XB-ART-17605Mech Dev October 1, 1996; 59 (2): 141-51.
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The mRNA encoding a beta subunit of heterotrimeric GTP-binding proteins is localized to the animal pole of Xenopus laevis oocyte and embryos.
In order to provide evidence for a potential role of heterotrimeric GTP-binding proteins in the transduction of developmental signals, we prepared cDNAs from Xenopus laevis embryos and looked for fragments amplified between primers located in conserved sequences of the different subtypes of beta subunit. Using the amplified fragment as a probe, we cloned a member of the beta subunit family. The deduced protein sequence of the amphibian cDNA is highly homologous to the beta 1 subtype and, accordingly, we have named the Xenopus gene XG beta 1. In situ hybridization and RNase protection assay revealed that XG beta 1 mRNA is confined to the animal hemisphere of the mature oocyte. This localization of XG beta 1 mRNA is established at stage V during oogenesis. Following fertilization, the maternal mRNAs cosegregate with animal cells during cleavage stages. At gastrulation, transcripts are expressed in the dorsal ectoderm layer that will give rise to the central nervous system. Thus, XG beta 1 mRNA belongs to the small family of localized maternal mRNAs; as a transducing protein, its restriction to a subset of embryonic cells could mediate the distinct responsiveness which contributes to the patterning of the embryo.
PubMed ID: 8951792
Article link: Mech Dev
Species referenced: Xenopus laevis
Genes referenced: gnb1 mt-tr trna
Article Images: [+] show captions
|Fig. 3. Northern blot analysis of adult eye, using a 165 bp cDNA probe for XC+%. Ten pg of total RNA were loaded and hybridized as described in experimental procedures. Positions of 285 and 18s RNA and RNA ladder are indicated. The size of XGH mRNA was estimated at 3.4 kb.|
|Fig. 5. Expression pattern of XGB1 in Xenopus observed after whole mount in situ hybridization. In situ hybridization was carried out as described in Section 4. (A-C) Cleared oocyte or embryos are positioned with the animal pole on top. They correspond, respectively, to mature oocyte, stage 2 embryo and morula. Note the restriction of the labeling to the animal pole. (D) Vegetal view of a gastrula. The staining is mainly localized in the dorsal region under the blastopore lip (bl), while the vegetal pole is unstained. (E) Dorsal view of a neurula showing specific labeling on the neural plate. (F) Early larval stage; head is on the left, dorsal on the top. An intense staining is observed in neural structures (br, brain; sp, spinal cord) and eye (e). All embryos were depigmented after in situ hybridization.|
|Fig. 6. Transverse sections from whole mount in situ hybridized embryos shown in Fig. 5. (A-C) These transverse sections arc presented with the animal pole on the top, and correspond, respectively, to mature oocyte, stage 2 embryo. and morula. Note that the staining is confined to the animal pole. (D) Section of a gastrula showing a specific labeling above the dorsal blastopore lip (dbl) in the ectoderm. (E) Transverse section of neurula showing the neural plate (np) staining. (F) Transverse section through the trunk of an early larva stage showing the labeling of the neural tube (nt). Abbreviations: gv, germinal vesicle; blc, blastocoele: not, notochord.|
|Fig. 7. Localization of XC/l transcripts during oogenesis between stages III and VI. Staged oocytes are positioned with the animal pole on top. (A-D) Control oocytes. Asymmetry of pigment and vitellus is established at stage IV. (E-H) Whole mount in situ hybridized and cleared oocytes. In situ hybridization was carried out as described in Section 4. Note that the XC/? labeling is homogeneous at stage III and IV, becomes heterogeneous at stage V and is asymmetric at stage VI. (I-L) Transverse sections from whole mount in situ hybridized oocytes. These sections show that the animal localization of XGFI transcript begin to be established at stage V.|
|Fig. 8. Localization of XGB1 transcripts by in situ hybridization on stage VI oocyte sections using s-32P-labeled RNA antisense probe. In situ hybridization was carried out as described in Section 4. Staged oocytes are positioned with the animal pole (AP) on top and the vegetal pole (VP) at the bottom. The position of the germinal vesicle (gv) is shown. Note that the XGfl labeling is exclusively localized in the animal pole.|
|Fig. 9. RNase protection assay on dissected oocytes and morulas. Mature oocytes were. cut into animal (AP) and vegetrd (VP) halves and animal blastomeres (AB) were separated from vegetal blastomeres (VB) in morulas. RNase protection was performed on total RNA isolated from two dissected oocytes or morulas. The amount of total RNA was, respectively, 6.8 pg for the animal pole and 2.6 flcg for the vegetal pole of oocytes, 9 pg for animal blastomeres and 5.4 pg for vegetal blastomeres of morulas. Lane DP corresponds to the digested probe in the absence of Xenopus RNA and lane P corresponds to 1110 undigested probe. Positions of the RNA ladder are indicated (0.53 kb and 0.4 kb). The same protected fragment (0.43 kb) was observed in animal poles of oocytes and animal blastomeres of morulas. The gel was exposed overnight and a Longer exposure failed to revealed a protected fragment in VP. The quality and integrity of total RNA were checked on a denaturing gel.|