Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo - PubMed (original) (raw)

. 2007 Dec;134(23):4147-56.

doi: 10.1242/dev.012385. Epub 2007 Oct 24.

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Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

Julien Y Bertrand et al. Development. 2007 Dec.

Abstract

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.

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Figures

Figure 1

Figure 1

Genes associated with multilineage hematopoiesis are expressed in the posterior blood island as early as 30 hpf. (A, B) By 30 hpf, expression of the pan-leukocyte markers cd45 and l-plastin are observed throughout the PBI. (G, H) By 36 hpf, the number of cells expressing each gene has increased in the PBI, and expressing cells begin to migrate throughout the embryo. Localized expression of lmo2 is observed in the PBI at 30 hpf (C) and 36 hpf (I). (D, J) Expression of gata-1 is also observed in cells within the vascular plexus of the PBI at both timepoints. (E, F, K, L) Expression of genes associated with myelopoiesis is also observed within the PBI, including pu.1 and mpx. Rare, pu.1+ cells are observed at 30 hpf in the PBI (E) that increase in number by 36 hpf (K), whereas mpx expression is not observed until 36 hpf (F, L), consistent with it being a marker of relatively mature myelomonocytic cell types. Lower panels are 20X magnification views of the PBI from upper, 10X whole-embryo photographs.

Figure 2

Figure 2

Coexpression of lmo2 and gata-1 reveals immature hematopoietic precursors in the PBI. (A) Fluorescence microscopy reveals cells within the vascular plexus of the PBI that expressed both gata-1:DsRed and lmo2:eGFP fluorochromes (arrowheads) in double transgenic animals. Blue boxes superimposed on embryonic photographs in upper panels denote the regions shown at 20× magnification in middle and 40× magnification in lower panels at 30, 36, and 48 hpf. Lower panels show a single, deconvolved Z slice, demonstrating coexpression of each transgene in single cells (arrowheads). (B) Cells coexpressing the gata-1:DsRed and lmo2:eGFP transgenes are prospectively isolated by flow cytometry. Double positive cells peak in number at 30 hpf, with approximately 160 cells per embryo (left panel). (C) Compared to purified primitive erythroblasts sorted by low levels of lmo2:eGFP and high levels of gata-1:DsRed (red gate in 30 hpf plot), cytological staining of purified 30 hpf lmo2+ gata-1+ cells (black gate) showed immature morphologies indicative of early hematopoietic progenitors.

Figure 3

Figure 3

Gene expression profiling of hematopoietic precursors in the PBI suggest multipotency. (A) Cells were purified from 30 hpf embryos by flow cytometry based on expression of gata-1:DsRed and lmo2:eGFP transgenes (DN – Double Negative; L - lmo2:eGFP+, gata-1:DsRed_−; LG - lmo2:eGFP+, gata-1:DsRed_+; G – lmo2:eGFPlow, gata-1:DsRed+; WKM – Whole Kidney Marrow) and subjected to RT-PCR. (B) FACS analysis shows a population that coexpresses the gata-1:DsRed and mpx:eGFP transgenes at 30 hpf (black gate). (C) Two-color FISH demonstrates that cells within the PBI at 30 hpf coexpress gata-1 and pu.1 (C, upper panels) and gata-1 and mpx (lower panels).

Figure 4

Figure 4

Functional studies demonstrate that gata-1+ lmo2+ cd41+cells are committed erythromyeloid progenitors. (A) Dissociated gata-1:DsRed+ lmo2:eGFP+ cells were purified from 36 hpf embryos by flow cytometry and transplanted into wild-type embryonic recipients. Transplanted cells were observed to home back to the PBI in host animals (right panel, 20X magnification). (B) In vivo fate mapping studies were performed by laser activation of caged rhodamine in cd41+cells in the 44 hpf AGM or 40 hpf PBI. Presumptive AGM HSCs were targeted as positive controls for thymus colonization (lower panels; outlined crescent-shaped structure). Boxed yellow and blue regions in upper panel denote close up areas shown in left panels for the AGM and right panels for the PBI, respectively. All animals are shown in lateral views, with heads oriented to the left and dorsal sides up. Dotted line at the left edge of lower boxes denotes outline of the eye for orientation. (C) Short-term culture of lmo2+ gata-1+ cells atop kidney stromal cells demonstrates erythroid (E) and myeloid (M) differentiation potential.

Figure 5

Figure 5

Erythromyeloid progenitors arise autonomously within the PBI. (A–D) Cells expressing GFP under control of the lmo2 promoter were lineage traced by uncaging a combination of caged rhodamine and FITC. GFP+ cells were targeted, either in the medial (bounded by somites 1–10; panel A) or most posterior (panel B) regions of the lmo2 expression domain between 13–15 somite stages. (C, D) Analysis of targeted progeny at 30 hpf showed no medially-derived cells in the PBI (C), whereas posterior-derived daughter cells were observed throughout the venous plexus of the PBI (D). Inset in (D) shows a close-up of the PBI in a second animal, with marked progeny found within the vascular plexus between the aorta (dashed red line) and caudal vein (dashed blue line). Primitive erythrocytes within each vessel are marked with asterisks, the daughters of posterior lmo2+ cells by white arrowheads.

Figure 6

Figure 6

Model of hematopoietic ontogeny in the zebrafish embryo. (A) Different regions of lateral plate mesoderm (LPM) give rise to anatomically distinct regions of blood cell precursors. Cartoon depicts a dorsal view of a 5 somite stage embryo. (B) Embryonic hematopoiesis appears to occur through four, independent waves of precursor production. Each wave is numbered based on the temporal appearance of functional cells from each subset.

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