Fig. 3.

Scleral ossicle development. (A) Alkaline phosphatase staining at HH 36.5 shows small, ellipsoidal condensations of variable size. Anterior is to the right. (B) Unstained embryo at HH 38 shows distinct trapezoidal condensations. (C) Ossicles continue to grow after the onset of mineralization and overlap one another extensively. The basic shape of the condensations is retained at post-hatching day 8 (P8). (D) Histology of condensation #12 at HH 36.5 shows a disorganized mass of cells among osteoid. (E) Ultrastructure of active osteoblasts at HH 36.5 shows a large nucleus, numerous mitochondria, cell–cell contacts, and distended rough endoplasmic reticulum, along with surrounding collagen fibers (asterisk). (F) Osteoblasts are polarized and arranged parallel to the long axis of the condensation at HH 36.5. Inset shows condensation under low magnification stained with toluidine blue. Asterisks indicate areas of active collagen secretion. (G) At HH 37, osteoblasts are separated by osteoid. (H) Ultrastructure of condensed cells, early HH 37. Intercellular spaces increase in size as more collagen fibers are laid down. (I) Whereas most cells move to the periphery of the condensation, some stellate-shaped cells (osteoid osteocytes, asterisk) remain surrounded by osteoid. (J) Osteoid osteocytes contain an extensive labyrinth of rough endoplasmic reticulum (asterisk) at HH 37. (K,L) The condensation expands in width via polarized secretion of osteoid. (M) The presumptive zone of overlap can be identified at HH 37.5. Condensation edges are marked by asterisks. Dashed line represents the conjunctival epithelium. (N) The zone of overlap expands at HH 38 as the condensations grow laterally towards one another via cell recruitment. Condensation edges are marked by asterisks. (O) Extensive overlap between two mineralized ossicles is present at post-hatching day 4 (P4). Ossicles outlined by black dashed lines. Scale bars: 1 μm (E,F,J), 2 μm (H,I), 20 μm (D,G,K,L), 50 μm (M,N), 100 μm (O); 1000 μm (A-C).