Fig. 2

Nob mutants encode entpd5, which is specifically expressed in osteoblasts. (A) Meiotic map of the nob^hu3718 locus. Recombinants per total number of mutants tested for each polymorphic marker are depicted in red, and markers used for mapping are in black. (B) Sequencing of nob^hu3718 revealed a Leu155>stop mutation. (C) Sequencing of nob^hu5310 revealed a Thr80>Ala mutation transversion. (D) Schematic representation of the predicted protein structures for Entpd5 and nob^hu3718. The asterisk indicates the position of the mutation in nob^hu5310. Gray bars, apyrase domains within Entpd5 (35). See also Fig. S1. (E) (Left and Center) Entpd5 and osterix are almost identically expressed in 3-dpf embryos, with osterix expression in the region of future teeth (arrows) being the single exception at this stage. (Right) Representative image of a 3-dpf entpd5:YFP transgenic embryo showing an expression pattern that matches endogenous entpd5 transcript distribution (Center). (F) Coexpression of entpd5:YFP and osterix:mCherry in the operculum of 3-dpf embryos. (G and H) entpd5 cDNA injection (CMV promoter) into nob^hu3718 mutants results in mosaic mineralization (G). Arrows point at cleithra in G; the arrowhead points to a nonmineralized osteoid in a partially mineralized cleithrum. Bar graphs in H represent percentages of rescued mutant embryos at 6 dpf (expressed as mean ± SEM, three independent experiments). (I) Alizarin red staining of a mutant nob embryo that had been injected with a cmv:dendra-t2a-entpd5 expression construct at the one-cell stage. The cleithrum is partially mineralized in the mutant (arrowhead); however, there are no dendra-positive cells immediately abutting the mineralized structure (arrow). (J) Representative example of an embryo partially rescued by endothelial-specific expression of entpd5 (kdr-like:entpd5).