ZFIN ID: ZDB-FIG-150317-25
Sawamiphak et al., 2014 - Interferon Gamma Signaling Positively Regulates Hematopoietic Stem Cell Emergence. Developmental Cell   31:640-653 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Prim-25 to Long-pec
PHENOTYPE:
Fish:
Condition:
Knockdown Reagents:
Observed In:
Stage: Long-pec

Fig. S4

Ifn-γ acts downstream of Notch during HSC emergence, related to fig. 4.

a-d) HSC deficiency in mib morphants is rescued by Ifng1-2 overexpression. HSCs in embryos not harboring the Tg(hsp:ifng1-2-V5) transgene, injected with mock (a), mib MO (b) and Tg(hsp:ifng1-2-V5) embryos injected with mib MO (c) are labeled by Tg(itga2b:EGFP) (green) and Tg(kdrl:HRAS-mCherry) (red) expression and marked by white arrowheads. d, HSCs per 500 µm dorsal aorta length. Values represent means +/- SEM. n=18- 33 embryos per group. ** p d 0.01, *** p d 0.001, n.s. not significant. e) Notch inhibition downregulates crfb17 and ifng1-2 expression. crfb17 and ifng1-2 transcripts assessed by in situ hybridization (Fig. 4n-q) were quantified as mean grey values. Red bars show mean+/- SEM. n=21-36 embryos per group. *** p d 0.001. f) Notch signaling inhibition does not appear to affect crfb13 or ifng1-1 expression. crfb13 and ifng1-1 expression in the AGM region of 36-38 hpf embryos treated with DMSO or DAPT starting at 24 hpf. The number of embryos showing the representative phenotype per total number of embryos observed is indicated in the lower left corner. Red brackets identify the dorsal aorta (DA), blue brackets identify the cardinal vein (CV). g-j) Ifn-γ signaling deficiency increases EC cycling rate. kdrl:HRAS-mCherry+ ECs (red) that exit the G0 phase, were marked by PCNA immunostaining (blue), in embryos injected with mock (g), ifng1-2 MO (h) and crfb17 MO (i). Reduction in the number of itga2b:EGFP+ HSCs (green) and, conversely, induction of PCNA+ cells within the dorsal aorta region, indicated by dashed lines, is evident in both ifng1-2 and crfb17 morphants. j. PCNA+ ECs per 500 µm dorsal aorta length. Values represent means +/- SEM. n=9-12 embryos per group. ** p d 0.01, *** p d 0.001. All images are lateral views, dorsal up and anterior to the left.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP la2Tg; s896Tg control Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO2-ifngr1 standard conditions Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO4-mib1 standard conditions Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg; s994Tg + MO4-mib1 heat shock Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
ifng1r WT control Prim-25 to Long-pec ventral wall of dorsal aorta ISH
WT chemical treatment: DAPT Prim-25 to Long-pec ventral wall of dorsal aorta ISH
ifngr1l WT control Prim-25 to Long-pec ventral wall of dorsal aorta ISH
WT chemical treatment: DAPT Prim-25 to Long-pec ventral wall of dorsal aorta ISH
mCherry la2Tg; s896Tg control Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO2-ifngr1 standard conditions Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO4-mib1 standard conditions Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg; s994Tg + MO4-mib1 heat shock Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab2-pcna IHC la2Tg; s896Tg control Long-pec ventral wall of dorsal aorta hematopoietic stem cell
IHC la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec dorsal aorta
IHC Long-pec ventral wall of dorsal aorta hematopoietic stem cell
IHC la2Tg; s896Tg + MO2-ifngr1 standard conditions Long-pec dorsal aorta
IHC Long-pec ventral wall of dorsal aorta hematopoietic stem cell
Phenotype Details
Fish Conditions Stage Phenotype
la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec endothelial cell cell population proliferation increased rate, abnormal
la2Tg; s896Tg + MO2-ifngr1 standard conditions Long-pec endothelial cell cell population proliferation increased rate, abnormal
la2Tg; s896Tg + MO4-mib1 standard conditions Long-pec ventral wall of dorsal aorta has fewer parts of type hematopoietic stem cell, abnormal
la2Tg; s896Tg; s994Tg + MO4-mib1 heat shock Long-pec ventral wall of dorsal aorta hematopoietic stem cell normal amount, normal
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, 31, Sawamiphak, S., Kontarakis, Z., Stainier, D.Y., Interferon Gamma Signaling Positively Regulates Hematopoietic Stem Cell Emergence, 640-653, Copyright (2014) with permission from Elsevier. Full text @ Dev. Cell