ZFIN ID: ZDB-FIG-190816-19
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:
Condition:
Knockdown Reagent:
Anatomical Terms:
Stage Range: 10-13 somites to Prim-5
PHENOTYPE:
Fish:
Condition:
Knockdown Reagent:
Observed In:
Stage Range: 20-25 somites to Prim-5

Fig. 3

Rap1b Cell-Autonomously Regulates HE Specification through Notch Signaling

(A) Confocal stack images of Tg(fli1a:GFP-rap1b) embryos at 14 (left) and 24 (right) hpf. Left, dorsal views with anterior to the left. Boxed area is enlarged at the bottom. Right, lateral view with anterior to the top. GFP-Rap1b is expressed in lateral plate mesoderm at 14 hpf and in blood vessels at 24 hpf.

(B) Expression patterns of runx1 in 24 hpf sibling (WT, upper) and rap1bncv124 (lower) embryos crossed without (GFP-Rap1b(‒), left column) or with (GFP-Rap1b(+), right column) Tg(fli1a:GFP-rap1b). Arrowheads indicate runx1-positive hemogenic ECs.

(C) Percentages of embryos showing normal (3 or more, blue) and decreased (less than 3, pink) numbers of runx1-positive hemogenic ECs in the AGM regions, as observed in (B). WT+GFP-Rap1b(‒), n = 31; rap1bncv124+GFP-Rap1b(‒), n = 28; WT+GFP-Rap1b(+), n = 23; rap1bncv124+GFP-Rap1b(‒), n = 27. ∗∗p < 0.01; p < 0.05.

(D) Confocal stack fluorescence images of trunk and tail regions of 20 hpf Tg(Tp1:GFP);(fli1a:DsRed) embryos injected with control MO (upper) and rap1b MO (lower). Left, merged images of GFP (green) and DsRed (red); middle, GFP images; right, enlarged merged (upper) and GFP (lower) images of boxed areas. Asterisks indicate notochord-derived Tp1:GFP signal.

(E) Relative fluorescence intensity of Tp1:GFP in the DsRed-labeled vascular cord, as observed in (D). Each dot represents an individual embryo. Error bars indicate means ± SD. Control MO, n = 10; rap1b MO, n = 7. ∗∗∗p < 0.001.

(F and H) Expression patterns of runx1 in 26 hpf control MO- and rap1bMO-injected embryos heat shocked at 14 hpf (F) and 22 hpf (H) for 45 min with either wild type or the Tg(hsp70:Gal4FF) background (NICD(‒), left column) and with the Tg(hsp70:Gal4FF);(UAS:NICD)background (NICD(+), right column).

(G and I) Percentages of embryos showing normal (blue) and decreased (pink) expressions of runx1 in the AGM regions of embryos heat shocked at 14 hpf (G) and 22 hpf (I), as observed in (F) and (H). (G) Control MO/NICD(‒), n = 22; rap1b MO/NICD(‒), n = 32; Control MO/NICD(+), n = 26; rap1b MO/NICD(+), n = 28. (I) Control MO/NICD(‒), n = 32; rap1b MO/NICD(‒), n = 26; Control MO/NICD(+), n = 20; rap1bMO/NICD(+), n = 17. p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s., not significant.

In (B), (F), and (H), boxed areas are enlarged in the insets. Scale bars: 200 μm (A, B, F, and H); 50 μm (D).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP ncv508Tg control 10-13 somites lateral plate mesoderm IFL
Prim-5 blood vessel IFL
um13Tg; um14Tg control 20-25 somites vascular cord IFL
um13Tg; um14Tg + MO4-rap1b control 20-25 somites vascular cord IFL
runx1 WT control Prim-5 ventral wall of dorsal aorta angioblastic mesenchymal cell ISH
WT + MO4-rap1b control Prim-5 ventral wall of dorsal aorta ISH
kca3Tg; kca4Tg + MO4-rap1b heat shock Prim-5 ventral wall of dorsal aorta ISH
ncv508Tg control Prim-5 ventral wall of dorsal aorta angioblastic mesenchymal cell ISH
ncv508Tg ; rap1bncv124/ncv124 control Prim-5 ventral wall of dorsal aorta ISH
Prim-5 ventral wall of dorsal aorta angioblastic mesenchymal cell ISH
rap1bncv124/ncv124 control Prim-5 ventral wall of dorsal aorta ISH
Prim-5 ventral wall of dorsal aorta angioblastic mesenchymal cell ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO4-rap1b control Prim-5 ventral wall of dorsal aorta runx1 expression decreased amount, abnormal
kca3Tg; kca4Tg + MO4-rap1b heat shock Prim-5 ventral wall of dorsal aorta runx1 expression amount, ameliorated
ncv508Tg ; rap1bncv124/ncv124 control Prim-5 ventral wall of dorsal aorta runx1 expression amount, ameliorated
rap1bncv124/ncv124 control Prim-5 ventral wall of dorsal aorta runx1 expression decreased amount, abnormal
Prim-5 ventral wall of dorsal aorta runx1 expression decreased distribution, abnormal
um13Tg; um14Tg + MO4-rap1b control 20-25 somites vascular cord EGFP expression decreased amount, abnormal
20-25 somites vascular cord Notch signaling pathway decreased occurrence, 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