Blistering and hemorrhages in homozygous my embryos and mice. (A) Exencephaly (denoted by arrowheads) in E13.5 my mutant. (B and C) Earliest visible hemorrhages in the choroid plexuses. Hemorrhage in the rostral (B) and caudal (C) choroid plexus of two representative E13.5 embryos. (Insets) Locations of the defects). (D) Blood-filled blisters on the surface of an E13.5 embryo, with an internal hemorrhage (arrowhead) in the caudal choroid plexus. (E) Hemorrhage on the E15.5 palate. (Inset) Lower magnification view of head. (F) Two hemorrhages on the head of an E15.5 embryo; the outer hemorrhage (arrowhead) appears to be nearly resolved, whereas the inner, subdermal one (arrow) appears to be recently formed. (G) Hemorrhage on the head of a newborn animal. (H) Adult head, showing reduced or absent eyes (arrows), reduced ear (arrowhead), and discoloration of fur (white asterisk). (I) Adult my mutant with discolored fur on the flank (the arrowhead denotes the border of discoloration).

Limb and organ defects in my embryos and mice. (A–E) Limb Defects. Shown are a fluid-filled blister on an E14.5 hind limb (A); a blood-filled blister encompassing distal structures of an E14.5 hind limb (B); a hemorrhage associated with malformation and dorsal flexure of the newborn hind limb (C); a malformed adult limb, showing reversal of flexure and soft tissue syndactyly (D); and a severely malformed adult limb (E). (F) Adult with unpigmented fur (arrow), typical of neural crest defects. (G) Section of an E15.5 heart, showing failure of atrial septation (arrow) and a defect in ventricular valves (arrowhead). Note that both ventricles drain into a single large chamber. (H) Newborn lungs, showing failure of proper lung septation. The arrows point to lobe fusion. (I) Section of newborn my lung, showing normal morphology. The arrows denote regions where the lung lobes have fused, and the arrowhead denotes a region with a limited adhesion between the two lobes.

my/Frem2 shows dynamic spatial and temporal gene expression. (A) Frem2 RNA expression at E9.5, including in the roofplate (RP) of the diencephalon, the midbrain/hindbrain boundary (MB/HB), and the branchial arch ectoderm (BAE). (B) Frem2 expression at E10.5, including the nephric duct (ND), precursor to the kidney, the ventral spinal cord (SC), and notochord (NC). (C) Dynamic expression of Frem2 transcript in the eye, including the lens vesicle (LV) and border of the retinal pigmented epithelium (RPE) at E10.5, the optic nerve (ON) at E11.5, and the conjunctival epithelium (CE) at E13.5. (D–G) Expression at E11.5. Shown are expression in the head in the rostral diencephalon (D) and future choroid plexuses (CP) of the telencephalon, the forming nasal epithelium (NE), and future palate (P) (D); expression in the trunk in the myotome (M), esophagus (E), the site where the heart is attached to the trunk (H), and bronchi in advance of the onset of branching morphogenesis that will generate the lung (L) (the arrows denote regions of expression in the limb ectoderm and mesenchyme; note that expression in the ventral spinal cord has been extinguished (E); in more caudal sections, expression is present in the developing kidneys (F); and expression is also present in other endodermal derivatives, such as the stomach (G).

Vasculature defects at sites of hemorrhage in my embryos and developmental gene and protein expression at sites of choroid plexus hemorrhages in E12.5 embryos. (A) Patterning of the choroid plexus is normal in my mutants, as indicated by appropriate BMP-7 RNA expression in neural cells adjacent to a hemorrhage (H) (magnification, ×10). (B) Expression of PECAM in a wild-type choroid plexus shows close association of the vasculature with the elaborating neural tissue (magnification, ×10). PECAM expression at hemorrhages (H) in the rostral (C) and caudal (D) choroid plexus of my embryos (the arrow denotes disorganized endothelial cells in the area of hemorrhage, and the arrowhead shows a normal vascular structure at the hemorrhage edge (magnification, ×20). (E–G) Markers of angiogenic signaling and vascular structure are expressed normally in regions of hemorrhages in my embryos. Flk-1, a VEGF receptor, remains expressed by endothelial cells that have failed to form proper vascular structures in the area of a hemorrhage (H) (magnification, ×20). The arrow indicates disorganized endothelial cells in close proximity to normal blood vessels (arrowhead) (E). The expression of Integrin-αV at the surface of neural and endothelial cells in the choroid plexus is shown in close proximity to a hemorrhage (H) (magnification, ×20). The arrowhead indicates a region of normal blood vessels. Cytoplasmic localization of this protein in some neural cells of the choroid plexus occurs in both mutant and wild-type samples (F). Laminin is deposited in the basement membrane of both neural and vascular cells at the choroid plexus. This expression is retained in areas where hemorrhaging (H) occurs (magnification, ×20). The arrow indicates the region of disrupted ECM at a hemorrhage, and the arrowhead indicates nearby blood vessels that are unaffected (G).