Figure 4. Overexpression of cxcr1... | Rockefeller University Press

Figure 4.

$Figure 4. Overexpression of cxcr1 increases HSPC residency time, mitotic rate and endothelial cell cuddling within the CHT. (a–e) Runx1:mCherry or Runx1:mCherry;kdrl:GFP zebrafish embryos were microinjected with DNA encoding hsp70l:cxcr1 or control DNA (hsp70l:GFP or empty vector), gene expression was induced by heat shock at 36 and 48 hpf, and HSPC colonization of the CHT was quantified by time lapse video microscopy from 52 to 72 hpf. (a) Time series plot showing the number of HSPCs in each group (n = 7 for Control and n = 5 for CXCR1). Colored bands represent 95% confidence intervals. The experiment was repeated twice with similar results; a representative experiment is shown. (b) Cumulative density function showing the fraction of HSPCs tracked in the CHT for any duration of time. The area under the curve between any two points on the x-axis represents the fraction of HSPCs continuously tracked for that length of time. Dashed lines represent the lower limit of the 10% of cells with the longest CHT residency time (CXCR1 in red, Control in green). n = 10 animals, 461 total tracked HSPCs for Control and n = 6 animals, 187 total tracked HSPCs for CXCR1, P = 0.029 (Student’s t test). The experiment was repeated twice with similar results; combined results are shown. (c–e) Tracked HSPCs were followed and mitotic events were enumerated. (c) A representative mitotic event in a Runx1:mCherry;kdrl:GFP transgenic injected with DNA encoding hsp70l:cxcr1. HSPCs undergoing mitosis were observed to reduce their migration (ci) and undergo nuclear cleavage (cii-ciii), often rotating in the process, followed by release of the daughter cell (cvii). Bars, 10 µm. The number of mitotic events per HSPC is plotted in (d; P = 0.0041, Student’s t test) and the number of mitotic events per HSPC per hour of CHT residency time for that HSPC is plotted in (e; P = 0.02, Wilcoxon’s rank sum test). n = 6 animals, 122 total tracked HSPCs for GFP/Control and n = 5 animals, 107 total tracked HSPCs for CXCR1. The experiment was repeated twice with similar results; a representative experiment is shown. (f–g) Runx1:mCherry;kdrl:GFP double transgenic embryos were injected with DNA encoding empty vector or hsp70l:cxcr1 and gene expression was induced at 36 and 48 hpf, followed by time lapse microscopy. (f) Representative still frames showing an HSPC (arrow) entering an endothelial cell pocket (fi-iii), followed by cuddling (fiv-xvii) and finally exit from the endothelial cell pocket (fxviii). Numbers in the bottom left corner of each frame represent hh:mm after fertilization; this track lasted for 8.5 h and produced one mitotic event (fvi, arrowhead). Bar, 20 µm. (g) The time that each tracked HSPC spent cuddled in the endothelial cell pocked is plotted as a percent of the overall track duration within the CHT (P = 6.28 × 10−7, Wilcoxon’s rank sum test). n = 5 animals, 354 tracked HSPCs for Control and n = 3 animals, 114 tracked HSPCs for CXCR1. The experiment was repeated twice with similar results; a representative experiment is shown.$

Overexpression of cxcr1 increases HSPC residency time, mitotic rate and endothelial cell cuddling within the CHT. (a–e) Runx1:mCherry or Runx1:mCherry;kdrl:GFP zebrafish embryos were microinjected with DNA encoding hsp70l:cxcr1 or control DNA (hsp70l:GFP or empty vector), gene expression was induced by heat shock at 36 and 48 hpf, and HSPC colonization of the CHT was quantified by time lapse video microscopy from 52 to 72 hpf. (a) Time series plot showing the number of HSPCs in each group (n = 7 for Control and n = 5 for CXCR1). Colored bands represent 95% confidence intervals. The experiment was repeated twice with similar results; a representative experiment is shown. (b) Cumulative density function showing the fraction of HSPCs tracked in the CHT for any duration of time. The area under the curve between any two points on the x-axis represents the fraction of HSPCs continuously tracked for that length of time. Dashed lines represent the lower limit of the 10% of cells with the longest CHT residency time (CXCR1 in red, Control in green). n = 10 animals, 461 total tracked HSPCs for Control and n = 6 animals, 187 total tracked HSPCs for CXCR1, P = 0.029 (Student’s t test). The experiment was repeated twice with similar results; combined results are shown. (c–e) Tracked HSPCs were followed and mitotic events were enumerated. (c) A representative mitotic event in a Runx1:mCherry;kdrl:GFP transgenic injected with DNA encoding hsp70l:cxcr1. HSPCs undergoing mitosis were observed to reduce their migration (c_i) and undergo nuclear cleavage (c_ii-c_iii), often rotating in the process, followed by release of the daughter cell (c_vii). Bars, 10 µm. The number of mitotic events per HSPC is plotted in (d; P = 0.0041, Student’s t test) and the number of mitotic events per HSPC per hour of CHT residency time for that HSPC is plotted in (e; P = 0.02, Wilcoxon’s rank sum test). n = 6 animals, 122 total tracked HSPCs for GFP/Control and n = 5 animals, 107 total tracked HSPCs for CXCR1. The experiment was repeated twice with similar results; a representative experiment is shown. (f–g) Runx1:mCherry;kdrl:GFP double transgenic embryos were injected with DNA encoding empty vector or hsp70l:cxcr1 and gene expression was induced at 36 and 48 hpf, followed by time lapse microscopy. (f) Representative still frames showing an HSPC (arrow) entering an endothelial cell pocket (f_i-iii), followed by cuddling (f_iv-xvii) and finally exit from the endothelial cell pocket (f_xviii). Numbers in the bottom left corner of each frame represent hh:mm after fertilization; this track lasted for 8.5 h and produced one mitotic event (f_vi, arrowhead). Bar, 20 µm. (g) The time that each tracked HSPC spent cuddled in the endothelial cell pocked is plotted as a percent of the overall track duration within the CHT (P = 6.28 × 10⁻⁷, Wilcoxon’s rank sum test). n = 5 animals, 354 tracked HSPCs for Control and n = 3 animals, 114 tracked HSPCs for CXCR1. The experiment was repeated twice with similar results; a representative experiment is shown.