ZFIN ID: ZDB-FIG-150323-7
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:
Anatomical Terms:
Stage: Long-pec
PHENOTYPE:
Fish:
Conditions:
Observed In:
Stage: Long-pec

Fig. 7

STAT3 Is Activated upon ifng1-2 Induction and Positively Regulates HSC Development

(A and B) ifng1-2 overexpression stimulates Stat3 activation in the AGM region. (A) Immunofluorescent staining of phosphorylated Stat3 (pStat3, green) in embryos without (A) and with (B) the hsp:ifng1-2 transgene. All embryos were heat shocked at 24 and 48 hpf and harvested at 52–54 hpf. The AGM region is recognizable by the vascular expression of kdrl:HRAS-mCherry (red).

(C–E) Ifng1-2 and Notch signaling regulate Stat3 activation. (C) Western blot analysis of pStat3, total Stat3, and actin expression in the dissected yolk-spanning region of the trunk. Dissected area is outlined by dashed lines in the embryo illustration; 36 hpf embryos were heat shocked for 5 and 8 hr for ifng1-2 and NICD overexpression, respectively. DMSO and DAPT treatments were done from 24–48 hpf. (D) Relative expression of pStat3 normalized to Actin. (E) Relative expression of total Stat3 normalized to Actin. Values represent means ± SEM, n = 3–4 independent experiments, n.s. p > 0.05, p d 0.05, p d 0.01.

(F–I) Stat3 inhibition causes a reduction in HSC numbers that cannot be restored by ifng1-2 overexpression. itga2b:EGFP+ (green) kdrl:HRAS-mCherry+ (red) HSCs (white arrowheads) in control embryos (not carrying the hsp:ifng1-2-V5 transgene) treated with DMSO (F) or Stat3 inhibitor (inh) (G) and Tg(hsp:ifng1-2-V5) embryos treated with Stat3 inh (H). All embryos were heat shocked at 24 and 48 hpf and imaged at 52–54 hpf. DMSO and Stat3 inh treatments started at 24 hpf. (I) Numbers of itga2b:EGFP+kdrl:HRAS-mCherry+ cells per 500 µm aortic length are shown as means ± SEM, n = 19–24 embryos, n.s. p > 0.05, p d 0.01.

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 chemical treatment: pharmaceutical Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg; s994Tg heat shock, chemical treatment: S3I-201 Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
mCherry la2Tg; s896Tg control Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg chemical treatment: pharmaceutical Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta hematopoietic stem cell IHC
la2Tg; s896Tg; s994Tg heat shock, chemical treatment: S3I-201 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 IHC
s896Tg; s994Tg heat shock Long-pec vasculature IHC
Long-pec ventral wall of dorsal aorta IHC
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-stat3 IHC s896Tg control Long-pec ventral wall of dorsal aorta
IHC s896Tg; s994Tg heat shock Long-pec ventral wall of dorsal aorta
Phenotype Details
Fish Conditions Stage Phenotype
la2Tg; s896Tg chemical treatment: pharmaceutical Long-pec ventral wall of dorsal aorta has fewer parts of type hematopoietic stem cell, abnormal
s896Tg; s994Tg heat shock Long-pec ventral wall of dorsal aorta has fewer 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