A Serrate-expressing signaling center controls Drosophila hematopoiesis

Abstract

The differentiation of Drosophila blood cells relies on a functional hierarchy between the GATA protein, Serpent (Srp), and multiple lineage-specific transcription factors, such as the AML1-like protein, Lozenge (Lz). Two major branches of Drosophila hematopoiesis give rise to plasmatocytes/macrophages and crystal cells. Serrate signaling through the Notch pathway is critical in the regulation of Lz expression and the specification of crystal cell precursors, thus providing a key distinction between the two lineages. The expression of Serrate marks a discrete cluster of cells in the lymph gland, a signaling center, with functional similarities to stromal signaling in mammalian hematopoiesis.

Figure 1

Notch signaling in hematopoiesis. (A) Wild-type first lymph gland lobe from a third instar larva raised at 29°C and stained with antibodies against Lz (green) and Serpent (red). Lz is expressed in a distinct subset of Srp⁺ hemocytes. (B) N^ts1 raised at 29°C and stained as in A. Lz protein is not expressed, but Srp (red) expression remains unaffected. (C) Su(H)^SF8/Su(H)^AR9 lymph gland stained with Lz antibody. Lz protein is not expressed. (D–F) hsp70-flp/FRT clones (arrows) of Notch pathway members are marked by the absence of βGal expression (red). Lz expressing cells (green) are excluded from mutant clones of N^55e11 (D), N^ts1 (E), and Su(H)^Δ47 (F). (G) hsp70-flp; Ay-Gal4, UAS-βGal/+. In this control “flp-out” experiment (Ito et al. 1997), βGal-expressing clones were induced in the first larval instar and analyzed in the third instar larval lymph gland. Nuclear βGal (green) marks cells in which Gal4 is expressed. Many examples of colocalization of Lz and βGal can be seen (inset). (H) hsp70-flp; Ay-Gal4, UAS-βGal/UAS-N^DN. In this genotype, βGal⁺ cells (green) also express N^DN. Lz (red) and N^DN (green) are mutually exclusive (inset), suggesting a requirement for Notch in the development of Lz⁺ cells. Additionally, the lymph gland size is reduced relative to wild-type controls. (I) Cell counts from the Notch clonal analysis described as in G and H. For wild-type clones (histogram 1), 20 lymph gland lobes were counted and of 1652 total Lz⁺ cells, 768 were also βGal⁺. N^DN clones were either counted from equivalent number of lymph gland lobes (histogram 2) or equivalent number of Lz⁺ cells (histogram 3). From 20 lymph gland lobes counted (histogram 2), of 153 total Lz⁺ cells, 148 did not express βGal and, therefore, N^DN. Similarly, of 1370 total Lz⁺ cells counted from 68 lymph gland lobes (histogram 3), only 55 expressed βGal. In either case, ∼96% of the Lz⁺ cells did not express βGal. (J) hsp70-flp; Ay-Gal4, UAS-βGal/+. Experimental conditions are the same as in G. Tunel staining (red) marks apoptotic cells. (K) hsp70-flp; Ay-Gal4, UAS-βGal/UAS-N^DN. Experimental conditions are the same as in H. Tunel staining (red) is similar to wild type (J). (L) Dorsal view of a wild-type stage 12 embryo. Anterior is to the left. Expression of Lz protein is observed in crystal cell precursors (circled) of the head mesoderm. Additional ectodermal expression of Lz (arrow) is unrelated to its role in hematopoiesis (Lebestky et al. 2000). Cell counts were performed for 30 bilateral clusters of Lz⁺ cells. (M) Dorsal view of a N^55e11 stage 12 embryo. Fewer Lz expressing crystal cell precursors (circled) are seen. In contrast, Lz expression in the ectoderm is expanded (arrow), due to the neurogenic phenotype of Notch, unrelated to its role in hematopoiesis. In C, G–H, and J–K, the dotted line marks the outline of the lymph gland lobe. Bars: A–C,G–H,J–M, 25 μm; D–F, 10 μm.

