Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Previous work has revealed that proteins that bind to bone morphogenetic proteins (BMPs) and inhibit their signalling have a crucial role in the spatial and temporal regulation of cell differentiation and cell migration by BMPs. We have identified a chick homologue of crossveinless 2, a Drosophila gene that was identified in genetic studies as a promoter of BMP-like signalling. Chick Cv-2 has a conserved structure of five cysteine-rich repeats similar to those found in several BMP antagonists, and a C-terminal Von Willebrand type D domain. Cv-2 is expressed in the chick embryo in a number of tissues at sites at which elevated BMP signalling is required. One such site of expression is premigratory neural crest, in which at trunk levels threshold levels of BMP activity are required to initiate cell migration. We show that, when overexpressed, Cv-2 can weakly antagonise BMP4 activity in Xenopus embryos, but that in other in vitro assays Cv-2 can increase the activity of co-expressed BMP4. Furthermore, we find that increased expression of Cv-2 causes premature onset of trunk neural crest cell migration in the chick embryo, indicative of Cv-2 acting to promote BMP activity at an endogenous site of expression. We therefore propose that BMP signalling is modulated both by antagonists and by Cv-2 that acts to elevate BMP activity.
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 posteriorlateralmesoderm (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.
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.
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 pδCS2Cv-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 ventralHNK1-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.
