Fig. 4.

Fibulin-1 deficiency does not alter the proliferation or survival of osteoprogenitors or increase osteoclast activity in the frontal bone of Fbln1 nulls. A and B show BrdU incorporation into DNA by immunohistological analysis of frontal bone sections from wild-type and Fbln1 null E17.5 embryos using an antibody to BrdU. Note that the Fbln1 null shows a discontinuous frontal bone compared to wild-type. Graphs in C and D, show % BrdU positive nuclei determined in the osteogenic front (brackets in A and B) and in the dermis adjacent to the osteogenic front. BrdU positive nuclei were counted in multiple sections from three E17.5 skulls per genotype. Error bars represent standard deviation of the mean; significance was determined by Fisher’s Exact test. E and F shows TUNEL analysis of wild-type and Fbln1 null E17.5 frontal bone sections. G and H show histological TRAP staining for osteoclasts in the trabecular region of frontal bone in wild-type and Fbln1 null E17.5 skulls. Graph in I, shows % TUNEL positive nuclei in the osteogenic front and trabecular bone region of the frontal bone in wild-type (n = 3) and Fbln1 null (n = 3) E17.5 skulls. Error bars represent standard deviation of the mean and significance determined by Fisher Exact test. Graph in J, shows the number of TRAP positive osteoclasts determined from the trabecular bone region of the frontal bone in wild-type (n = 3) and Fbln1 null (n = 3) E17.5 skulls. Error bars represent standard deviation of the mean and significance determined by Students t-test. Graphs in K, L, and M, show levels of Ctsk, Dcstamp and Ocstamp mRNA determined by nanostring analysis of RNA isolated from E17.5 wild-type (WT) and Fbln1 null (KO) calvariae. The graphed values show mean relative amounts of mRNA based of WT (n = 3) and KO (n = 3). P values were calculated using NanoStriDE. D, dermis; FB, frontal bone; OF, osteogenic front; Oc, osteoclast; RBC, red blood cells; Cathepsin K, Ctsk.