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Specification of Drosophila Hematopoietic Lineage by Conserved Transcription Factors

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Science  07 Apr 2000:
Vol. 288, Issue 5463, pp. 146-149
DOI: 10.1126/science.288.5463.146

Abstract

Two major classes of cells observed within theDrosophila hematopoietic repertoire are plasmatocytes/macrophages and crystal cells. The transcription factor Lz (Lozenge), which resembles human AML1 (acute myeloid leukemia– 1) protein, is necessary for the development of crystal cells during embryonic and larval hematopoiesis. Another transcription factor, Gcm (glial cells missing), has previously been shown to be required for plasmatocyte development. Misexpression of Gcm causes crystal cells to be transformed into plasmatocytes. The Drosophila GATA protein Srp (Serpent) is required for both Lz and Gcm expression and is necessary for the development of both classes of hemocytes, whereas Lz and Gcm are required in a lineage-specific manner. Given the similarities of Srp and Lz to mammalian GATA and AML1 proteins, observations in Drosophila are likely to have broad implications for understanding mammalian hematopoiesis and leukemias.

Hematopoietic stem cells give rise to all blood cell lineages in mammals (1). Molecular decisions that differentiate one lineage from another are regulated by unique protein complexes constituted of general as well as lineage-specific transcription factors (2). Gene inactivation studies in mice have identified an important role for a number of hematopoietic transcription factors. For example, GATA-1 is required for erythroid development (3), GATA-2 for definitive hematopoiesis (4), and GATA-3 for T cell development (5). Interestingly, the DrosophilaGATA homolog serpent (srp) is required for embryonic blood cell development (6). Another mammalian gene, encoding the AML1 protein, is the most frequent target of chromosomal translocations in acute myeloid leukemias (7). AML1, like GATA-2, is essential for all definitive hematopoiesis (8). However, the relation between GATA proteins and AML1 in mammals is unclear. The DrosophilaLz protein shares 71% identity to AML1 within the Runt domain and regulates the expression of multiple transcription factors during eye development (9). Here, we describe the role of Lz inDrosophila hematopoiesis and investigate its relation to Srp as well as to another transcription factor, Gcm (10, 11), in the generation of Drosophila blood cell lineage.

Hemocytes of the Drosophila embryo are derived from the head mesoderm (12) (Fig. 1, A to C). The hemocyte precursors express the GATA factor Srp (6, 13) and give rise to two classes of cells: plasmatocytes and crystal cells (14). Plasmatocytes spread throughout the endolymph (Fig. 1, B and C) and act as macrophages, whereas crystal cells contain crystalline inclusions and are involved in the melanization of pathogenic material in the hemolymph (15). These cells can be first recognized in the late embryo, where they form a cluster around the proventriculus (Fig. 1, C and G). Crystal cells are made clearly visible by the Black cell (Bc) mutation, which causes premature melanization of the crystalline inclusions (16).

Figure 1

Drosophila hematopoiesis and the requirement of lozenge for crystal cell development. (A to D) Schematic representation of hematopoiesis during embryonic (A to C) and larval (D) development. Dorsal is up, anterior to the left; br, brain; dv, dorsal vessel; e, esophagus; lb, labial segment; m, maxillary segment; pv, proventriculus. In (A), a stage 5 embryo, the anterior endoderm (ae) is shown in light purple, the hemocyte anlagen (he) in green, and the lymph gland anlagen (lg) in blue. In (B), a stage 11 embryo, CCPs (red) are specified from hemocyte precursors (gray). Lymph gland precursors (blue) are visible in the ventral-lateral trunk mesoderm. In (C), a stage 17 embryo, CCPs cluster around the proventriculus (pv). The lymph glands (blue) are visible along the dorsal vessel. The plasmatocytes (gray) are dispersed within the endolymph. In (D), a third-instar larva, crystal cells (red) and plasmatocytes (gray) circulate freely throughout the hemolymph. All hemocytes are produced from the lymph glands (blue). (E toH) lzts1; Bc/+ flies raised at permissive (25°C) or nonpermissive (29°C) temperature. In (E), a third-instar larva raised at 25°C displays a normal distribution of crystal cells (arrowhead). In (F), a third-instar larva raised at 29°C lacks crystal cells. In (G), a stage 17 embryo raised at 25°C, crystal cells (arrowhead) are clustered around the proventriculus. In (H), a stage 17 embryo raised at 29°C, crystal cells are absent. Scale bars, 300 μm (E and F), 50 μm (G and H).

