Notch signaling distinguishes 2 waves of definitive hematopoiesis in the zebrafish embryo

Abstract

Recent studies have revealed that definitive hematopoiesis in vertebrates initiates through the formation of a non-self-renewing progenitor with limited multilineage differentiation potential termed the erythromyeloid progenitor (EMP). EMPs are specified before hematopoietic stem cells (HSCs), which self-renew and are capable of forming all mature adult blood lineages including lymphoid cells. Despite their differences, EMPs and HSCs share many phenotypic traits, making precise study of their respective functions difficult. Here, we examine whether embryonic specification of EMPs requires Notch signaling as has been shown for HSCs. In mindbomb mutants, which lack functional Notch ligands, we show that EMPs are specified normally: we detect no significant differences in cell number, gene expression, or differentiation capacity between EMPs purified from wild-type (WT) or mindbomb mutant embryos. Similarly N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a chemical inhibitor of Notch receptor activation, has no effect on EMP specification. These studies establish that HSCs are the only hematopoietic precursor that requires Notch signaling and help to clarify the signaling events underlying the specification of the 2 distinct waves of definitive hematopoiesis.

Figure 1

HSCs, but not EMPs, express Notch pathway genes. qPCR analysis of notch1b (top left), notch3 (bottom left), and the Notch target her6 (bottom right) on 26-hpf EMPs and 36-hpf HSCs, with whole kidney marrow (WKM) as reference standard. The y-axis indicates percentage relative to WKM, defined as 100% in all examples. Results are the average of 3 experiments; error bars represent standard deviation.

Figure 2

EMPs, but not HSCs, are specified in mib mutant embryos. Expression of the HSC transcripts cmyb in wild-type (Ai) and mib (Aii) and runx1 in wild-type (Bi) and mib (Bii) at 30 hpf. Expression of the EMP transcript pu.1 at 24 hpf in wild-type (Ci) and mib (Cii) and 26 hpf in wild-type (Di) and mib (Dii) and mpx at 36 hpf in wild-type (Ei) and mib (Eii) and 48 hpf in wild-type (Fi) and mib (Fii). Embryos are shown with anterior to the left and dorsal up. Dashed line boxes describe the embryonic regions (AGM: Ai-ii, Bi-ii; PBI: Ci-ii, Di-ii; and CHT: Ei-ii, Fi-ii) shown in magnified images to the right of each whole embryo view. Magnification: ×100 in whole embryo images; ×300 in AGM, PBI, and CHT images.

Figure 3

Lack of Notch signaling does not affect EMP number or gene expression. (A) lmo2^higata1⁺ dual-positive cells (EMPs) are present in wild-type (top left) and mindbomb (top right) embryos at 30 hpf. EMPs (in boxed gate) are a fraction of the total live cells in the embryo at this time point. Resorting shows > 97% purity of isolated EMP populations (bottom row). (B) Gene expression of sorted 28-30 hpf WT and mib EMPs was examined by qPCR and compared with WKM as a reference. HSC (runx1), thrombocytic (cd41), erythroid (gata1), and myeloid (pu.1, mpx) transcript levels were determined. The y-axis indicates expression relative to WKM, defined as 100% in all examples. Levels reflect the average of 3 experiments; error bars represent standard deviation.

Figure 4

Similar differentiation capacity of WT and mib mutant EMPs in vitro. lmo2^hi, gata1⁺ 30-hpf EMPs from WT and mib embryos were isolated by FACS and plated on zebrafish kidney stroma (ZKS). Samples from WT (A) and mib (B) cultured cells were cytocentrifuged and stained after 3 (3 left columns) or 5 (3 right columns) days in culture. Cells were stained for morphology with May-Grünwald/Giemsa (2 left and 2 right columns). o-Dianisidine, a chemical stain for hemoglobin, was used to assess erythroid differentiation (middle column). (C) Differential cell counts for immature blasts (yellow), erythroid (red), and myeloid (blue) cell types yielded statistically similar percentages from WT or mib EMPs (*P = .988; **P = .802). The y-axis indicates percentages of cultured cells. Levels reflect the average of 3 experiments. Statistical analyses were performed by a 2-sample, unequal-variance t test with 2-tailed distribution.