Wessely O and De Robertis EM (2000), The Xenopus homologue of Bicaudal-C is a locali...

XB-ART-11186

Development 2000 May 01;12710:2053-62. doi: 10.1242/dev.127.10.2053.

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The Xenopus homologue of Bicaudal-C is a localized maternal mRNA that can induce endoderm formation.

Wessely O , De Robertis EM .

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In Xenopus, zygotic transcription starts 6 hours after fertilization at the midblastula transition and therefore the first steps in embryonic development are regulated by maternally inherited proteins and mRNAs. While animal-vegetal polarity is already present in the oocyte, the dorsoventral axis is only established upon fertilization by the entry of the sperm and the subsequent rotation of the egg cortex. In a screen for maternal mRNAs whose stability is regulated by this cortical rotation, we isolated the Xenopus homologue of the Drosophila gene Bicaudal-C (xBic-C). It encodes a putative RNA-binding molecule expressed maternally and localized predominantly to the vegetal half of the egg. Upon fertilization and cortical rotation, xBic-C mRNA is displaced together with the heavy yolk towards the future dorsal side of the embryo. In UV-ventralized embryos, xBic-C is polyadenylated less than in untreated embryos that undergo cortical rotation. Overexpression of xBic-C by injection of synthetic mRNA in whole embryos or in ectodermal explants leads to ectopic endoderm formation. This endoderm-inducing activity is dependent on the presence of the RNA-binding domain of the protein. In contrast to the two other known maternally encoded endoderm inducers, Vg1 and VegT, xBic-C ectopic expression leads specifically to endoderm formation in the absence of mesoderm induction.

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Species referenced: Xenopus laevis
Genes referenced: a2m actl6a bicc1 cat2 fabp2 gdf1 krt12.4 ncam1 odc1 tbx2 vegt

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	Fig. 3. Expression analysis of x-Bic-C. (A-K) Localization of xBic-C mRNA analyzed by in situ hybridization. (A) Albino oocytes at stages II, III and V. (B) In situ hybridization of a stage V oocyte section, inset shows a magnification of the punctate staining in the oocyte vegetal cortex. Low levels of xBic-C transcripts are found in the animal cytoplasm, but not in the germinal vesicle nucleus. (C) Double in situ hybridization showing vegetal expression of xBic- C and of the germ plasm marker Xcat-2 (Forristall et al., 1995); the arrowhead indicates Xcat-2. Although both genes are localized to the vegetal pole, their distribution is clearly different. (D) Regularly cleaving embryos with a strong dorsoventral polarity at the 4-cell stage. Note the dorsal (light blastomeres) displacement of xBic-C mRNA upon cortical rotation. (E,F) Hemisections of stage 8 and 10.5 embryos on a plane perpendicular to the dorsoventral axis. (G,H) Stage 12 hybridized as a whole mount and as a hemisection; note zygotic expression in the dorsal lip (arrowhead). (I) Stage 24, dorsal view; (J) stage 30, lateral view; staining is seen in the pronephros, the pronephric duct and the posterior spinal cord. (K) Transverse section of a stage 30 embryo showing the staining in the pronephros anlage as well as in the floorplate. (L) RT-PCR analysis of the expression of xBic-C at different developmental stages using ODC as loading control.
	Fig. 4. xBic-C mRNA is displaced towards the dorsal side of the embryo by midblastula. In situ hybridization was performed on sagittal sections of embryos with strong dorsal/ventral polarity at stage 8. (A,B) xBic-C antisense probe, two different embryos are shown. (C) xBic-C sense probe. The arrowheads mark the rotation of the pigment indicating the future dorsoventral axis. While no signal for the xBic-C sense control could be detected, some staining in the animal region was present in the case of xBic-C. Note that the xBic-C signal is stronger in dorsal endoderm than in ventral endoderm. (D) RT-PCR analysis of stage 8 embryos dissected into animal, marginal and vegetal thirds showing that in contrast to Vg1, low levels of xBic-C mRNA can be detected in the animal pole; EF1-α shows equal loading of the RNA.
	Fig. 5. Overexpression of xBic-C mRNA leads to excessive endoderm formation. Synthetic RNA for xBic-C (160 pg) was injected marginally into each of the 4 blastomeres of 4-cell-stage embryos. (A,B) Phenotypic appearance at stage 26; injected embryos did not undergo proper gastrulation movements. (C) RT-PCR analysis of similar embryos at stage 26 showed elevated levels of endodermin (Edd) expression, but a decrease in the mesodermal markers α-actin and α-globin. NCAM was less decreased. (D,E) In situ hybridization of Edd on hemisections of embryos injected as described above and harvested at stage 10.5. (F) Control and xBic-C- injected embryos were dissected at blastula stage (stage 9) into animal cap, equatorial and vegetal region. Explants were kept in culture until sibling embryos reached stage 18 and then analyzed by RT-PCR using Sox17β as a marker for endoderm, cytokeratin for epidermis and α-actin for mesoderm. Note that upon overexpression of xBic-C the endodermal germ layer extends into the animal cap. EF1α indicates equal loading of RNA.
	Fig. 6. Ectodermal explants expressing xBic-C form dorsal endoderm. Either 160 pg (B,E) or three two-fold dilutions of xBic-C (160, 80, 40 pg; C,D) of xBic-C mRNA were injected into each animal blastomeres at the 4-cell stage, dissected at stage 9 and analyzed by in situ hybridization at stage 35 with Edd (A,B, 160 pg injected) or by RT-PCR at stage 10.5 (C), stage 24 (D) and stage 35 (E). Injections of xBic-C mRNA caused the induction of dorsal endodermal markers, but not of the induction of the posterior (small intestine) marker IFABP.

References [+] :

Adinolfi, Novel RNA-binding motif: the KH module. 1999, Pubmed