Kloc M et al. (1989), The maternal store of the xlgv7 mRNA in full-gr...

XB-ART-26341

Development 1989 Dec 01;1074:899-907.

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The maternal store of the xlgv7 mRNA in full-grown oocytes is not required for normal development in Xenopus.

Kloc M , Miller M , Carrasco AE , Eastman E , Etkin L .

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We have attempted to analyze the function of a maternal mRNA xlgv7 which is distributed as an animal-vegetal gradient in stage 6 oocytes using a combination of antisense oligodeoxynucleotide injection into oocytes followed by in vitro maturation and fertilization. Injection of 20 ng of the antisense oligodeoxynucleotide resulted in the destruction of the xlgv7 mRNA to undetectable levels. Upon maturation and fertilization the resulting embryos develop with no specific defects suggesting that the maternal store of xlgv7 in stage 6 oocytes is not required and that the embryo can develop solely with the maternal store of the xlgv7 protein. Also, these results demonstrate the feasibility of this approach in destroying a specific maternal RNA and assaying its effect on development.

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Species referenced: Xenopus laevis
Genes referenced: h4c4 igf2bp3 palm3

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	Fig. 1. Gradient of xlgv7 mRNA distribution in stage 6 oocytes. Poly(A)+ RNA from total oocytes (lane 1), animal pole (lane 2), middle region (lane 3), and vegetal pole (lane 4) was separated on formaldehyde-agarose gels, blotted, and hybridized to random prime labeled xlgv7 cDNA and Xenopus histone H4 probe. The arrow points to the xlgv7 mRNA and the H4 represents the histone H4 transcript. Approximately 2 jig of RNA was loaded per lane except in lane 1 in which a larger quantity (approximately 4jig) was loaded.
	Fig. 2. In situ hybridization with xlgv7 probe. Sagittal sections of stage 6 oocytes hybridized to control (sense) xlgv7 transcript (A); B and C show stage 2, 3 and 6 oocytes hybridized with the antisense strand of xlgv7; D is a longer exposure of C. Arrowheads mark the position of animal pole in stage 6 oocytes. Magnification xl5 (darkfield).
	Fig. 3. Northern blots and ethidium bromide staining of total RNA (20 ng per lane) from stage 6 oocytes injected with different amounts of sense and antisense oligodeoxynucleotides. Panels A and B are different exposures of the same blot hybridized to random labeled xlgv7 probe. Lane 1, RNA from sham-injected control oocytes; Lane 2, RNA from oocytes injected with 60 ng of antisense no. 01; Lane 3, RNA from oocytes injected with sense no. 1 oligonucleotide. Oocytes were incubated for 2h post-injection. Panel B also shows the ethidium bromide staining of the same gel before blotting. Panel C. Lane 1, RNA from control sham-injected oocytes; Lane 2, RNA from oocytes injected with 50ng of antisense; Lane 3, RNA from oocytes injected with 10 ng of antisense oligodeoxynucleotides no. 2.2. Panel C also shows the ethidium bromide staining pattern of the gel before blotting and the same blot re-probed with Xenopus histone H4 random labeled probe. The 18s refers to the position of 18S rRNA on the gel.
	Fig. 4. Hybridization of oligodeoxynucleotides to Northern blot of poly (A)+ RNA from noninjected stage 6 oocytes. Strips from two independent blots containing 20 ftg and 5/*g of poly (A)+ RNA probed with antisense oligonucleotide (01) showing specific hybridization to xlgv7 mRNA (arrow). (S) Under the same conditions control sense oligonucleotide (1) does not hybridize to oocyte poly (A)+ RNA. Similar results were obtained with the 2.2 antisense probe.
	Fig. 5. A and B. Northern blot of total RNA (40^g per lane) from matured oocytes (A), and 2-cell-stage embryos (B) originating from the oocytes injected with 20 ng of oligonucleotides. The blots were hybridized with random labeled xlgv7 and Xenopus histone H4 probes. Lanes Al and Bl, RNA from control oocytes that were sham injected; Lanes A2, 4 and B2, RNA from oocytes injected with antisense (2.2) oligonucleotide; Lanes A3 and B3, RNA from control oocytes injected with sense (no. 1) oligonucleotide. Arrow points the position of xlgv7 transcript.
	Fig. 6. Maturation and development of oocytes injected with sense (S) and antisense (AS) oligodeoxynucleotides.
	Fig. 7. Resynthesis of xlgv7 mRNA in embryos. Northern blot of total RNA (25 /*g per lane) from stage 25 embryos originating from oocytes injected with oligodeoxynucleotides (20 ng per oocyte). The blot was probed with xlgv7 and reprobed with Xenopus histone H4 random-labeled probes. Lane 1, RNA from sham-injected embryos; Lane 2, RNA from oocytes injected with antisense DNA (no. 2.2); Lane 3, RNA from embryos injected with sense DNA (no. 1). Arrow marks the position of xlgv7 transcript.