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Fig. 4. Overexpression of ADMP2. (A) Northern blot of RNA from stage 32 sibling embryos of the ones shown in panels C–F and H–J. The figure shows the results of hybridizations for globin, muscle actin (m.actin) and EF1α (loading control). The expression of globin was increased by overexpression of ADMP2, ADMP or BMP7 RNA (lanes 2, 4 and 6 compared to lane 1), while that of m.actin was virtually eliminated by the same treatment (lane 2, 4 and 6 compared to lane 1). The level of m.actin expression in the BMP7-injected embryos was rescued to normal levels by co-injection of tBR RNA (lane 7 compared to lane 1). However, in the ADMP2 or ADMP-injected embryos, co-injection of tBR RNA failed to rescue m.actin expression (lanes 3 and 5 compared to lane 1). (B) The phenotype and globin expression by in situ hybridization in ADMP2-overexpressed embryos. The expression of a blood marker, globin, is normally detectable in the ventral-most region of the tailbud stage embryo at stage 32 (white arrowheads in top embryo). Overexpression of ADMP2 caused a typical DAI 0 phenotype (two bottom embryos). The expression of globin was restricted in the leading edge mesoderm of these embryos (white arrowheads) at the equivalent of stage 32. (C–J) Rescue experiments by co-injection of tBR RNA. Overexpression of either ADMP2, ADMP or BMP7 RNA caused a similar DAI 0 phenotype (C–E compared to B). However, only the phenotype caused by overexpression of BMP7 was rescued by co-injection of tBR RNA (G–I), suggesting that ADMP2 and ADMP do not bind the BMP receptor. (G) The phenotype of tBR RNA-injected embryos. All the embryos shown are at stage 32. |
