (C) In situ hybridisation with Sox10 of an embryo overexpressing Cv-2. The domain of Sox10 expression extends further caudally on the side overexpressing Cv-2 (right) than the control side (left). Insets in A and C show the distribution of GFP-positive cells, demonstrating the domain and efficacy of the electroporation prior to in situ hybridisation analysis; left side of the neural tube is the control; right side is the electroporated side. (D,E) Transverse sections through embryo (in C) as indicated by lines, with immunofluorescence labelling of HNK1. (D',E') Corresponding bright-field images in which the Sox10 signal is more easily seen. In the more anterior section (D,D') there is an increase in the number and distance of migration of HNK-1-positive migratory crest cells on the transfected side (black arrow) compared with the control side (white arrow). Sox10 signal is not detected in the ventral HNK1-expressing neural crest, perhaps owing to a decreased level of expression during ventral migration. In the more posterior section (E,E') Sox10- and HNK-1-positive crest cells have initiated migration on the Cv-2 electroporated side (black arrow) but not on the untreated side.