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. 2012 Sep 4;109(38):15354–15359. doi: 10.1073/pnas.1203000109

Fig. 1.

Fig. 1.

ADMP inhibits head formation through repression of head inducers. (A–I) Embryos were radially injected in the marginal zone at the four-cell stage with morpholino antisense oligo (MO): Control MO (80 ng/embryo), BMP4 and BMP7 MOs (mix of 25 ng each/embryo) or ADMP MO (80 ng/embryo). Embryos were stained by in situ hybridization for neural markers (mix of xAg1, xRx-1, Krox20, and HoxB9) at late neurula stage (A–C), for xRx-1 at early neurula stage (D–F; anterior view), or for Sizzled at gastrula stage (G–I; ventral view, dorsal up; Nieuwkoop and Faber stage 10.5). Number of embryos (n) was ≥18, derived from two independent experiments; for each class the frequency of the shown phenotype was >80%. (J–O) Similar in situ hybridization to A–I of embryos injected with ADMP mRNA (15 pg/embryo) or BMP2 mRNA (15 pg/embryo). Insets in J–L show the close-up of head/anterior region in ventral view. Number of embryos (n) was ≥20, derived from two independent experiments; for each class the frequency of the shown phenotype was >80%. (P) Cerberus (Cer) and DKK-1 expression was monitored by qPCR on dorsal and ventral halves of embryos injected with ADMP, BMP4/7, or control morpholino (CoMO). Expression levels are normalized to EF1a mRNA. (Q–T) Expression of head-organizer markers in anterior endodermal cells is inhibited by ADMP. Four-cell stage embryos were injected with ADMP MO (80 ng) or control MO (CoMO, 80 ng). At stage 11, embryos were fixed, removed of the blastocele roof to reveal the underlying endoderm (for Q and R), and processed for in situ hybridization for the indicated markers. Number of embryos (n) was ≥20, derived from two independent experiments; for each marker the frequency of the shown expression was >70%. For additional markers, refer to Fig. S1 G–I. (U, Left) Thirty-two–cell-stage embryos shown from animal pole. Embryos were injected with the Smad1-responsive reporter ID1-lux in dorsal “C1” blastomeres. Targeting to anterior endoderm was confirmed at gastrula by coinjected GFP mRNA (Lower Left picture). (Right) Luciferase was determined on extracts from stage 10.5 embryos coinjected with control or ADMP MO.