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Fig. 3
Mmp2 Inhibition Causes Abnormal HSPC Accumulation in the VDA
(A) In vivo imaging for Runx1:GFP confirmed altered HSPC development in the VDA with Prinomastat (20 ?M) and ARP (10 ?M) (12?36 hpf; n ? 20 embryos/condition).
(B) In vivo imaging for Runx1+144:GFP phenocopied effects seen in 3A.
(C) Qualitative phenotypic distribution of embryos from (B) scored with normal or abnormal Runx1+144:GFP expression in the VDA at 36 hpf (n value as in A).
(D) FACS analysis of Runx1+144:GFP+/Flk:dsRed+ HSPCs showed no significant difference with MMP inhibitor exposure (12?36 hpf; 5 embryos/sample, ?3 replicates/condition).
(E) Representative confocal images of Tg(runx1+23(144?378):egfp/kdrl:dsred) embryos, showing: (top) double-positive HSPC clusters in the VDA with ARP treatment (12?54 hpf) compared with control; (bottom) high magnification of a select cluster (n > 10 embryos/condition).
(F) Absolute counts of Runx1+144+/Flk+ HSPC clusters (of >3 cells) in the VDA in embryos from (E) showed a significant increase in total clusters after ARP treatment (n > 10 embryos/condition; ?p < 0.05).
(G) Absolute counts of Runx1+144+/Flk1+ HSPC clusters (of >3 cells) in the VDA in embryos from (E) showed a significant increase in cells per cluster with ARP treatment (n > 10 embryos/condition; ?p < 0.05).
(H) Still images from the time-lapse analysis (2:45?3:10 min time stamp; see Movies S3 and S4) showed altered budding and egress of Runx1+144+Flk1+ HSPCs in the presence of ARP exposure (12?54 hpf; 2 replicates/condition) compared with controls.
Arrows mark HSPC clusters; brackets mark dorsal/ventral boundaries of the VDA and CV. Error bars denote mean ± SD. Scale bars, 100 ?m (A and B) and 50 ?m (E and H).