Mutants of ldd239 display granulopoiesis and angiogenesis defects. ((A)–(D)) WISH analysis of mpx expression at 18 hpf and 22 hpf. In ldd239 mutants, mpx has an obviously decreased expression in the ICM compared with wild-type siblings at 18 hpf ((A) and (B), black arrows) and 22 hpf ((C) and (D), black arrows). Black arrows indicate the mpx+ cells in the ICM. White arrows represent the mpx+ cells in the A-LPM which have no significant difference between siblings and mutants ((C) and (D), white arrows). ((E)–(J)) WISH assays of fli1 and flk1 expression at 18 hpf and 22 hpf. Expression of fli1 has no difference between siblings and mutants at 18 hpf ((E) and (F)). Expression of fli1 and flk1 at 22 hpf show deficient angiogenesis and defects are better shown in higher magnifications ((G)–(J), black arrows). ((K)–(N)) Expression of lyz ((K) and (L)) and l-plastin ((M) and (N)) are normal in the A-LPM of mutants at 22 hpf. ((O)–(T)) Hematopoietic progenitor marker, scl ((O) and (P)), erythrocyte progenitors marker, gata1 ((Q) and (R)), and mature erythrocyte marker, hemoglobin hbae1, expression of all markers have no change in mutants at 22 hpf.

Identify plcg1 is indeed responsible for phenotypes of ldd239. ((A)–(F)) Plcg1 mRNA can rescue the ldd239 defect. Injection of plcg1 mRNA can restore the decreased mpx expression in mutant ICM region at 22 hpf compared with mutants ((C) and (D), black arrows). Simultaneously, deficient angiogenesis can also be rescued by overexpression of plcg1 ((E) and (F), black arrows). ((G) and (H), (K) and (L)) Plcg1 morphants have the same phenotypes as ldd239 mutants. Plcg1 morphants have a decreased mpx expression like ldd239 mutants ((G) and (H), black arrows) and also have a deficient angiogenesis marked by flk1 deficient expression ((K) and (L), black arrows). ((I) and (J)) WISH for l-plastin. Expression of l-plastin ((I) and (J)) has no change at 22 hpf in plcg1 morphants.

Loss-of-function of plcg1 affects granulopoiesis independent of plcg1′s role in angiogenesis and in a cell-autonomous manner. ((A)–(D)) WISH analysis of flk1 and mpx expression. In vegfa morphants, flk1 expression is disrupted at 22 hpf ((A) and (B)) indicating defects in angiogenesis. However the mpx expression has no change ((C) and (D)), suggesting granulopoiesis in the ICM is independent of angiogenesis. ((E)–(H)) WISH analysis of gata1 and pu.1 expression. In vegfa morphants, expression of gata1 ((E) and (F)) and pu.1 ((G) and (H)) are not affected. ((I)–(L)) Rescuing experiment with Tol2(flk1: plcg1-2a-mCherry). Fluorescence microscopy images of the trunk of 22 hpf ldd239 mutants injected at one cell-stage with Tol2(flk1:plcg1-2a-mCherry) transgene driving expression of mCherry ((I), red). Injection of Tol2(flk1:plcg1-2a-mCherry) construct can rescue the vascular defect ((J) and (K)) while it cannot rescue decreased mpx expression (L). ((M)–(P)) Rescuing experiment with Tol2(mpx: plcg1-2a-mCherry). Fluorescence microscopy images of the trunk of 22 hpf ldd239 mutants injected at one cell-stage with Tol2(mpx:plcg1-2a-mCherry) transgene driving expression of mCherry ((M), red). Injection of Tol2(mpx:plcg1-2a-mCherry) construct can rescue the decreased mpx expression ((N) and (O)) while it cannot rescue the deficient angiogenesis (P). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Loss-of-function of plcg1 does not affect initiation of granulocytes. ((A)–(H)) Expression analysis of granulocyte progenitor markers pu.1, cebpa and gfi1.1 in plcg1 mutants. Expression of pu.1 ((A) and (B)) and cebpa ((C) and (D)) have no difference between siblings and mutants. Another markers gfi1.1 also has indistinguishable expression at 18 hpf ((E) and (F)) and 20 hpf ((G) and (H)) between siblings and mutants.

Disruption of plcg1 plays an important role in terminal maturation of granulocytes. ((A) and (B), (A′) and (B′)) Sudan black staining of plcg1 mutants. In the mutants, the staining is significantly lower ((B) and (B2), black arrow) than that in wild-type siblings ((A) and (A′), black arrow). ((C) and (D)) Confocal scanning of live wild-type sibling and mutant embryos. The equivalent scanned area is shown ((A) and (B), dotted squares). In one scanning layer, some round and big cells similar to granulocytes could be detected and the numbers of these group cells are equal between wild-type siblings ((C), white arrows) and mutants ((D), white arrows). notochord: nc. yolk sac: yc. (E) Average numbers of granulocytes in the boxed region between siblings and mutant embryos. ((F) and (G)) Higher magnification of granulocytes between siblings and mutant embryos. Red arrows represent granules of granulocyte (F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

WISH analysis of mpx and flk1 expression at 32 hpf. mpx ((A) and (B)) had a decreased expression within trunk region of mutants. Expression of flk1 is also reduced in mutants (D) when compared to wild-type siblings (C).

WISH analysis of plcg1 expression during early development stages. Results show that plcg1 is expressed maternally (A). At 22 hpf, in the trunk plcg1 is expressed mainly in vascular and ICM region ((J), black arrow).

Apoptosis is not responsible for decreased mpx positive cells in ICM. ((A) and (B)) TUNEL assays of ldd239 wild-type siblings and mutants. In the mutants, increased apoptosis signals can be detected compared with wild-type siblings ((A) and (B), black arrows), but the staining signals mainly appeared in the skin of embryos, not in the ICM region. ((C) and (D)) Overexpression of bcl2-gfp mRNA cannot rescue decreased mpx expression in plcg1 mutants (D).

Loss-of-function of plcg1 does not affect the balance between erythropoiesis and myelopoiesis. ((A)–(D)) Expression of gata1 ((A) and (B)) and pu.1 ((C) and (D)) have no difference in plcg1 mutants compared with siblings at 18 hpf. ((E)–(H)) WISH for mpx. Sumo123 MOs cannot rescue mpx expression in plcg1 mutants. Wild-type siblings injected with sumo MO have increased mpx expression compared with control ((E) and (G), black arrows). But in plcg1 mutants, sumo MOs fail to rescue mpx expression ((F) and (H)).