Rizzoti K et al. (1998), A constitutively activated mutant of galphaq do...

XB-ART-14147

Mech Dev 1998 Aug 01;761-2:19-31.

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A constitutively activated mutant of galphaq down-regulates EP-cadherin expression and decreases adhesion between ectodermal cells at gastrulation.

Rizzoti K , Paquereau L , Shaw A , Knibiehler B , Audigier Y .

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We have examined the expression and function of the heterotrimeric GTP-binding protein Gq during early Xenopus embryogenesis. Abundant XGalphaq transcripts were detected in oocytes and early embryos by Northern blot analysis. In situ hybridization revealed that these transcripts are confined to the animal hemisphere of the mature oocyte and to the presumptive ectoderm of cleaving embryos. Microinjection at the two-cell stage of alphaq and Q209Lalphaq, a constitutively activated mutant, causes a disruption in ectodermal cell adhesion at late gastrulation. Dissociation/reaggregation experiments performed on animal cap explants clearly demonstrate that the Q209Lalphaq-induced phenotype occurs after reaggregation of the explants with a time-course similar to that observed in whole embryos. RT-PCR experiments performed on the explants from Q209Lalphaq-injected embryos revealed a selective decrease in the amount of EP-cadherin mRNA. Co-injection of EP-cadherin RNA, but also E-cadherin RNA, rescued the disaggregated phenotype. These data emphasize the functional link between Gq protein-coupled signalling pathways and cadherin molecules in the ectodermal layer during the morphogenetic movements of gastrulation.

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Species referenced: Xenopus laevis
Genes referenced: cdh1 cdh3 eef1a2 gal.2 prss1 tbx2

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	Fig. 1. Temporal expression of XGaq during Xenopus development. Northern blot analysis was carried out on 15 mg of total RNA from mature oocytes (OVO) and from embryos staged according to Nieuwkoop and Faber (1967). (A) The blot was hybridized with a 32P-labelled probe from the entire coding region of XGaq cDNA and autoradiographed for 5 h. The size of XGaq mRNAs was estimated from migration of the RNA ladder and is indicated on the left. (B) The blot was hybridized with a 32P-labelled probe from the entire coding region of EF1a cDNA. This elongation factor is ubiquitously expressed in the Xenopus embryo and its levels increase from the midblastula transition at stage 8 (Krieg et al., 1989).
	Fig. 2. Spatial expression of XGaq mRNA during Xenopus development. Whole mount in situ hybridization was performed with an antisense RNA probe corresponding to the coding region. Oocytes and embryos are positioned with the animal pole up. (A) Mature oocyte. Note the restriction of labelling to the animal pole. (B) Stage 2 embryo. (C) Morula. Cells of the animal region express high levels of XGaq mRNA. (D) Blastula. (E) Vegetal view of a gastrula. The staining is mainly localized to the animal pole while the vegetal pole is unstained. (F) Dorsal view of a neurula showing specific labelling on neurectoderm.
	Fig. 3. Phenotypic effect of microinjecting aq and Q209Laq RNAs into both blastomeres at the two-cell stage. (A) In vitro translation of aq and Q209Laq mRNAs and trypsin digestion of translated products. (B) Neurulas from control embryos (B), from embryos injected with 1 ng of aq RNA (C) and from embryos injected with 2.5 pg of Q209Laq RNA (D). The removal of the vitelline membrane clearly increases the extent of cell dissociation from the ectodermal layer.
	Fig. 4. Micrographs of sections prepared from embryos injected with b-galactosidase alone or together with 1.25 pg Q209Laq RNA (see Section 4). Embryos were fixed at late gastrula stages, stained with X-Gal, embedded in paraffin-wax and sectioned. (A) A transverse section of a b-galactosidase-injected embryo. Staining reveals that only the ectodermal layer received the injected RNA. (B) A magnification () of the ectoderm region. Note that the ectodermal layer is normal. (C) A transverse section of Q209Laq-injected embryo. (D) A magnification () of the ectoderm region. Note that the ectodermal layer shows signs of dissociation.
	Fig. 5. Phenotype observed in animal cap explants from b-galactosidase-, Q209Laq- and aq-injected embryos. Animal caps were isolated at stage 8 and cultured for 20 h, by which time control embryos had reached stage 14. (A) Control explants from b-galactosidase-injected embryos. (B) Explants from embryos injected with 2.5 pg Q209Laq RNA. (C) Explants from embryos injected with 4 ng aq RNA. Explants from Q209Laq- and aq-injected embryos contain numerous dissociated cells in suspension, while cell patches protrude from their surface.
	Fig. 6. Cellell adhesion assay with animal cap explants from b-galactosidase- and Q209Laq-injected embryos. Animal cap explants were isolated at the blastula stage and dissociated in Ca2 + -free and Mg2 + -free buffer. (A) Dissociated explants from b-galactosidase-injected embryos. (B) Dissociated explants from embryos injected with 1.25 pg Q209Laq mRNA. Dissociated explants were transferred to a Ca2 + - and Mg2 + -containing buffer and incubated for 20 h, by which time control embryos had reached stage 14. Explants from b-galactosidase-injected embryos photographed after 12 h (C) or 20 h (E) of incubation. Explants from Q209Laq-injected embryos photographed after 12 h (D) or 20 h (F) of incubation.
	Fig. 7. EP-cadherin and E-cadherin expression in animal cap explants from b-galactosidase- and Q209Laq-injected embryos. Total RNA was extracted from 7 animal caps. Five micrograms of total RNA were reversed transcribed, amplified and blotted as described in Section 4. The blot was hybridized with 32Plabelled probes from the coding region of EP-cadherin, E-cadherin and EF1a cDNAs. (A) Southern blot of one representative experiment. (B) Quantification of the amplified fragments blotted on the membranes. The radioactivity was measured with a phosphoimager. The results are expressed as the ratio of radioactivity in the Q209Laq-lane to the radioactivity in the b-galactosidase-lane. The values represent the mean + SEM obtained in eight separate experiments, involving a total number of 75 explants from each set of injected embryos.
	Fig. 8. Rescue of the Q209Laq-induced phenotype by microinjection of EP-cadherin or E-cadherin RNA. Embryos were injected with 1.25 pg of Q209Laq RNA alone or co-injected with various amounts of cadherin RNAs (see Section 4). Animal caps were isolated at stage 8 and cultured for 20 h. (A) Explants from b-galactosidase-injected embryos. (B) Explants from Q209Laq-injected embryos. (C) Explants from embryos co-injected with Q209Laq and 0.1 ng of EP-cadherin RNA. (D) Explants from embryos co-injected with Q209Laq and 0.5 ng of EP-cadherin RNA. (E) Explants from embryos co-injected with Q209Laq and 0.1 ng of E-cadherin RNA. (F) Explants from embryos co-injected with Q209Laq and 0.5 ng of E-cadherin RNA.