CD41+ cmyb+ precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis

Abstract

Development of the vertebrate blood lineages is complex, with multiple waves of hematopoietic precursors arising in different embryonic locations. Monopotent, or primitive, precursors first give rise to embryonic macrophages or erythrocytes. Multipotent, or definitive, precursors are subsequently generated to produce the adult hematopoietic lineages. In both the zebrafish and the mouse, the first definitive precursors are committed erythromyeloid progenitors (EMPs) that lack lymphoid differentiation potential. We have previously shown that zebrafish EMPs arise in the posterior blood island independently from hematopoietic stem cells (HSCs). In this report, we demonstrate that a fourth wave of hematopoietic precursors arises slightly later in the zebrafish aorta/gonad/mesonephros (AGM) equivalent. We have identified and prospectively isolated these cells by CD41 (itga2b) and cmyb expression. Unlike EMPs, CD41(+) AGM cells colonize the thymus to generate rag2(+) T lymphocyte precursors. Timelapse imaging and lineage tracing analyses demonstrate that AGM-derived precursors use a previously undescribed migration pathway along the pronephric tubules to initiate adult hematopoiesis in the developing kidney, the teleostean equivalent of mammalian bone marrow. Finally, we have analyzed the gene expression profiles of EMPs and AGM precursors to better understand the molecular cues that pattern the first definitive hematopoietic cells in the embryo. Together, these studies suggest that expression of CD41 and cmyb marks nascent HSCs in the zebrafish AGM, and provide the means to further dissect HSC generation and function in the early vertebrate embryo.

Figure 1

Expression of CD45 and c-myb mark regions of definitive hematopoiesis in the zebrafish embryo. (A–E) CD45 expression initiates in the PBI by 24 hpf. (B) By 30 hpf, rare cells begin to appear between the axial vessels in the AGM (ventral to the yolk tube extension). (C–E) Robust expression is observed in both the PBI/CHT and AGM regions from 36 hpf onwards. (F–J) c-myb expression marks cells within the AGM slightly before CD45 is expressed. (H–J) After 36 hpf, c-myb expression is observed in a similar pattern to CD45 throughout the AGM and CHT. All animals are displayed anterior to the left and dorsal side up. All panels are 20X magnification views of the trunk and tail.

Figure 2

A CD41:eGFP transgene marks cells in the AGM, along the pronephric ducts, and in the thymic lobes. (A) Overview of regions shown in close-up fluorescent images. Purple region denotes left thymic lobe, blue region the left pronephric tubule, and red region the AGM (space between axial vessels). (B–E) CD41 is expressed in the first thymic immigrants beginning at approximately 48 hpf. GFP⁺ cells increase in number until 78 hpf, after which GFP expression disappears. (F–I) GFP⁺ cells appear along the pronephric tubules beginning at approximately 32 hpf, and increase in number over time. Dotted lines denote the boundaries of the duct. (J–N) Within the embryo, GFP⁺ cells are first observed in the AGM region at approximately 27 hpf, and increase in number over time. After 48 hpf, the AGM region greatly expands as the aorta and vein move apart. The upper dotted line denotes the ventral wall of the dorsal aorta, the lower line the dorsal wall of the cardinal vein. GFP⁺ ductal cells appear ventrolateral to cells within the demarcated AGM region. Images are merged fluorescence and Nomarski photographs at 20X magnification. Embryos positioned anterior to the left and dorsal side up.

Figure 3

Transplantated CD41:eGFP⁺ cells colonize the thymus and caudal hematopoietic tissues. (A) Photograph of 72 hpf larva, demarcating regions shown in close-up views in B (20X; Nomarski/fluorescence merge). Purple region denotes left thymic lobe and orange region the CHT. (B) One day following transplantation, recipient animals showed robust colonization of thymi (left panel) and the CHT (right panel). Transplanted CD41⁺ cells also carried a gata-1:DsRed transgene to visualize erythroid differentiation. Yellow cells in the CHT express both transgenes.

