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Figure 3: In vivo functional assays in Xenopus embryos suggest that BAMS-associated mutations behave as gain-of-function alleles. (a) Expression of smchd1 relative to 18S rRNA by qPCR. Data represent means ± s.d. of triplicates. (b) In late tailbud stages, smchd1 expression is restricted to the head region and the neural tube. (c) To target the head structures, dorsal–animal blastomeres in eight-cell-stage Xenopus embryos were injected with synthesized mRNA (120 pg for all panels except k). These cells are fated to give rise to head structures, as shown by dextran lineage tracing. D, dorsal; V, ventral. (d–f) Representative stage 45 tadpoles that are uninjected (d) or injected with SMCHD1WT (e) or SMCHD1A134S (f) mRNA. Those injected with SMCHD1A134S mRNA display craniofacial anomalies and smaller eyes in comparison to control tadpoles and those injected with SMCHD1WT mRNA. Scale bar, 0.3 mm. All images were acquired at the same magnification. (g) Immunoblot of stage 12 embryonic extracts from control and injected embryos showing expression of exogenous human SMCHD1. (h) Eye diameter is significantly reduced in embryos overexpressing BAMS-associated mutants (blue) relative to siblings overexpressing wild-type SMCHD1 (black) or embryos overexpressing an FSHD2-associated mutant (open circles). (i,j) In situ hybridization for rx2a, six1 and twist1, demarcating the eyes, placodes and neural crest, respectively, in embryos injected with SMCHD1WT (i) or SMCHD1A134S (j) mRNA. Images are representative of 9 of 10, 7 of 10, and 10 of 10 embryos for each probe. The dotted lines outline nasal placodes (middle) and the eye (right). Numbers label streams of migrating cranial neural crest. Scale bars, 0.2 mm (same magnification for each comparison of i to j). (k) Measurements of eye diameter for Xenopus embryos injected with 0.5 or 1 ng of mRNA encoding wild-type SMCHD1 or a BAMS-associated mutant show that SMCHD1 overexpression causes dose-dependent craniofacial anomalies. Biological variation between clutches of tadpoles is seen in the data presented in h and k. For both h and k, n indicates the number of embryos analyzed, data are shown as means ± s.d. and P values were calculated by Kruskal–Wallis test followed by Dunn's post test. |
