Holt CE et al. (1994), Cadherin-mediated cell interactions are necessa...

XB-ART-20573

Proc Natl Acad Sci U S A 1994 Nov 08;9123:10844-8.

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Cadherin-mediated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm.

Holt CE , Lemaire P , Gurdon JB .

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Muscle progenitors in Xenopus interact in a community of 100 or more cells to activate their myogenic genes and the muscle differentiation pathway. We examine whether the cell adhesion molecule cadherin is involved in this process. Injections of dominant negative N-cadherin RNA into the region of 2- to 4-cell embryos that will give rise to muscle suppress MyoD expression in muscle progenitor cells. By contrast, Xbra expression is unaffected and levels of Xwnt-8 message rise with increasing doses of dominant negative cadherin RNA. MyoD inhibition in embryos injected with the dominant negative cadherin mRNA is rescued by coinjection of full-length cadherin RNA, showing that the inhibition of MyoD occurs through the cadherin pathway. These results show that cadherin-mediated cell interactions play a critical role in the signaling events required for muscle progenitor cells to differentiate, as judged by their stable activation of MyoD.

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Species referenced: Xenopus laevis
Genes referenced: cdh1 cdh2 myc myod1 ncam1 tbxt wnt8a
???displayArticle.antibodies??? Myod1 Ab1 Ncam1 Ab2 Somite Ab1

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	FIG. 1. Schematic diagram of the cadhenn constructs (A) and summary ofexperimental design (B). (A) Normal full-length N-cadrin (top) has a large extracellular domain, a signal sequence (sig), a single transmembrane (TM) domain, and a short cytoplasmic (cyto) domain. The dominant negative form N-cadAE (middle) has had most of the extracellular domain deleted. The nonfunctional mutant N-cadAE/ C(T6) (bottom) has had both the extracellular and cytoplasmic domains deleted (16). The two mutants contain a Myc tag (myc). (B) Cadherin RNA was injected into the dorsal region of embryos at the two sites shown or at four sites (mirror image to those shown). Whole embryos were processed for Xbra in situ hybridization at stage 11 and for MyoD immunostaining, Northern blot analysis, and [3Hluridine autoradiography at stages 16-18. Excised pieces of dorsolateral (DL) mesoderm were probed for MyoD expression at stages 16-18.
	FIG. 2. MyoD expression in dorsolateral mesoderm explants from uniqjected control embryos (A and B) and dominant negative N-cadherin RNA-injected embryos (Cand D). A and C show staining with Hoechst 33258 to reveal the nuclei, and B and D show immunostaining for MyoD. In control explants (B) 30-60% of the nuclei are positive for MyoD, whereas MyoD staining is absent in dominant negative RNA-injected (600 pg of RNA) explants (D). Explants are equivalent to controls at stages 16-18. (Bars = 150 um.)

	FIG. 4. Cells expressing the dominant negative N-cadherin protein are transcriptionally active. (A) Stage 16 embryo immunostained with c-Myc antibody to reveal cells expressing high levels of the Myc-tagged mutant protein (black and dark gray cells). (B) Two blue Myc-positive cells (dark gray) showing autoradiographic grains clustered above the nuclei (arrows) after [3H]uridine labeling. Embryos received 600-pg injections of N-cadAE RNA. (Bar = 80 um in A and 10 Um in B).
	FIG. 5. Two Northern blots showing decreasing levels MyoD RNA in response to increasing amounts of injected dominant negative N-cadherin (N-cadAE) RNA. Conversely, Xwnt-8 activity rises with increasing doses of injected dominant negative RNA. RNA from two embryos was loaded per lane. Levels of elongation factor EF-la activity are similar across all the lanes.
	FIG. 6. Rescue of MyoD protein expression with full-length E-cadherin. Transverse sections of uninjected control at stage 16 (A) and E-cadherin-rescued embryo at stage 18 (B). Positively stained nuclei are present throughout the myotomes (m). The morphology and staining of the E-cadherin embryo are indistinguishable from controls. Two hundred picograms of N-cadAE RNA was coinjected with 1.5 ng of E-cadherin RNA (B). np, Neural plate; nt, neural tube; no, notochord. (Bar = 100 ,um.)
	FIG. 7. Northern blot showing rescue of MyoD message by injection of full-length E-cadherin RNA. Coinjection of E-cadherin RNA (1.5 ng) with the dominant negative N-cadAE RNA (200 pg) restores MyoD message to normal levels. Coinjection of the dominant negative RNA (200 pg) with N-cadherin RNA (1 ng) does not rescue as well as E-cadherin here, but other Northern blots have shown some increase in MyoD RNA. Xwnt-8 activity is similar in all lanes. The increases seen in Xwnt-8 activity in Fig. 5 do not occur here because the lowest dose of dominant negative RNA was used (200 pg). EF-la activity was the same in lanes 1-4 (data not shown).

References [+] :

Angres, Differential expression of two cadherins in Xenopus laevis. 1991, Pubmed, Xenbase