ZFIN ID: ZDB-FIG-150323-1
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:
Fish:
Condition:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Prim-25 to Long-pec
PHENOTYPE:
Fish:
Condition:
Knockdown Reagent:
Observed In:
Stage: Long-pec

Fig. 1

Ifng1-2 and Its Receptor Crfb17 Positively Regulate HSC Development

(A) Schematic drawing of construct for heat-shock-inducible expression of ifng1-2. The area imaged and analyzed in all experiments (red box) is shown in the embryo illustration.

(B and C) Immunofluorescent labeling of V5-tagged Ifng1-2 (blue) in the vicinity of the AGM of control animals lacking the hsp:ifng1-2-V5 transgene (B) compared with Tg(hsp:ifng1-2) animals (C). The AGM region is recognized by Tg(kdrl:HRAS-mCherry) expression (red) in the vasculature.

(D and E) Increased number of HSCs at the ventral wall of the dorsal aorta and in the cardinal vein lumen upon ifng1-2 overexpression. HSCs (white arrowheads) in control (D) and Tg(hsp:ifng1-2-V5) (E) embryos were fluorescently labeled by Tg(itga2b:EGFP) (green) and Tg(kdrl:HRAS-mCherry) (red) expression.

(F) Number of HSCs per 500 µm aortic length. Values represent means ± SEM, n = 21–22 embryos. p d 0.05. All embryos were heat-shocked at 24 and 48 hpf and imaged at 52–54 hpf. Of note, the numbers of Tg(itga2b:EGFP)+;Tg(kdrl:HRAS-mCherry) (only green) pronephric duct cells appear unaffected by Ifng1-2 overexpression.

(G–I) Impaired HSC development in crfb17-/- embryos. Control (crfb17+/+) (G) and crfb17-/- (H) siblings were imaged at 52–54 hpf, and itga2b:EGFP+kdrl:HRAS-mCherry+ HSC (white arrowheads) numbers were analyzed prior to genotyping. (I) HSCs per 500 µm aortic length. Values represent means ± SEM, n = 8–14 embryos, p d 0.01.

(J–L) Ifng1-2 knockdown recapitulates the HSC phenotype of crfb17 mutants. itga2b:EGFP+kdrl:HRAS-mCherry+ HSC (white arrowheads) in mock (1% phenol red in nuclease-free distilled water)-injected (J) or ifng1-2 MO-injected (K) embryos imaged at 52–54 hpf. Numbers of HSCs per 500 µm aortic length is shown in (L). Values represent means ± SEM, n = 32–40 embryos, p d 0.001.

(M–O) Reduction of runx1 expression upon Ifng1-2 knockdown is restored by ifng1-2 induction. Expression of the HSC marker runx1 in mock-injected (M) and ifng1-2 MO-injected embryos without (N) and with (O) the hsp70:ifng1-2-V5 transgene.

(P and Q) ifng1-2 overexpresssion is unable to rescue Crfb17 knockdown. runx1 expression in crfb17 MO-injected embryos without (P) and with (Q) the hsp70:ifng1-2-V5 transgene. All embryos were heat shocked at 24 hpf, and runx1 expression was assessed by in situ hybridization at 36 hpf. The number of embryos showing the representative phenotype per total number of embryos analyzed is indicated in the lower left corner. Red brackets identify the dorsal aorta (DA); blue brackets identify the cardinal vein (CV). All images are lateral views, dorsal up, anterior to the left.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP ifngr1s986/s986; la2Tg; s896Tg control Long-pec posterior cardinal vein hematopoietic stem cell IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
ifngr1s987/s987; la2Tg; s896Tg control Long-pec posterior cardinal vein hematopoietic stem cell IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg control Long-pec posterior cardinal vein hematopoietic stem cell IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec posterior cardinal vein hematopoietic stem cell IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg; s994Tg heat shock Long-pec posterior cardinal vein hematopoietic stem cell IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
mCherry ifngr1s986/s986; la2Tg; s896Tg control Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
ifngr1s987/s987; la2Tg; s896Tg control Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
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 vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg; s994Tg heat shock Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
s896Tg control Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
s896Tg; s994Tg heat shock Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
runx1 WT control Prim-25 hematopoietic stem cell ISH
WT + MO1-ifng1 standard conditions Prim-25 hematopoietic stem cell ISH
WT + MO2-ifngr1 standard conditions Prim-25 hematopoietic stem cell ISH
s994Tg + MO1-ifng1 heat shock Prim-25 hematopoietic stem cell ISH
s994Tg + MO2-ifngr1 heat shock Prim-25 hematopoietic stem cell ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
ifngr1s986/s986; la2Tg; s896Tg control Long-pec hematopoietic stem cell decreased amount, abnormal
Long-pec ventral wall of dorsal aorta has fewer parts of type hematopoietic stem cell, abnormal
ifngr1s987/s987; la2Tg; s896Tg control Long-pec hematopoietic stem cell decreased amount, abnormal
Long-pec ventral wall of dorsal aorta has fewer parts of type hematopoietic stem cell, abnormal
la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec hematopoietic stem cell decreased amount, abnormal
la2Tg; s896Tg + MO1-ifng1 standard conditions Long-pec ventral wall of dorsal aorta has fewer parts of type hematopoietic stem cell, abnormal
la2Tg; s896Tg; s994Tg heat shock Long-pec hematopoietic stem cell increased amount, abnormal
Long-pec posterior cardinal vein has extra parts of type hematopoietic stem cell, abnormal
Long-pec ventral wall of dorsal aorta has extra parts of type hematopoietic stem cell, 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, 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