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Abstract
The homeodomain factors Msx1 and Msx2 are expressed in essentially identical patterns in the epidermis and neural crest of Xenopus embryos during neurula stages. Disruption of Msx1 and Msx2 RNA splicing with antisense morpholino oligonucleotides shows that both factors are also required for expression of the neural crest gene Slug. Loss of Msx1 can be compensated by overexpression of Msx2 and vice versa. Loss of Msx factors also leads to alterations in the expression boundaries for neural and epidermal genes, but does not prevent or reduce expression of epidermal keratin in ventrolateral ectoderm, nor is there a detectable effect on dorsal mesodermal marker gene expression. These results indicate that Msx1 and Msx2 are both essential for neural crest development, but that the two genes have the same function in this tissue. If Msx genes have important functions in epidermis or axial mesoderm induction, these functions must be shared with other regulatory proteins.
Fig. 1. Expression of Msx1 and Msx2 in Xenopus embryos. (A) Glyoxal
RNA gel blots of 0.75 μg per lane of total RNA isolated from different
embryonic stages probed with Msx1 and Msx2. Nieuwkoop-Faber stages
are as indicated. Both transcripts appear first at early gastrula, level off at
early neurula and are maintained at roughly constant amounts through
mid tailbud stages. Ethidium bromide staining of the 18S ribosomal band
is shown to control for equal RNA loading. (B) Whole mount in situ
hybridization to late gastrula (St. 12.5) and early (St. 14) and mid-neurula
(St. 16) embryos. The expression patterns of Msx1 and Msx2 are very
similar. Expression of Slug is shown for comparison. Dorsal views, with
anterior towards the top of the figure.
Fig. 2. Animal cap assay showing dependence of Msx and other neural and neural plate
gene expression on canonical Wnt and modulated BMP signaling.
Ectoderm was isolated from mid/late blastula stage embryos
(St. 7-8) injected at the one-cell stage with 300 pg of
Wnt3a RNA and variable amounts of chordin RNA, ranging from zero
to 5 ng, cultured to early neurula (St. 14) and processed for Northern
blot analysis using methylmercury hydroxide agarose gel
electrophoresis. Note that compared to the other NC genes
AP2a, Slug and Sox9, Msx genes are up-regulated significantly by
Wnt3a alone and also are not fully extinguished by the highest dose of
chordin, which activates neural plate genes such as Sox2. UI, uninjected
animal caps; W, whole embryo. Ethidium bromide staining of 18S
ribosomal RNA is shown to control for equal RNA loading.
Fig. 3. Effectiveness and
specificity of antisense
morpholino oligonucleotides
(MOs). Fertilized
eggs were injected with
30 ng of Msx1 (M1) or 10
ng Msx2 (M2) splice-targeted
MOs, which were
then cultured to early neurula (St. 14) along with uninjected embryos (UI)
and processed for Northern blot analysis with Msx1 and Msx2 probes.
Msx1 and Msx2 transcripts were reduced to a baseline of approximately
20% of control levels, as measured by densitometry of X-ray films,
respectively. Neither MO affected the non-homologous RNA, demonstrating
specificity. Ethidium bromide staining of 18S ribosomal RNA is
shown to control for equal RNA loading.
Fig. 4. Loss of Msx function. Two-cell
stage embryos were injected at a single
site in one blastomere with 15 ng M1, 5
ng M2, along with 250 pg β-galactosidase
RNA as a lineage tracer and cultured to
early neurula (St. 13), then fixed, stained
with X-gal and processed for whole mount
in situ hybridization with probes for the
neural crest marker gene Slug. Dorsal
views, with the hybridization signals represented
by purple color and the lineage
tracer by light blue. The injected side is on
the left in all cases and anterior is towards
the top of the Figure. Both M1 and M2
greatly reduced the expression of Slug.
The Slug expression domains were also
shifted laterally in some cases. Near-normal
levels of Slug expression were rescued
in a majority of embryos by coinjection
of Msx1 mRNA (5 pg, with 5 ng
M2) and Msx2 RNA (10 pg, with 15 ng
M1). An uninjected embryo is shown for
comparison (UI).
Fig. 5. Effect of combined Msx1/Msx2 knockdown on the dorsal/
ventral axis. Embryos were injected into one blastomere at the two-cell
stage with a mixture of 15 ng M1 and 5 ng M2 (A,B) or twice this dose at
the one-cell stage (C). Fluorescein-labeled standard control MO was used
as a tracer in all injections except the one which was used for hybridization
to Xnot, in which case β-galactosidase was used as the tracer. (A)
Expression at stage 14 of the neural plate marker Sox2 was expanded
laterally, while the epidermal boundary, indicated by XK81, retreated to
a corresponding degree. The axial midline is indicated by the dashed
vertical line. (B) Notochord expression at stage 14 of chordin and Xnot1
were not noticeably affected by Msx knockdown. The lateral expression
domains of Xnot1 were largely eliminated, however. Uninjected controls
are shown for comparison (UI). (C)Embryos injected at the one-cell stage
into the ventral marginal zone with 30 ng M1 + 10 ng M2 were cultured
to stage 10.5, fixed and probed for chordin expression. Vegetal views,
with dorsal towards the top of the figure. No significant difference was
observed between injected and uninjected controls (UI).
