Heasman J et al. (1994), A functional test for maternally inherited cadh...

XB-ART-21748

Development 1994 Jan 01;1201:49-57.

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A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage.

Heasman J , Ginsberg D , Geiger B , Goldstone K , Pratt T , Yoshida-Noro C , Wylie C .

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We report here on the consequences of reducing the expression of EP-cadherin at the earliest stages of Xenopus development. Injection of oligodeoxynucleotides antisense to maternal EP-cadherin mRNA into full-grown oocytes reduced the mRNA level in oocytes, and the protein level in blastulae. Adhesion between blastomeres was significantly reduced, as seen in whole embryos, and in assays of the ability of blastomeres to reaggregate in culture. This effect was especially conspicuous in the inner cells of the blastula and included the disruption of the blastocoel. The severity of the EP-cadherin mRNA depletion and of the disaggregation phenotype was dose dependent. This phenotype was rescued by the injection into EP-cadherin mRNA-depleted oocytes of the mRNA coding for a related cadherin, E-cadherin, that is normally expressed at the gastrula stage in the embryonic ectoderm.

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Species referenced: Xenopus laevis
Genes referenced: cdh1 cdh3 eef1a2 myod1 ocm tbx2

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	Fig. 1. Northern blot of RNA from 2.5 oocyte equivalents per lane after injection of 4 ng sense oligo; and 4 ng, 2 ng and 1 ng antisense 61 oligo; 2 ng and 1 ng of antisense oligo 62 and uninjected control, respectively. The antisense oligos deplete EP-cadherin mRNA (arrow) in a dose-dependent fashion. This blot was stripped and reprobed for EF1a RNA, to indicate RNA loading.
	Fig. 2. Northern blot of RNA from 2.5 blastula equivalents per lane after injection of 0.5 ng, 1.0 ng, 2.0 ng of antisense oligo, and 160 pg and 1.6 ng E-cadherin mRNA, respectively, into the oocytes. The blot was probed first for EP-cadherin mRNA, then stripped and reprobed for E-cadherin mRNA. No resynthesis of EP-cadherin mRNA is seen by the blastula stage after injection of antisense oligo into the oocyte. E-cadherin mRNA injected into the oocyte remains stable at least until the blastula stage.
	Fig. 3. (A) The external surface of blastulae injected as oocytes with 1 ng antisense oligo. Despite the apparently normal external surface, the inner cells are highly disaggregated (B) compared to controls (C). The control used here was injected with an oligo that depletes maternal MyoD mRNA, and is indistinguishable from those injected with 1 ng antisense + 2 ng sense oligo, or 2 ng sense oligo only. (A) Bar, 0.43 mm; (B,C) bar, 0.35 mm
	Fig. 4. (A,B) Scanning electron micrographs of blastulae derived from antisense (A) and control (B) oocytes, cut open to reveal the disrupted blastocoel after EP-cadherin RNA depletion. Bar, 0.17 mm
	Fig. 5. (A) A newly dissected blastula after antisense oligo injection into the oocyte, together with a control embryo (1 ng antisense + 2 ng sense oligo). After 4 hours (B) in 1´ MBSH the control embryo has reaggregated whereas the antisense-injected embryo remains a disaggregated mass. After 12 hours (C) both batches of embryos have reaggregated and are indistinguishable. Both have exogastrulated due to the high ionic strength of the saline (1´ MBSH) used to allow reaggregation. Antisense-injected blastulae that are left undissected will gastrulate (D) and form abnormal tailbud embryos (E). These have a variety of abnormalities, but form dorsoventral and anteroposterior axes. Control embryos at the same stage are shown in F. Bar, 0.55 mm.
	Fig. 6. (A) Blastulae derived from oocytes injected with 2 ng sense oligo, dissected open at the late blastula stage. (B) An embryo at the same stage after 1 ng antisense oligo injection to show the loss of interblastomere adhesion. The embryos shown in C were also injected with the antisense oligo, but 24 hours later were injected with 1.6 ng E-cadherin mRNA. Normal blastula morphology has largely been restored. (D-F) Equivalent embryos fixed, sectioned and stained with a pan-cadherin antibody. Cadherin can be seen around the blastomeres in the 2 ng sense oligo-injected embryos (D). It is missing from the surfaces of the blastomeres after 1 ng antisense oligo (E) and present again after 1 ng antisense oligo followed by Ecadherin mRNA (F). Notice that the blastocoel is obliterated in E, and restored in F. Each picture is from an equivalent region of the blastula, and shows animal and marginal cells. In all cases, the antibody stains nuclei and the vitelline membrane nonspecifically. (A-C) Bar, 0.55 mm; (D-F) Bar, 0.05 mm.
	Fig. 7. Bar charts to show the rates of reaggregation of isolated groups of 50 animal blastomeres disaggregated in Ca2+- and Mg2+- free saline, and allowed to reaggregate in OCM for the two time periods shown. A score of 3 indicates that the aggregates had become smooth spheres, while 0 indicates no reaggregation. For further details of scoring system, see Turner et al. (1992). EPcadherin mRNA depletion causes reduced blastomere aggregation, compared with sense-injected controls. This is rescued by the injection of E-cadherin mRNA. In all cases, addition of anti-M4B glycolipid further reduces aggregation.