FIG. 7.

Upregulation of runx1 and modulation of apoptosis through inhibition of Jak reverses the effects of pdcd2 knockdown on erythropoiesis. (A) WISH of runx1 expressing cells in the arterial and AGM regions of treated embryos at 30 hpf. Treatment with AG490 resulted in increased runx1 expression in the arterial and AGM regions (upward arrowheads in the AG490 panel), and possibly expansion of the anterior myelopoietic compartment (downward arrowheads). Note the dramatic rescue of runx1 expression in the ICM with the combination of AG490 and 5ssMO compared to the combination of vehicle and 5ssMO on top. (Vehicle n=35 (71%); AG490 n=28 (86%); 5ssMO n=34 (91%); and 5ssMO⁺ AG490 n=36 (14%) showed the displayed phenotypes). (B) Q-PCR analyses of runx-1 expression relative to β-actin in the AGM region of pdcd2 morphants after AG490 treatment (mean±s.e.m, *P<0.05). Scale bar, 500 μm. (C) DAF staining of hemoglobinated cells in WT, or 5ssMO-injected embryos at 48, or 96 hpf treated with either vehicle, 50 μM AG490, pdcd2 5ssMO, or combinations of 5ssMO and AG490. Numbers and percentages of embryos representing the demonstrated DAF⁺ staining are for WT with vehicle at either 48 or 96 hpf, n=23 (100%), WT with AG490 at 48 hpf, n=18 (100%), and at 96 hpf, n=31 (100%). For 5ssMO treated with vehicle at 48 hpf, n=20 (40%), and at 96 hpf, n=24 (21%), and 5ssMO treated with AG490 at 48 hpf, n=22 (68%), and at 96 hpf, n=14 (86%). The effects of combined 5ssMO⁺ AG490 treatments were significant when compared to individual treatment (P<0.01). (D) Left, representative images from the 4 groups of TUNEL-stained 30-hpf embryos. Insets demonstrate the AGM/PBI region where hematopoietic cells were counted. Right, quantification of the antiapoptotic effects of Jak inhibition in pdcd2 morphant embryos. Sixteen embryos were analyzed for each treatment from the AGM/PBI region highlighted in (A) in 3 experiments with 3 investigators blinded to treatment performing the counts for TUNEL-positive cells. Data for each embryo are derived from a series of Z sections spanning the depth of the embryo, and then numbers of apoptotic cells were averaged from counts of 3 investigators. (*P<0.01; **P<10⁻⁴; bars s.e.m.). Scale bar, 500 μm. Color images available online at www.liebertpub.com/scd