Figure 1.
Eafs are required for specification of erythroid cells.
(A) Erythroid defects in eafs morphants were detected by o-dianisidine staining for hemoglobin. (B) Eafs morphants displayed erythroid defects, indicated by reduced mRNA of βe3 globin. (C) Eafs morphants displayed reduced protein of βe3 globin.
Figure 2.
Erythroid defects in eafs morphants are specific.
(A) At 24 hpf to 26 hpf, the expression detection of control or eafs-MO injected embryos (8 ng per embryo) processed by WISH for tissue specific genes: somites (A1, A2), notochord (A3, A4), vasculature (cdh5: A9, A10; flk1: A11, A12), and primitive progenitors (c-myb: A5, A6; scl: A7, A8, indicated by black arrow). (B) At the 8 somites stage, primitive hematoposis in eafs morphants, indicated by primitive progenitor genes (scl: B1, B2; lmo2: B3, B4). (C) At the 10 somites stage, progenitors for blood (gata2: C1, C2; lmo2: C3, C4; runx1: C7, C8; c-myb: C15, C16), progenitors for vasculature (fli: C11, C12), progenitors for both blood and vasculature (scl: C5, C6), more mature erythroid progenitors (gata1: C9, C10), and more mature myeloid progenitors (Pu.1: C13, C14). A1–A12, C13–C16, lateral view, anterior to the left. B1–B4, C1–C12, dorsal view, anterior to the up.
Figure 3.
Eafs act upstream of c-myb in primitive hamatopoiesis.
(A) In situ hybridization of βe3 globin showing rescue of erythroid differentiation defects in eafs morphants by knockdown of c-myb, the numbers of embryos displayed reduced βe3 globin in total detected embryos was shown in (A2, A3). (B) βe3 globin expression in eafs morphants (B2) and in,c-myb morphants (4 ng per embryo) (B3) and in morphants injected with combined eafs-MO and c-myb-MO (8 ng eafs-MO per embryo and 2 ng c-myb-MO) (B4). (C) Morphology of representative embryos injected with c-myb-MO (C2), and mesoderm pattern indicated by expression of pax2a and myoD in c-myb morphants at the bud stage (C4). (D) C-myb function downstream of eafs in primitive hematopoiesis. Early hematopoietic progenitors, lmo2, displayed obviously reduced expression from the 6 somites stage both in c-myb morphants (4 ng per embryo) (D2) and in morphants injected with combined c-myb-MO and eafs-MO (8 ng eafs-MO/per embryo and 2 ng c-myb-MO) (D3). Gata1 and scl also displayed obviously reduced expression in embryos injected with combined eafs-MO and c-myb-MO (D6, D9) as in embryos injected with c-myb-MO single (D5, D8) at the 10 somites stage. A1–A3, D1–D9, dorsal view, anterior to the up; C3, C4, dorsal view, anterior to the left. B1–B4, C1, C2, lateral view, anterior to the left.
Figure 4.
C-myb suppressed specification of mature erythroid cells, and the phenotype of c-myb gain of function is specific.
(A) Different dosage of c-myb on specification of mature erythroid cells (βe3 globin: A2, 50 pg per embryo; A4, 200 pg per embryo) and on erythroid progenitors (gata1: A6, A8). (B) In situ hybridization of βe3 globin showed that erythroid differentiation was blocked in embryos with ectopic c-myb expression (50 pg per embryo) (B2), the number of embryos displayed reduced βe3 globin was shown in (B2), and the in situ hybridization of c-myb shown its ectopic expression in corresponding embryos (B4). (B) Hematopoietic progenitor cells including gata1 (C1, C2) and scl (C3, C4), and other mesoderm cells including pax2a and myoD (C5, C6) specified and maintained normally in embryos with ectopic c-myb expression. A1–A8, B1, B2, C1–C4, dorsal view, anterior to the up; B3, B4, C5, C6, lateral view, anterior to the left.
Figure 5.
Knockdown Wnt signaling in hs:dnTCF-GFP embryos by heat shock at the bud stage resulted in reduced progenitor cells and accelerated differentiation of erythroid cells.
(A) Scheme of using hs:dnTCF-GFP fish to knockdown Wnt signaling in embryos. (B) Reduced progenitor blood cells, labeled by c-myb (B1, B2) and gata1 (B3, B4), but accelerated erythroid cells differentiation, labeled by βe3 globin (B5, B6, B9, B10) and band3 (B7, B8) displayed in hs:dnTCF-GFP positive embryos, and black arrow indicate the increased expression of βe3 globin expression in hs:dnTCF-GFP positive embryos (B10) compared to its control siblings (B9). Other mesoderm, labeled by pax2a and myoD (B9, B10, B11, B12), was normal in hs:dnTCF-GFP positive embryos. B1, B2, B7–B12, lateral view, anterior to the left; B3–B6 dorsal view, anterior to the up.
Figure 6.
Knockdown Wnt signaling in eafs morphants by transiently inducing dn-Tcf expression rescued defects of c-myb expression and erythroid cells specification.
(A) Scheme of rescuing experiments in eafs morphants by using hs:dnTCF-GFP embryos. (B) Increased c-myb expression and reduced βe3 globin expression were restored in eafs morphants by transiently inducing dn-Tcf in embryos at the bud stage. B1–B8, dorsal view, anterior to the up.