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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
.
???displayArticle.abstract??? 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.
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).
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