ZFIN ID: ZDB-FIG-190816-21
Rho et al., 2019 - Rap1b Promotes Notch-Signal-Mediated Hematopoietic Stem Cell Development by Enhancing Integrin-Mediated Cell Adhesion. Developmental Cell   49(5):681-696.e6 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage Range: Prim-5 to Day 4
PHENOTYPE:
Fish:
Observed In:
Stage Range: Prim-5 to Day 4

Fig. 1

Rap1b Is Not Essential for Vascular Development and Integrity but Is Required for HSC Development

(A) Confocal stack fluorescence images of trunk vasculature in sibling (WT; upper) and rap1bncv124 (lower) embryos and larvae at 24, 36, 48, and 60 hpf with the Tg(fli1a:lifeact-mCherry) background. Lateral views with anterior to the left. The cross-sectional images of the area indicated by dotted green lines on the images are shown on the right side. Asterisks indicate lumens of DA (upper) and posterior cardinal vein (lower).

(B) Expression patterns of endothelial marker genes kdrl (left column), cdh5 (middle column), and fli1a (right column) in sibling (WT; upper) and rap1bncv124 (lower) embryos at 36 hpf.

(C) Representative bright field images of the heads of 2 dpf rap1bncv124embryos without (upper panels) or with (lower panels) intracranial hemorrhage. Lateral and dorsal view images are shown in the left and right columns, respectively. Dotted circles indicate the region of cranial hemorrhage.

(D) Quantification of intracranial hemorrhage in sibling (WT) and rap1bncv124 embryos at 2 dpf. Percentages of normal embryos (blue) and those showing intracranial hemorrhage (pink). WT, n = 69; rap1bncv124, n = 88. n.s., not significant.

(E) Expression patterns of the HSC marker cmyb in 36 hpf sibling (WT) and rap1bncv124 embryos. Arrowheads indicate cmyb-positive HSCs.

(F) Confocal stack fluorescence images of trunk axial vasculature in 36 hpf sibling (WT; upper) and rap1bncv124 (lower) embryos with the Tg(cmyb:GFP);(fli1a:lifeact-mCherry) background. Lateral views with anterior to the left. Merged images of cmyb:GFP (green) and fli1a:lifeact-mCherry (magenta). Arrowheads indicate cmyb:GFP- and fli1a:lifeact-mCherry double-positive HSCs located in the ventral side of the DA.

(G) Number of cmyb:GFP- and fli1a:lifeact-mCherry double-positive HSCs in the aorta-gonad-mesonephros (AGM) regions of 36 hpf sibling (WT) and rap1bncv124 embryos, as observed in (F). Each dot represents an individual embryo. Error bars indicate means ± SD. WT, n = 18; rap1bncv124, n = 17. ∗∗p < 0.01.

(H) Expression patterns of the lymphoid lineage marker rag1 in the 4 dpf sibling (WT) and rap1bncv124 embryos. Lateral (left column) and dorsal (right column) views with anterior to the left. Dotted circles indicate the region of the thymus.

(I) Quantification of the rag1-positive thymus area in the 4 dpf sibling (WT) and rap1bncv124 embryos, as observed in (H). Each dot represents an individual thymus. Error bars indicate means ± SD. WT, n = 12; rap1bncv124, n = 19. ∗∗∗p < 0.001.

In (B) and (E), boxed areas are enlarged in the insets. Scale bars: 50 μm (lateral view in A); 20 μm (cross-sectional image in A); 200 μm (B, C, E, and H); 100 μm (F).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
cdh5 WT control Prim-25 endothelial cell ISH
rap1bncv124/ncv124 control Prim-25 endothelial cell ISH
fli1 WT control Prim-25 endothelial cell ISH
rap1bncv124/ncv124 control Prim-25 endothelial cell ISH
kdrl WT control Prim-25 endothelial cell ISH
rap1bncv124/ncv124 control Prim-25 endothelial cell ISH
mCherry rap1bncv124/ncv124 ; ncv7Tg control Prim-5 dorsal aorta IFL
Prim-5 posterior cardinal vein IFL
Prim-5 trunk vasculature IFL
Prim-25 dorsal aorta IFL
Prim-25 posterior cardinal vein IFL
Prim-25 trunk vasculature IFL
Long-pec dorsal aorta IFL
Long-pec posterior cardinal vein IFL
Long-pec trunk vasculature IFL
Pec-fin dorsal aorta IFL
Pec-fin posterior cardinal vein IFL
Pec-fin trunk vasculature IFL
myb WT control Prim-25 ventral wall of dorsal aorta hematopoietic stem cell ISH
rap1bncv124/ncv124 control Prim-25 ventral wall of dorsal aorta ISH
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell ISH
rag1 WT control Day 4 thymus T cell ISH
rap1bncv124/ncv124 control Day 4 thymus ISH
Day 4 thymus T cell ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
rap1bncv124/ncv124 control Prim-25 ventral wall of dorsal aorta myb expression decreased amount, abnormal
Prim-25 ventral wall of dorsal aorta myb expression decreased distribution, abnormal
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell decreased amount, abnormal
Day 4 thymus rag1 expression decreased amount, abnormal
rap1bncv124/ncv124 ; ncv7Tg control Prim-5 trunk vasculature morphology, normal
Prim-25 trunk vasculature morphology, normal
Long-pec blood vessel structure, normal
Long-pec trunk vasculature morphology, normal
Pec-fin trunk vasculature morphology, normal
zf169Tg ; ncv7Tg ; rap1bncv124/ncv124 control Prim-25 ventral wall of dorsal aorta hematopoietic stem cell decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 49(5), Rho, S.S., Kobayashi, I., Oguri-Nakamura, E., Ando, K., Fujiwara, M., Kamimura, N., Hirata, H., Iida, A., Iwai, Y., Mochizuki, N., Fukuhara, S., Rap1b Promotes Notch-Signal-Mediated Hematopoietic Stem Cell Development by Enhancing Integrin-Mediated Cell Adhesion, 681-696.e6, Copyright (2019) with permission from Elsevier. Full text @ Dev. Cell