Fig. 3. Antibodies against pSMAD1 provide evidence of BMP signaling throughout lens development. pSMAD1 immunostaining is in red and nucleic acids are stained green with TOTO-1. (A) At HH10, the nuclei of cells in the lens placode (LP) and the portion of the optic vesicle (OV) in contact with the lens placode stain with antibodies against pSMAD1. B. The nuclei of cells throughout the lens vesicle at HH18 (E3) stain for pSMAD1. Staining is weaker in the adjacent prospective corneal epithelium (CE). C. At HH22 (E4) strong nuclear pSMAD1 staining is localized to cells at the equator of the lens and at the margin of the optic cup, the prospective iris and ciliary epithelia. Nuclear pSMAD1 levels are barely detectable in the lens epithelium and in the more mature lens fiber cells. D. At HH33 (E7) pSMAD1 staining localizes to the nuclei of cells at the lens equator (EQ), the earliest lens fibers. More mature fiber cells deeper in the lens have low to undetectable levels of pSMAD1 staining. Inset: nuclei and cytoplasmic vesicles stained for pSMAD1 in elongating lens fiber cells. This section was not stained for nucleic acids. Scale bars: A-D 25 µm, inset 10 µm.