Palma V et al. (2001), Calcium mediates dorsoventral patterning of mes...

XB-ART-8200

Curr Biol 2001 Oct 16;1120:1606-10. doi: 10.1016/s0960-9822(01)00479-1.

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Calcium mediates dorsoventral patterning of mesoderm in Xenopus.

Palma V , Kukuljan M , Mayor R .

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Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.

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Species referenced: Xenopus laevis
Genes referenced: cacna1s dpys

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Figure 1. Expression patterns of Xα1S in the Xenopus laevis embryo. (a) A clone containing a 2 kb insert with an 89% homology to human α1S was isolated. The GenBank accession number for Xα1S is AF421880. Nested RT-PCR experiments were conducted by using RNA isolated from whole embryos. RT-PCR analysis of the expression of Xα1S and Xβ3 in oocytes (OO); embryos at blastula (St. 7), gastrula (St. 10), neurula (St. 18), and early tadpole (St. 35) stages; RT−, no reverse transcription. EF1α primers were used as controls. It should be noticed that this RT-PCR is not quantitative. (b) Schematic representation of dissection procedures. (c) Upper panels: whole-mount ISH analysis of the expression of Xα1S [22]. Left to right: stage 10 embryo, vegetal view showing the localization of the transcript in the dorsal mesoderm, above the blastopore lip; Xα1S is present in presomitic mesoderm (ps) and somites (s) at stages 18 and 26 (dorsal views, anterior to the left); the inset shows somitic localization of Xα1S in a tissue section. Lower panels: Xα1S ISH in explants dissected at stage 10 and cultured until the equivalent of stage 18; Xα1S mRNA is present in DMZ but not VMZ; some labeling is appreciated in lateral mesoderm (LMZ). (d) Upper panels: DMZ explants and AC exhibit specific DHP binding. Left to right, control DMZ explant preincubated with 20 μM nitrendipine; DMZ, VMZ, and AC preincubated for 5 min with 250 nMST of Bodipy-DHP (Molecular Probes). Insets show a transmitted light image of the corresponding explants. Lower panels: DMZ explants and AC display higher Ca2+ concentration than VM. Left to right: control, unloaded DM explant; explants of DMZ, VMZ, and AC incubated for 60 min with 10 μM of Fluo-3 (Molecular Probes); experiments representative of three similar measurements (four explants each)