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Fig. 2

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ZDB-IMAGE-130205-12
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Figures for Huitema et al., 2012
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Fig. 2 Nob mutants encode entpd5, which is specifically expressed in osteoblasts. (A) Meiotic map of the nobhu3718 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 nobhu3718 revealed a Leu155>stop mutation. (C) Sequencing of nobhu5310 revealed a Thr80>Ala mutation transversion. (D) Schematic representation of the predicted protein structures for Entpd5 and nobhu3718. The asterisk indicates the position of the mutation in nobhu5310. 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 nobhu3718 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).

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