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???displayArticle.abstract???
Injection of Xenopus myoD mRNA into Xenopus embryos leads to only a modest activation of myogenic markers. In contrast, we show that injected mouse myoD mRNA leads to a potent activation. We postulate that XMyoD is under negative control in frog embryos, but because of slight sequence differences, mouse MyoD fails to see the negative signal. Whereas mMyoD is constitutively nuclear, XMyoD is largely cytoplasmic except in a region of the embryo that includes the location where mesoderm induction occurs; there, it is nuclear. At MBT, endogenous XmyoD mRNA is expressed ubiquitously in the frog embryo. Our results suggest that this expression would lead to cytoplasmic XMyoD protein. Among other events, muscle induction might remove this negative regulation, allow MyoD to enter the nucleus, and establish an autoregulatory loop that could commit cells to myogenesis.
???displayArticle.pubmedLink???
7926732
???displayArticle.link???Genes Dev
Figure 1. (a) Trans-activation of muscle-specific genes by frog
and mouse MyoD proteins in animal cap explants. Both cells of
two-cell embryos were injected near the animal pole with various
amounts of capped, nonpolyadenylated synthetic RNAs
encoding MyoD proteins. Animal cap explants were dissected
from control and injected embryos after MBT and cultured in
neutral saline until sibling embryos reached late neurula stages.
Total cellular RNA was then prepared, reverse-transcribed, and
analyzed by multiplex PCR for the presence of EFla and XmyoD
RNAs, and by single-primer pair PCR for cardiac actin RNA.
After amplification, all multiplex PCR samples were subjected
to a HindIII digest, which results exclusively in cleavage of the
PCR product derived from the injected XmyoD(H3) RNA. This
engineered variant behaves identically to wild-type XMyoD,
but its levels of expression can be distinguished from endogenous
MyoD because of the presence of the HindIII site (see
Materials and methods). PCR fragments generated from EF-la
(EFI-a) or endogenous XmyoD RNAs contain no HindlII restriction
site. Injected myoD RNAs are not detected by primers specific
for XmyoD-coding regions. (Lane 1) Mock RT/PCR, no
template; (lane 2) uninjected control embryos. Embryos were
injected with the given amount of RNAs encoding the following
proteins: (lane 3) mMyoD-VP16 (0.03 ng); (lane 4) coinjection of
mMyoD-VP16 (0.03 ng) and XMyoDb(H3) (1.5 ng); (lane 5)
XMyoDb(H3) (1.5 ng); (lane 6) coinjection of mMyoD (0.75 ng)
and XMyoDb(H3) (1.5 ng); (lane 7) mMyoD (0.75 ng); (lane 8)
XMyoDb(H3) (3 ng). (b) Injected embryos contain similar levels
of overexpressed frog and mouse MyoD proteins. Embryos were
injected with 2 ng of synthetic RNAs lacking the SV40 poly(A)
signal, which encode MTXmyoDb and MTmmyoD, respectively.
At late gastrula, whole cell protein extracts were prepared
from animal halves of embryos and analyzed for exogenous
protein in Western blots with the Myc tag-specific mAB
9El0. (Lane 1) In vitro-translated MTXMyoDb protein (10-sec
exposure); protein extracts in the following lanes (90-see exposure)
represent MTXmyoDb RNA-injected embryos (lane 2) uninjected embryos (lane 3), and MTrnmyoD RNA-injected embryos (lane
4). Protein from one embryo half was loaded per lane (vegetal halves contained minor amounts of overexpressed proteins at equal
abundance; data not shown). Coomassie staining of these extracts on a second gel run in parallel reveals equal total protein contents
of the different extracts.
Figure 2. The MyoD-positive feedback loop does not operate at
MBT. At the late blastula stage (NF9), animal caps were dissected
from control embryos (A) or embryos that were injected
at the two-cell stage with 0.01 ng of RNA encoding mMyoD-
(bHLH~VP16 protein and lysed immediately for RNA purification
(B). RT/PCR analysis was performed in parallel with the
primer pairs detecting either the injected mmyoD(bHLH)-VP16
RNA {lanes 1) or endogenous mRNAs (lanes 2-4). (Lanes 2)Multiplex
PCR for EF-Ia and XmyoDa and XmyoDb RNAs; (lanes 3)
cardiac cx-actin RNA; (lanes 4) GS17 RNA. The signals for
XMyoDa and XMyoDb represent general MBT-specific transcription
of these genes.
Figure 3. High levels of endogenous XmyoD mRNAs are not
sufficient to maintain expression of XmyoD genes through autoactivation
in uninduced animal cap explants. At the late blastula
stage, animal caps were dissected from control embryos
{A,BI or embryos injected with [0.01 ngl RNA encoding
mMyoD(bHLH~VP16 protein (C-E), cultured in neutral saline
(A,C-E) or in the presence of activin A (0.5 ng/ml; B) until
siblings had reached neural fold (NF 17; A-C1, tailbud (NF30; DI,
or hatching stages {NF36; E). RNA was prepared from each stage
and analyzed by RT/PCR with primers specific for the injected
mmyoD(bHLH)-VP16 RNA (lanes 11 endogenous EF-la and
XmyoDa and XmyoDb RNAs (lanes 2; multiplex PCR), and
cardiac a-actin RNA (lanes 3) {see Materials and methodsl.
