Vasiliev OL et al. (1997), A novel marker of early epidermal differentiati...

XB-ART-15528

Int J Dev Biol 1997 Dec 01;416:877-82.

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A novel marker of early epidermal differentiation: cDNA subtractive cloning starting on a single explant of Xenopus laevis gastrula epidermis.

Vasiliev OL , Lukyanov SA , Belyavsky AV , Kazanskaya OV , Zaraisky AG .

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To understand the molecular mechanism underlying in the earliest steps of the embryonic ectoderm subdivision into epidermis and neuroectoderm, it would be important to isolate differentially expressed genes in presumptive neuroectoderm and epidermis at the gastrula stage, the period of the divergence of the two adjacent ectodermal compartments. Meanwhile, the most direct approach for such a task, i.e. subtractive enrichment of cDNA from neuroectodermal and epidermal explants with differentially expressed gene sequences, was difficult to realize because of the high number of explants needed for this technique. In the present paper we report a novel effective and quite simple method of cDNA subtractive enrichment, based on amplification of cDNA in vitro by polymerase chain reaction (PCR) and allowing to use a very small amount of initial cDNA samples. With this method we have cloned cDNA of a novel gene of Xenopus laevis, which was named XEP-1 for its specific expression in the presumptive epidermis starting from the midgastrula stage.

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Species referenced: Xenopus laevis
Genes referenced: hesx1 otogl2 trio

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	Fig. 1. A scheme of subtractive cloning of epidermis-specific sequences, (see text for details).
	Fig. 2. Southern analysis of enrichment of the twice.subtracted ((neural" sample with 8 neural specific cDNA. XANF-1. «Neural. sample was blocred on a nitrocellulose filter before and after two founds of subtraction and hybridized with 32p labeled probe for XANF-l. Note that only 10 ng of the tWice subtracted sample displayed the same intensifY of hybridization signal as did 1JJmof unsubtracted cDNA
	Fig.3. Nucleotide and deduced amino acid sequencesof XEP-1cDNA. TheA TGat the position 126-128 represents the putative start codon of rhe XEP-7-encoded protem. Leader peptide IS underlined. Asterisk indicates the termination codon.
	Fig. 4. Temporal pattern of XEp-1 expression. Total RNA samples were reversed transcribed and subjected to 31 cycles of PCR with oligonucleotide primers for XEP-1 and for Na+, K+ -ATPase a-subunit (as a control). PCR products were separated by eletrophoresis in 1.5% agarose gel with ethidium bromide and were photographed. Developmental stages' blastula (stages 8-9), early gastrula (stages 10-10.5), mid gastrula (stages 11-12), early neurula (stages 13-14), late neurula (stages 18-20).
	Fig. 5. In situ hybridization with XEP-1 digoxygenin labeled probe. Whole-mount in Situ hybridization was performed with albino-embryos collected at the early neurula stage (stage 14). Treated embryos were embedded in paraffin. sectioned and stained with Eosin. Note that hybridization signal presents only in the outer epidermal cells, but not In the bordering neural plate cells. ep, epidermis; np, neural plate; no, notochord,' sm, somite. Arrows Indicate the borders between eplderm!s and neural plate. Bar: 50 um.