In larval stages, hemocytes are produced from a separate organ called the lymph gland (15, 17). Precursors of this gland first appear during embryogenesis in the dorsal mesoderm of the thoracic segments (18) (Fig. 1B). Later, these precursors migrate dorsally, forming a tight cluster adjacent to the dorsal vessel, the larval circulatory organ (Fig. 1C). The larval lymph glands form a bilateral chain of cell clusters (“lobes”) flanking the dorsal vessel (Fig. 1D).

In the temperature-sensitive allelelzts1 , crystal cells develop normally at 25°C (Fig. 1, E and G). However, crystal cell development is completely blocked at 29°C (Fig. 1, F and H). Consistent with earlier genetic analysis (19), crystal cells are missing inlz null mutant alleles (20). Plasmatocytes develop normally in number and pattern in lz null embryos (21).

Temperature shifts of lzts1; Bcflies showed that Lz function during stages 10 to 14 of embryogenesis is essential for crystal cell development (22). Crystal cells formed in the embryo do not persist into late larvae, and Lz function is continuously required during the late larval stages for further crystal cell development. The time scale for de novo crystal cell development in the larva is about 4.5 hours (22).

Lz is first detected in a small cluster of cells within the embryonic head mesoderm in a bilaterally symmetric pattern (Fig. 2A) (23). Lz expression remains localized in bilateral clusters of 20 to 30 cells within the head mesoderm (Fig. 2, B and C). At later stages, these crystal cell precursors (CCPs) form a loose cluster around the proventriculus (Fig. 2D). These cells have smooth, round morphology with large nuclei (Fig. 2E). The CCPs form a subset of the Srp-expressing hemocyte precursors (Fig. 2, F to H).

Figure 2

Expression of Lz during embryonic hematopoiesis. (A to C) Dorsal views. In (A), a stage 10 embryo shows in situ localization of lzmRNA. In (B), a stage 12 lz-gal4/+; UAS-NlacZ /+ embryo shows immunolocalization of a nuclear form of β-Gal in a lzpattern. In (C), a stage 13 embryo shows immunolocalization of Lz protein. (D to H) lz-gal4/+; UAS-NlacZ/+embryos. Immunolocalization of β-Gal protein is shown (brown in D and E, green in F and H). In (D), a stage 17 embryo, the black arrowhead denotes the Lz-expressing cluster of crystal cells around the proventriculus. A white arrowhead points to expression of Lz in the gnathal region, which is unrelated to the expression in the CCPs and has no known phenotypic consequences. In (E), a stage 17 embryo at higher magnification shows Lz expression. Note the large nuclei and the round morphology of the crystal cells. In (F), a stage 13 embryo shows Lz expression (green). In (G), the same section as in (F) shows immunolocalization of Srp (red); an arrowhead marks the position of a CCP cluster, and an arrow points to circulating plasmatocytes. In (H), a merged image of (F) and (G) displays colocalization of Lz and Srp (yellow). (I) Stage 11lz-gal4; UAS-lacZ embryo showing immunolocalization of phospho-Histone H3A (black) and cytoplasmic β-Gal (brown). A white arrowhead marks a nondividing β-Gal–expressing cell; a black arrowhead marks colocalization of phospho-Histone H3A (black) and β-Gal (brown) in a dividing cell. (J) Stage 13lz-gal4; UAS-lacZ embryo showing expression of the plasmatocyte-specific marker Croquemort (red) in circulating embryonic plasmatocytes. An arrow points to Croquemort expression in a plasmatocyte that lacks expression of β-Gal. Arrowheads point to plasmatocytes that express both Croquemort and β-Gal (green). Scale bars, 50 μm (A to D and F and J), 8 μm (E and I).

