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Fig. 2. pnr/tin-dependent and independent phases of Doc expression. (A-D) Expression of Doc proteins (green) in the Mef2-labeled mesoderm (red) in wild-type and tin³⁴⁶ mutant embryos. Images are merged optical sections of embryonic trunk mesoderm (lateral views), which occasionally include ventrolateral ectoderm and amnioserosa (as). (A,B) Doc expression initiates normally in the early dorsal mesoderm in tin mutants at stage 10. (C) At stage 11 strong Doc expression is seen in the dorsal-most areas of the mesoderm in wild-type embryos (arrowheads; arrow indicates somatic mesodermal Doc). (D) In tin mutant embryos, mesodermal Doc fades away during stage 11, except for longitudinal gut muscle founders (in dorsoposterior region of germ band) and the lateral somatic Doc clusters (arrow). (C',D') Detection of Pnr and Mef2 protein in wild-type and tin³⁴⁶ mutants at stage 11. tin mutants (D') do not express pnr in the mesoderm, as is seen in wild-type embryos (white arrowheads, C'). Ectodermal Pnr expression (partially present in these projections; green arrowheads) is not affected. (E-J) Detection of Doc (green) and Tin protein (red) in wild-type and pnr^VX6 mutant embryos. (E,F) At stage 10, normal Doc expression is seen in pnr mutants. (G,H) Doc protein fades away during stage 11 in pnr mutant embryos (arrowheads) and Tin levels also begin to decrease. (I) Expression of UAS-pnr only in the mesoderm via 2xPE-twi-GAL4 can restore high levels of Doc expression in the cardiogenic mesoderm of pnr mutants (arrowheads). (J) No significant rescue of cardiogenic Doc expression is observed if the Dpp-pathway is activated by mesodermal UAS-tkv^Q253D expression (arrowheads). (K,L) Ventral view of stage 10-11 wild-type embryo and (L) of 2xPE-twi-GAL4; UAS-pnr embryo stained with anti-Doc2+3 (green) and anti-phospho-Smad1/PMad antibodies (red). Ectopic expression of pnr in the mesoderm causes striped ectopic Doc expression in the mesoderm along with ectopic Mad phosphorylation.

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