Steinbach OC et al. (1998), Temporal restriction of MyoD induction and auto...

XB-ART-14138

Dev Biol 1998 Oct 15;2022:280-92. doi: 10.1006/dbio.1998.8993.

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Temporal restriction of MyoD induction and autocatalysis during Xenopus mesoderm formation.

Steinbach OC , Ulshöfer A , Authaler A , Rupp RA .

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In Xenopus, the activation of the myogenic determination factors MyoD and Myf-5 in the muscle-forming region of the embryo occurs in response to mesoderm-inducing factors (MIFs). Different members of the FGF, TGF-beta, and Wnt protein families have been implicated in this process, but how MIFs induce the myogenic regulators is not known. For MyoD, the induction process may serve to locally stabilize a transient burst of ubiquitous transcription at the midblastula transition, possibly by triggering MyoD's autocatalytic loop. Here we have sought to distinguish separate activating functions during MyoD induction by analyzing when MyoD responds to different MIF signaling or to MyoD autoactivation. We show that MyoD induction depends on the developmental age of the induced cells, rather than on the type or time point of inducer application. At the permissive time, de novo MyoD induction by Activin requires less than 90 min, arguing for an immediate response, rather than a series of inductive events. MyoD autoactivation is direct, but subject to the same temporal restriction as MyoD induction by MIF signaling. Further evidence implicating MyoD autocatalysis as an essential component of the induction process comes from the observation that both autocatalysis and induction of MyoD are selectively repressed by a dominant-negative MyoD mutant. In summary, our observations let us conclude that MyoD's expression domain in the embryo results from an interplay of timed changes in cellular competence, pleiotropic signaling pathways, and autocatalysis.

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Species referenced: Xenopus
Genes referenced: mif myod1

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	FIG. 1. Developmental time scale and flow diagram of experimental manipulations. Developmental time is given as hours post fertilization (hpf). NF, Nieuwkoop and Faber stages. The mesodermal competence period, represented by the black bar, lasts from about 3.5 to 11.25 hpf. Where applicable, embryos were injected into the animal hemisphere at the two-cell stage with capped, synthetic transcripts. The application regimens for soluble reagents are described in the respective figure legends. Animal caps were routinely cut shortly after the midblastula transition (MBT), i.e., between 6.75 and 7.5 hpf and cultured until successively later time points (F), at which time they were lysed and subjected to quantitative RT/PCR analysis.
	FIG. 2. The onset of MyoD induction. Columns show the average increase of XMyoD steady-state mRNA levels as a function of time. RNA samples were from control embryos or from animal cap explants injected either with synthetic transcripts encoding XmyoD (400 pg) or mouse MyoD (200 pg) or with a combination of bFGF (50 pg) and XWnt-8 (10 pg) transcripts. In this series of experiments, Activin induction was triggered by application of a 1:4 dilution of Activin-containing tissue culture supernatant at 7 hpf. For all five conditions tested, myoD induction occurs between 9.75 and 10.5 hpf. The relative XMyoD induction is calculated as mRNA increase of induced over control explants of the same developmental age, after normalization to H4 mRNA as internal standard (see Materials and Methods for details). (n 5 3–5 independent experiments; error bars, SD). Note the broken x-axis and the different y-axis scale for the 24 hpf time point.
	FIG. 3. MyoD activation is a fast response to Activin, but requires protein synthesis. (a) Addition of soluble Activin to animal caps at 9.75 hpf leads to an increase in XMyoD mRNA levels within 45 to 90 min. Explants, which were previously incubated with cycloheximide (CHX) from 9.25 to 9.75 hpf, show no increase in XMyoD mRNA. (b) mMyoD protein, translated from microinjected RNA, promotes autoactivation in the absence of protein synthesis (CHX treatment from 9.25 to 9.75 hpf). (c) MyoD autoactivation is direct. In this experiment, autocatalysis is triggered by the dexamethasone- inducible MD-GR protein, which has been preexpressed by mRNA injection at the two-cell stage (100 pg/embryo). After protein synthesis has been blocked by CHX treatment as in a, MD-GR is activated through dexamethasone application at 9.75 hpf. Note that MD-GR is completely inactive without dexamethasone (n 5 4 for each panel).
	FIG. 4. Dominant-negative XWnt-8 blocks MyoD activation by bFGF/XWnt-8, but not by Activin or autocatalysis. RT/PCR analysis of relative XMyoD mRNA levels in animal caps, which were preloaded with synthetic transcripts as indicated below the panels (numbers refer to the injected RNA dose in pg/embryo). dnXWnt-8, dominant-negative XWnt-8 (Hoppler et al., 1996). For the experiment in column 3 (*), dnXWnt-8 RNA was injected at the two-cell stage, followed by coinjection of bFGF/XWnt-8 RNAs at the eight-cell stage, to provide a head start for the intereference mutant. Panels show MyoD mRNA levels at: a, 12 hpf (i.e., shortly after activation); or b, 24 hpf (tail bud stage; during muscle differentiation) (n 5 3).
	FIG. 5. MT6-MyoD(bHLH)-enR blocks MyoD induction in animal caps. RT/PCR analysis of XMyoD (a– c) or XMyf-5 (d) mRNA levels in animal caps, preloaded with synthetic transcripts as indicated below the panels (numbers refer to the injected RNA dose in pg/embryo). Panels show: a, MT6-MyoD(bHLH)-enR inhibits MyoD autoactivation; b, MT6-MyoD(bHLH)-enR interferes with Activin-induction of MyoD; c, Activin induction of MyoD is rescued by coinjection of MT6-MyoD(bHLH)-enR and wild-type MyoD protein; d, Activin induction of XMyf-5 is unaffected by MT6-MyoD(bHLH)-enR; e, Synergism of autocatalysis and Activin induction. Activin protein was applied as a 1:25 dilution of Activin containing tissue culture medium (Sokol et al., 1990); mMyoD protein was provided by RNA injection (200 pg/embryo) (n 5 4).
	FIG. 6. MT6-MyoD(bHLH)-enR inhibits MyoD induction in vivo. RNA in situ hybridization with an antisense XMyoDb probe at midgastrula (NF 11), shortly after MyoD induction. (a) Uninjected control embryos. Embryos were injected equatorially into one cell at the two-cell stage with 100 pg lacZ mRNA as lineage tracer, together with transcripts encoding the following enR-fusion proteins: (b) MT6-MyoD(bHLH)-enR (50 pg/embryo); (c) MT6-E12basic(bHLH)-enR (50 pg/embryo); (d) NLSMT6-enR (200 pg/embryo). Asterisks mark the location of b-galactosidase-positive cells, indicating the injected region. Inserts show representative close-ups of MyoD and b-galactosidase staining.