Colocalization with a mitotic marker suggests that Lz-expressing cells can divide (Fig. 2I). Interestingly, not all of the daughter cells from these divisions will become crystal cells. This is inferred from the observation that lz-lacZ expression is also seen in a group of plasmatocytes (Fig. 2J) that do not express lzmRNA or Lz protein. We interpret the expression of lz-lacZin these cells to be due to the long half-life of β-galactosidase protein that is left over from the parent cell. This is also observed with additional, independent lz promoter fusions to lacZ (20). Thus, Lz is expressed in a small subset of hemocyte precursors that may undergo cell division. All crystal cells resulting from these precursors maintain Lz expression. The few daughter cells that will differentiate into plasmatocytes do not express Lz protein.

In the larval lymph gland, Lz expression is initiated in a small number of cells during the second larval instar (Fig. 3A) (24). The number of cells expressing Lz steadily increases during the third larval instar, reaching 50 to 100 cells per lobe (Fig. 3, B and C). Lz-expressing cells are scattered uniformly throughout the large, primary lobe of the lymph gland, whereas the smaller secondary lobes do not express Lz. Similar to the embryonic head mesoderm, all lymph gland cells express Srp, but only a small subset of them express Lz (Fig. 3, D to F). Interestingly, the Lz-expressing cells appear to down-regulate Srp when compared to the surrounding non–Lz-expressing hemocyte precursors (inset, Fig. 3, D and E).

Figure 3

Expression of Lz during larval hematopoiesis in lz-gal4/+; UAS-NlacZ/+ (A,B, D to F) and wild-type (C) larvae. Immunolocalization of β-Gal is shown as a reporter for Lz (green) and Srp (red). Lz expression is revealed within a single anterior lobe of a second-instar larval lymph gland (A) and within a single anterior lobe of a third-instar larval lymph gland (B). Immunolocalization (with antibody to Lz) of Lz protein expression in a single anterior lobe of a third-instar larval lymph gland is shown in (C). Expression is similar to lz reporter expression seen in (B). A 1-μm confocal optical section of a single lobe of a third-instar larval lymph gland is shown in (D); an arrowhead points to a Lz-expressing cell (magnified in inset), and an arrow points to a cell lacking Lz expression. In (E), the same section as in (D) shows Srp protein expression; an arrowhead points to a cell expressing both Srp and Lz (magnified in inset), and an arrow points to a cell expressing high levels of Srp but no Lz. In (F), a merged image of (D) and (E) displays colocalization of Lz and Srp. Scale bars, 25 μm (A), 50 μm (B to D).

Immunolocalization studies (25) of circulating hemocytes in third-instar larvae suggest that the expression of Lz protein is maintained in circulating crystal cells (21). Given that crystal cells are missing in lz mutants, this demonstrates an autonomous requirement for Lz in crystal cell development. As observed for embryonic hemocytes, Lz-expressing precursors give rise to all crystal cells and a small subset of plasmatocytes, as evidenced by morphology as well as expression of the plasmatocyte marker Croquemort (21). However, Lz protein is not observed in any circulating larval plasmatocytes.

An allele of srp (srpneo45 ) specifically abolishes Srp expression in embryonic hemocytes (6). Because this allele also eliminates lz mRNA expression, Srp function is required for the expression of Lz (Fig. 4, A and B). This finding establishes that srp functions upstream of lz during embryonic hematopoiesis. The lethality of srp precludes the analysis of Lz expression in larval lymph glands of srpmutants. However, as in the embryo, Srp is expressed earlier than Lz in the larval hemocyte precursors, which suggests that srpacts upstream of lz during both developmental stages.