Figure 4. Intracellular localization of
overexpressed MyoD proteins. Capped,
nonpolyadenylated synthetic RNAs (2 ngl
were injected into the animal pole region
of two-cell embryos. At late gastrula
stages (NF11 1/2--12), embryos were fixed
and subjected to whole-mount immunocytochemistry
using either the Myc tag-specific
mAb 9El0 (all pictures except n) or
the XMyoD-specific mAb D7F2 (n only;
Hopwood et al. 1992). Bound primary antibodies
were detected with alkaline phosphatase-
conjugated goat anti-mouse mAb
and NBT/BCIP staining. RNAs encoded
the following proteins: (a-c} Myc-tagged
XMyoDa; (d-t] Myc-tagged XMyoDb; {g-i)
Myc-tagged mMyoD; (Jl uninjected control
embryos; (k,1} Myc epitope tag only.
(n) Animal pole region from an embryo injected
with non-Myc-tagged XmyoDb
RNA and stained with XMyoD-specific
mAb. (a,d, gl Lateral view of injected embryos
with animal pole at top and yolk
plug at the bottom. High magnification
view of blastocoel roof explants: (b,e,h,k)
epithelial cell layer; (n) sensorial cell layers.
(c,f,i,1) High magnification view of
marginal zone explants. Note the staining
of nuclei in deep layer cells of embryos
injected with RNAs encoding Myc-tagged
XMyoD proteins (c,f, arrows). (m) Schematic
overview of gastrula regions displaying
differential intracellular localization of
XMyoD. Horizontal lines above the yolk
plug indicate the approximate positions of
involuting marginal zone (IMZ) and noninvoluting
marginal zone (NIMZ). The
shading represents presumptive mesoderm
in the deep layers of the IMZ (redrawn
after Keller 1991).
Figure 5. Transition zone between nuclear and cytoplasmic
MyoD. Myc-tagged XmyoD RNA was injected and assayed as in
Fig. 4. The photo shows an area extending from the animal pole
(ap) to the marginal zone (mz) of a stained, dissected gastrula
stage whole mount (enlargement of Fig. 4o).
Figure 6. A threshold for cytoplasmic retention of XMyoD protein.
Capped RNAs, which either contain or lack the SV40 polyadenylation
signal, were transcribed from the vector pCS2 +
MTXMyoDb in vitro and injected into the animal hemisphere
of two-cell embryos. At gastrula stages, embryos were fixed and
subjected to immunocytochemistry with mAb 9El0 for detection
of the overexpressed Myc-tagged XMyoDb protein. The
right embryo was injected with 2 ng of RNA lacking the SV40
polyadenylation signal; the embryo in the middle represents
and uniniected control; the left embryo was injected with 0.5 ng
of RNA containing the SV40 polyadenylation signal. Embryos
were stained in parallel for 30 min. {Inset) High magnification
view of the animal pole region of the embryo that was injected
with RNA containing the SV40 polyadenylation signal. Exogenous
protein is present in both cytoplasm and nuclei.
Figure 7. Ectopic activation of myogenic
marker genes by overexpression of MyoD
proteins. Embryos were injected with 0.25
ng of RNAs that contain the SV40 polyadenylation
signal and encode either
mMyoD (b-d,g) or XMyoDb (e--f,h) protein.
Expression of muscle-specific proteins was
analyzed at stage NF34 (heartbeat; Nieuwkoop
and Faber 1967) by immunocytochemistry.
All embryos, except those
shown in b, were cleared in 1:2 BABB. {a)
Uninjected sibling embryos, which were
stained for MyHC (bottom) or 12/101
epitope (top). Staining is restricted to the
somitic mesoderm and, in the case of
MyHC, also to the heart. (b) Injected with
mmyoD; stained with anti-myosin heavy
chain (MyHC). (c) Injected with mmyoD;
stained with anti-MyHC. (d) Injected with
rnmyoD; stained with mAb 12/101. {e) Injected
with XmyoD; stained with anti-
MyHC. (f) Injected with XmyoD; stained
with mAb 12/101. Every embryo shows at
least some, and in many cases, extensive,
expression of MyHC or 12/101 outside the
somitic mesoderm, g and h are blow-ups of
injected embryos shown in d and f. Both
show 12/101 epitope-positive cells in ectopic
positions, whose elongated morphology
resembles that of differentiated myocytes
in the somitic mesoderm. In general,
myotomal staining became visible at least
twice as fast as staining in ectopic places.
