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Fig. 7
shha:GFP-expressing basal epidermal cells extend cellular protrusions through incompletely assembled basement membrane to contact Runx2+ progenitor osteoblasts. (A-P) GFP (white), laminin (magenta) and Runx2 (green) antibody stained fin sections from 96 hpa shha:GFP fish. All images are 1 airy unit (∼1 µm) single optical confocal sections, except D, which is a structured illumination microscopy (SIM) image representing an ∼100 nm section. (A-D) Longitudinal section showing the distal regenerate where the shha-expressing basal epidermis is split into two clusters and Hh/Smo signaling is activate in both pObs and basal epidermis. (C) An overlay showing Runx2 staining together with A and B. (D) A high-magnification SIM image of the boxed area in C. (E-G) A proximal field from the same longitudinal section shown in A-D. (H-P) Transverse sections representing three positions along the proximal-distal axis from a single regenerating ray. (H-J) An extreme distal section beyond the distal extent of Runx2+ pOb pools. (K-M) Section from a position where shha:GFP-expressing basal epidermis is split into two clusters on each side of the regenerating fin. (N-P) A further proximal section where shha expression has initiated in epidermal cells but prior to their division into split clusters. The dashed boxes in I, L and O mark regions shown at higher magnification in J, M and P, respectively. White arrows indicate cellular protrusions from shha:GFP+ cells that contact or enshroud Runx2+ osteoblasts. White asterisks mark gaps in the basal lamina. Magenta arrows show a continuous laminin-containing basement membrane that physically separates the basal epidermis from Runx2+ pObs. Scale bars: 5 µm in D,J,M,P; 50 µm in C,G,I,L,O.