Figure 4

Functional relations among Srp, Lz, and Gcm. (A and B) In situ localization oflz mRNA expression (arrowhead) in the head mesoderm of stage 13 srpneo45/+ (A) andsrpneo45 / srpneo45 (B) embryos. Lz expression is absent insrpneo45/srpneo45 embryos. (C) Immunolocalization of Lz protein in stage 11gcmN7-4 embryo. Lz expression is unaffected ingcm mutants. (D to F) Stage 11gcm-lacZ/+ embryo. In (D), Gcm (green) is expressed in a majority of the hemocyte precursors; an arrowhead points to a gap in Gcm expression. In (E), Srp protein (red) is expressed in all hemocyte precursors. In (F), a merged image of (D) and (E) shows that Srp and Gcm do not colocalize in a small field of cells (arrowhead) presumed to be the CCPs. (G toI) Stage 13 gcm-lacZ/+ embryo. In (G), Gcm (green) is expressed in the majority of hemocyte precursors. In (H), Lz protein (red) is expressed in the CCPs. In (I), a merged image of (G) and (H), Lz and Gcm are mutually exclusive. (J toL) Stage 14 lz-gal4/+; UAS-lacZ/+; UAS-gcm embryo. In this genotype, Gcm protein is misexpressed in CCPs. In (J), Lz (green) is expressed in the head mesoderm. In (K), Croquemort (red) is expressed in plasmatocytes. In (L), a merged image of (J) and (K) shows that misexpression of Gcm results in expression of Croquemort in the Lz-expressing cells of the head mesoderm, indicating transformation of CCPs into plasmatocytes. (M) Stage 17lz-gal4/+; UAS-NlacZ/+ embryo, showing the smooth round morphology of the crystal cells. (N) Stage 17lz-gal4/+; UAS-lacZ/+; UAS-gcm embryo. The Lz-expressing cells that also express Gcm show altered morphology resembling circulating plasmatocytes. (O) Third-instar lz-gal4/+; Bc/UAS-gcm larva. Misexpression of Gcm in larval CCPs causes a complete loss of mature crystal cells in the hemolymph. (P) Model for the generation of hematopoietic lineages in theDrosophila embryo. Srp is expressed earliest in the hemocyte precursor pool. Most of the Srp-expressing cells begin to express Gcm and differentiate as plasmatocytes. A small subset of Srp-expressing cells begin to express Lz; these cells are still mitotic and give rise to all the crystal cells and a very small fraction of the plasmatocytes. Scale bars, 50 μm (A and B, D to I, J to L), 8 μm (M and N), 200 μm (O).

The transcription factor Gcm promotes glial cell fate, and it also functions downstream of Srp in plasmatocyte differentiation (10). Lz expression is unaffected in gcm mutants (Fig. 4C). Gcm expression (26) is initiated in a number of Srp-expressing hemocyte precursors (Fig. 4, D to F), but Gcm is excluded from the CCPs (Fig. 4, G to I). Consistent with their cell fate, the small subset of plasmatocytes derived from Lz-expressing progenitors do initiate Gcm expression (21).

We misexpressed Gcm in the CCPs to assess whether exclusion of Gcm from these cells is essential for proper fate determination. This resulted in the transformation of CCPs into plasmatocytes (Fig. 4, J, K, L, and N). The converted cells exhibit morphological characteristics of plasmatocytes (Fig. 4N) and express Croquemort (Fig. 4, J to L). Moreover, in third-instar larvae, misexpression of Gcm in CCPs prevents the development of all crystal cells (Fig. 4O). These results suggest that the restricted expression of Gcm is required for the developmental program of embryonic plasmatocytes, and that its misexpression can override Lz-mediated crystal cell differentiation during both embryonic and larval hematopoiesis. The converse experiment of Lz misexpression in the entire hemocyte pool under the control of a heat shock promoter did not convert plasmatocytes into crystal cells (20). Vertebrate homologs of Gcm have been identified (27), but any role in hematopoiesis has not been investigated.

We arrive at a model of Drosophila hematopoiesis (Fig. 4P) in which a pool of Srp-positive hemocyte precursors gives rise to a large population of Gcm-positive cells and a smaller subpopulation of Lz-positive cells. Our results support a genetic hierarchy in which Srp, a Drosophila GATA factor, acts upstream of both Gcm and Lz, two mutually exclusive, lineage-specific transcription factors in hematopoiesis. Although the description of this hierarchy is incomplete in terms of the breadth of molecules involved, it does provide a theoretical framework for understanding how early hematopoietic progenitors in the embryo can differentiate and assume distinct cell fates.

Recent findings have identified similarities between mammalian andDrosophila innate immunity (28). Given the similarities of Srp and Lz to mammalian GATA and AML1 proteins, the results reported here suggest a conservation of the molecular basis for blood cell lineage commitment in mammalian and Drosophilahematopoiesis.

  • * These authors contributed equally to this report.

  • To whom correspondence should be addressed. E-mail: banerjee{at}mbi.ucla.edu

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