Hematopoietic stem cells in Drosophila

Abstract

The Drosophila lymph gland, the source of adult hemocytes, is established by mid-embryogenesis. During larval stages, a pool of pluripotent hemocyte precursors differentiate into hemocytes that are released into circulation upon metamorphosis or in response to immune challenge. This process is controlled by the posterior signaling center (PSC), which is reminiscent of the vertebrate hematopoietic stem cell niche. Using lineage analysis, we identified bona fide hematopoietic stem cells (HSCs) in the lymph glands of embryos and young larvae, which give rise to a hematopoietic lineage. These lymph glands also contain pluripotent precursor cells that undergo a limited number of mitotic divisions and differentiate. We further find that the conserved factor Zfrp8/PDCD2 is essential for the maintenance of the HSCs, but dispensable for their daughter cells, the pluripotent precursors. Zfrp8/PDCD2 is likely to have similar functions in hematopoietic stem cell maintenance in vertebrates.

Fig. 1.

Four types of clones are identified in wild-type third instar larval lymph glands. (A) Wild-type clones. Proportion of MARCM clones generated at different time points after fertilization. Numbers of glands with each clone type and glands from mosaic animals with no clones are shown. Percentage (y axis) and numbers of each clone type (on column) are indicated. (B) Zfrp8 mutant clones. No type 1 clones are detected, and the percentage of type 2 clones is reduced. (C-G′) Confocal projections of mid-third instar lymph glands. (C,C′) Type 1 are cohesive clones, typically stretching from the medulla into the cortex and across all or some of the three to four cell layers (see intensity of GFP), occupying 10-30% of the lobe. (D-E′) Two examples of mixed population type 2 clones, located in different parts of the lymph gland, encompassing 4-8% of the lobe. (F,F′) Type 3 clones mainly consist of cells scattered in the cortex. (G,G′) Type 4 clones consist of one to eight cells, always in the cortex. (H) Schematics of third instar larval lymph gland primary lobes with different types of typical clones (green), based on multiple clones. (For more information, see Fig. 2.) PSC, posterior signaling center (red); MZ, medullary zone (gray); CZ, cortical zone (light blue).

Fig. 2.

Distribution of wild-type and Zfrp8 mutant lymph gland clones. (A,A′) The largest type 1 clones generated in early embryogenesis (2-6 hours) might be derived from a primordial cell. (B-C) Type 1 clones characteristically originate in the central part of the medulla (outlined with yellow dotted line) and extend into the cortex (between yellow and white dotted lines). (C,D) All type 1 clones investigated (10/10) and some of the type 2 clones (4/10) contain cells that are in immediate contact with the PSC. (E-G′) The majority of wild-type and mutant type 2 clones are located in the cortex, but can also be found in medullar cells next to the cortex. (H-J′) Wild-type and Zfrp8 mutant type 3 clones develop scattered patterns in the cortical zone of mid-(H,H′) and late (I-J′) third instar glands. (K-L′) Type 2 and 4 wild-type and Zfrp8 clones in the two lobes of one lymph gland. Clones are shown in green, PSC is marked by nuclear Antp staining (A,C-E,G,H,J, red), and the cortical zone is marked by membrane P1 (J, red), and cytoplasmic PPO staining (B,F,I,K,L, red). Scale bars: 20 μm.

Fig. 3.

Lack of Zfrp8 affects HSCs but not prohemocyte pluripotency. (A-B′) Confocal crossections of wild-type (A,A′) and Zfrp8 mutant (B,B′) clones (white in A,B; outlined in A′,B′) encompassing prohemocytes (no marker), plasmatocytes (P1, red) and crystal cells (PPO, green). Scale bars: 20 μm. (C) Hematopoiesis in the Drosophila lymph gland. During embryogenesis primordial cells form hematopoietic stem cells (HSCs) and hemocyte precursors (HP). HSCs undergo an asymmetric division, self renewing and giving rise to HPs, which undergo a number of mitotic divisions, migrate towards cortex and undergo gradual differentiation (Jung et al., 2005; Mandal et al., 2007). We call committed cells at intermediate stages of differentiation prohemocytes (PHs). Advanced PHs (PH^P) express the early cortical marker Pxn, and during the third instar larval stage differentiate into plasmatocytes (PM), crystal cells (CC) and lamellocytes (LM). Zfrp8 functions in HSC maintenance.

Fig. 4.

Hemocyte differentiation in lymph glands. Wild-type (A-B′) and Zfrp8 mutant (C-D′) lymph glands. Pxn (red) is expressed in the cortex of wild type (A) and Zfrp8 mutant (C) second instar lymph glands. The medulla of wild-type glands (A-B′) is defined by the absence of Pxn, whereas the cortex shows a sharp gradient of staining. Zfrp8 mutants show relatively normal Pxn staining in second instar lymph glands (C,C′) and rapid loss of medulla cells in early third instar glands (D,D′), where virtually all cells are Pxn-positive (note shallow gradient of staining attaining inner cortex levels of wild type). Scale bars: 20 μm.