Fig. 2

Bmp Expression and Signaling Is Dependent on Acsl4a

(A and B) Whole-mount mRNA in situ hybridization of bmp transcripts in control embryos and embryos injected with acls4a MO (500 fmol) or acsl4a mRNA (1.25 ng). In acls4a morphants, the ventral-to-dorsal gradient of (A) bmp2b and (B) bmp7a is attenuated (in 87% and 78%, respectively) while acls4a overexpression enhances bmp expression (in 77% and 75%, respectively). n = 4; 88–119 embryos. Animal pole view, dorsal to the right (30% epiboly).

(C) Western blot of C-terminally phosphorylated Smad1/5/8 (pSmad^C-Term) and total Smad1/5/8 (Smad^Total) from shield-stage control embryos and embryos injected with 750 fmol acsl4a MO (left). Quantification of three (sphere) or five (shield) experiments (right). Data are represented as mean ± SE. p < 0.05 p < 0.001; one sample t test with Bonferroni correction (sphere stage MO = 500 fmol, shield stage MO = 500–750 fmol).

(D) Whole-mount immunostain against pSmad^C-Term at 80% epiboly. Staining is reduced in morphants (500 fmol) when compared to control siblings (98%; n = 5; 155 embryos). Animal pole view, dorsal to the right (80% epiboly). Inset is a 2× magnification of the ventral-most region.

(E) Acsl4a is needed in the Bmp ligand-receiving cell for Smad activation. (Left) Schematic of transplantation experiment. (Right) pSmad^C-Term immunofluorescence in transplanted cells in ventral equatorial regions of host embryos (80% epiboly). Top panel: wild-type cells transplanted into wild-type embryos have normal pSmad^C-Term staining (26/26 embryos, three independent experiments). Middle panel: Morphant cells transplanted into wild-type embryos lack pSmad^C-Term staining, whereas adjacent cells have normal pSmad^C-Term staining (16/16 embryos, n = 3). Bottom panel: Wild-type cells transplanted into morphants have pSmad^C-Term staining, whereas adjacent morphant cells lack pSmad^C-Term signals (17/17 embryos, n = 3). Wild-type cells transplanted into morphants show reduced pSmad^C-Term staining compared to wild-type cells transplanted into wild-type hosts (compare bottom and top panels). FL, fluorescein dextran.

See also Figure S3.