Msx1 and Msx2 have shared essential functions in neural crest but may be dispensable in epidermis and axis formation in Xenopus.
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.
PubMed ID: 16586351
Article link: Int J Dev Biol.
Grant support: Intramural NIH HHS
Genes referenced: chrd msx1 msx2 not snai2 sox2 sox9 tfap2a wnt3a xk81a1
Morpholinos referenced: msx1 MO2 msx2 MO1 msx2 MO2
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|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).|