Figure 4

Fate-mapping demonstrates that AGM CD41:eGFP⁺ cells seed the thymus to become rag-2⁺ thymocytes. (A) Upper left panel shows one CD41:eGFP⁺ cell immediately after rhodamine uncaging at 40 hpf (arrowhead). Ten cells were uncaged per embryo, and thymic lobes (areas within dotted lines in lower panels) analyzed at 4 dpf. Rhodamine⁺ cells were observed in the thymic lobes, along with GFP⁺ cells that were not uncaged (lower left panel). Control animals where regions outside of the AGM were laser uncaged never showed rhodamine⁺ cells in the thymus (right panels). (B) Similar uncaging experiments using CD41:eGFP, Rag-2:eGFP double transgenic animals show labeled thymic immigrants are lymphoid. CD41:eGFP⁺ cells were laser targeted at 40 hpf in the AGM and thymi analyzed at 5 dpf, when thymic cells no longer express the CD41 transgene (left panel; asterisks mark CD41⁺ thrombocytes in circulation) and when nascent thymocytes robustly express the rag-2 transgene (right panel). (C) Targeted CD41:eGFP⁺ cells migrate to the thymus and express the rag-2 transgene. Left panel shows GFP expression in a representative thymic lobe, middle panel clones of rhodamine⁺ cells, and right panel a merged imaged including Nomarski overlay. (D) Confocal imaging of targeted thymic immigrants. Left panel shows a maximum projection of the entire thymic lobe, and shows a rhodamine⁺ GFP⁻ cell migrating into the thymus via a posterior thymic duct (arrowhead). Right panel shows a single Z slice through the thymus showing expression of GFP and rhodamine.

Figure 5

Comparison of gene expression profiles between purified HSCs and EMPs. (A) PBI (green box) or AGM (red box) regions were dissected from embryos at 30 or 42 hpf, respectively. (B) EMPs were purified from PBI preparations by flow cytometry based on coexpression of Gata-1:DsRed and lmo2:eGFP transgenes (green gate). (C) HSCs were purfied from AGM preparations by flow cytometry based on CD41:eGFP⁺ Gata-1:DsRed⁻ expression (red gate). (D) QPCR was performed for a variety of lineage-affiliated genes. Whole kidney marrow (WKM; blue bars) was used as the reference standard for all analyses of EMP (green bars) and HSC expression (red bars). (E) c-myb⁺ FSC^hi HSCs (orange gate) were purified from dissected trunks of 42 hpf c-myb:eGFP embryos for QPCR analyses (orange bars).

Figure 6

Hematopoietic precursors migrate along the pronephric tubules. (A) CD41:eGFP (left panel; asterisk marks region of GFP fluorescence from circulating thrombocytes) and c-myb:eGFP (middle panel) transgenes are expressed in cells along each pronephric tubule. The anterior pronephric tubules are marked by a gata-3:AmCyan transgene (right panel). Dorsal views of animals with anterior side up. (B) c-myb:eGFP⁺ cells (arrowheads) are localized upon gata-3:AmCyan⁺ pronephric tubules (T). (C) Timelapse imaging demonstrates c-myb:eGFP⁺ cells migrate along the pronephric tubules in an anterior direction. Two GFP⁺ cells (dotted red circle) were observed to migrate approximately 190μm (blue arrow) over 30h. Embryos imaged dorsal side up, anterior to the right.

Figure 7

Characterization of hematopoietic precursors on the pronephric tubules. (A) FITC was uncaged in 5 CD41:eGFP⁺ cells (arrows) at 4 dpf along the left pronephric tubule. (B) Animals were fixed three days later and analyzed for uncaged FITC. Ductal cells migrated from where they were targeted on the left pronephric tubule (green arrowheads, left panel) to the left anterior pronephros (red arrowhead). Contralateral anterior pronephri were not colonized (right panel). (C) By 65 hpf, AGM expression of the c-myb:eGFP transgene becomes restricted to the pronephric tubules. c-myb:eGFP⁺ tubular cells (T) were purified away from GFP⁺ neural cells (N) in dissected trunks by flow cytometry and analyzed for hematopoietic gene expression (right panel).

Figure 8

Model of hematopoietic stem cell migration in the zebrafish embryo. (1) HSCs (green) appear between the dorsal aorta and cardinal vein. (2) Some HSCs translocate to the pronephric tubules (orange) and often back again to between the vessels (3). (4) Some HSCs enter circulation to seed the developing CHT and thymic anlage. (5) HSCs migrate anteriorly along each duct to generate the first hematopoietic cells in the developing kidney. Nascent HSCs can be visualized by expression of the c-myb, CD41, and CD45 genes (bottom panel).