Fig. 1. BMP signaling regulates Pax gene expression in dorsal and intermediate domains. (A) Identification of virally infected cells by immunofluorescence using an antibody to the viral gag protein. (B-E) Electroporation constructs generate different levels of BMP signaling. Electroporations into the left side of the neural tube included a GFP expression plasmid to mark transfected cells. (B,C) Low levels of activated BMPR expression generate weak BMP target gene activation. (B) Expression of activated BMPR-Ib driven by the EGFP vector is barely detectable over endogenous expression of BMPR-Ib (stage 24). An alternate section (C) shows correspondingly weak activation of Msx protein expression, a target of BMP signaling. Inset shows GFP, which labels transfected cells, indicating that the construct is present. (D,E) High levels of activated BMPR expression generate strong BMP target gene activation. (D) Expression of activated BMPR-Ib driven by pMiW-III vector results in expression readily detected above the endogenous expression pattern (stage 24 embryo, detection reaction stopped prior to clear detection of endogenous BMPR-Ib expression). This results in a correspondingly robust MSX response in alternate sections (E). (F-H) Pax7 expression is activated by BMP signaling. (F) Wild-type Pax7 expression in a stage 23 embryo. Ectopic Pax7 expression (arrowheads) is apparent in virally infected (G, stage 24) and electroporated (H, stage 24) embryos. The unusual morphology in H was caused by apoptosis (see Materials and Methods). (I-M) Expression of Pax6 is regulated by BMP signaling. The vertical bar denotes the normal region of high-level Pax6 expression in the intermediate region of the neural tube. (I) Wild-type Pax6 expression in a stage 24 sample. (J) Pax6 expression is repressed by high levels of BMP signaling (arrowheads; inset shows that Pax6 repression is limited to transfected cells). High signaling was generated by electroporation of pMiWIII-activated BMPR-Ib; sample is stage 24. (K,L) High level expression of Pax6 in intermediate cells is downregulated by moderate levels of BMP signaling. Moderate signaling was generated by electroporation of pCAGGS-activated BMPR-Ia; sample is stage 24, insets show correlation of alterations with transfected cells. (M) Ventral Pax6 expression is upregulated in response to BMP signaling (arrowhead; viral infection, stage 23).