Figure 2

Serrate/Notch signaling is necessary and sufficient for crystal cell development. (A) Wild-type first lymph gland lobe from a third instar larva. Lz protein (green) is expressed in a subset of Srp⁺ hemocyte precursors (red). (B) Ser^Bd3/+. Lz protein is absent, but Srp (red) is unaffected. (C) Whole-mount third instar Bc/+ larva. Crystal cells in circulation and in sessile populations along the dorsal body wall are visible due to precocious melanization in the Black cells (Bc/+) genotype. (D) Bc/+; Ser^Bd3/+. Mature crystal cell development is largely suppressed by the dominant Ser mutation. (E–F) hsp70-flp/FRT clones of Ser^Rx82. Clones (arrow) are marked by the absence of βGal (red) expression. Lz⁺ cells (green) are generally excluded from mutant clones (E). However, rare examples (4 such cells seen of 92 Lz⁺ cells counted) of Ser⁻/Ser⁻ cells at the edge of a mutant clone were found to express Lz (F, arrowhead). (G–M) Ectopic development of crystal cells upon misexpression of N^act or Ser. (G) Wild-type lymph gland stained for Lz protein (red). (H) hsp70-Gal4/UAS-N^act. Transient misexpression of N^act during the third larval instar causes a dramatic increase in the number of Lz⁺ cells. (I) hsp70-Gal4/+; UAS-Ser/+. Similar misexpression of Ser causes a more moderate increase in Lz⁺ cells. (J) Wild-type second lobe of the third instar lymph gland contains very few Lz⁺ cells (arrow). (K) hsp70-Gal4/UAS-N^act second lobe. A large number of Lz⁺ cells are seen upon ectopic activation of the Notch pathway. (L–M) Sustained misexpression of Ser can create ectopic signaling centers. (L) Second lobe from hsp70-flp; Ay-Gal4, UAS-βGal/+ lymph gland is outlined. Random flp-out clones express βGal (green), but no Lz⁺ cells are seen (red). (M) hsp70-flp; Ay-Gal4, UAS-βGal/+; UAS-Ser/+. In this genotype, βGal⁺ cells (green) also express Ser, causing ectopic development of Lz⁺ cells (red). Bars: A,B,E,F, 10 μm; C,D, 100 μm; G–M, 25 μm.

Figure 3

Serrate expression defines a signaling center. (A) Ser protein-expressing cells are seen concentrated near the posterior end of the lymph gland (lower left corner). Additionally, dispersed cells throughout the gland also express Ser protein. (B–D) Identical flattened confocal images of the PSC region of Ser-βGal lymph gland showing Ser-βgal expression (red, B), Ser protein expression (green, C), and merged image (D). Ser protein expression and Ser-βGal colocalize in the Posterior Signaling Center (PSC) adjacent to a pericardial cell (arrowhead). (E–G) Identical flattened confocal images of a first lobe, stained for Srp (red, E), Ser-βGal (green, F), and merged (G). Ser-βGal-expressing cells colocalize with Srp in the PSC (arrowhead). (F, inset) Ser⁺ cells that appear to be migrating from the PSC (arrow). (H–I) The expression patterns of Lz protein (green) and Ser⁺ cells marked by either Ser protein (red, H), or Ser-βGal (red, I) are mutually exclusive. Multiple examples of Lz⁺ cells in close proximity to βGal⁺ cells can be seen. Visualization of the lower level of Ser-βGal expression in cells inside the lobe requires increased laser power on the confocal microscope, used here but not in E–G. (J) Expression of Lz (green) in the lymph gland is spatially distinct from the PSC marked by high-level expression of Ser-βGal (red). (K) N^ts1; Ser-βGal/+. Ser expression (arrowheads) in the PSC is unaffected in the N^ts1 mutant. Two bilaterally symmetric anterior lobes (arrowheads) are shown in this panel. (L) hsp70-flp; Ay-Gal4, UAS-GFP/UAS-N^DN. Experimental conditions are the same as in Figure 1H. Cells expressing GFP (green) also express N^DN and are preferentially observed in the PSC region (arrowhead). Apoptotic cells (red) are not observed in the PSC region. (M,N) Flattened confocal images of Ser-βGal expression (red) marking the PSC and BrdU incorporation (green) in a first lobe of third instar lymph gland. Ser-βGal and BrdU are mutually exclusive in the PSC region (arrowhead). BrdU was incorporated for either 1 h in vitro (M), or for 18 h in vivo (N). Bars: A–G,J–N, 25 μm; H–I, 10 μm.

Figure 4

Activation of the Notch pathway precedes Lz expression. (A) Ser-βGal (green) and Srp (red) expression in second instar larval lymph glands. (B) Ser protein expression is also observed within the smaller posterior lymph gland of a third instar larva. Lz is rarely expressed (Fig. 2J). (C) lz^r15; Ser-βGal/+. Ser expression (green) is unaffected in a lz null mutant background. DAPI (blue) marks nuclei within the lymph gland. (D–E) N^ts1; 12XSu(H)–βGal/+ first lobe from third instar lymph gland. The 12XSu(H)–βGal transgene marks cells receiving the Notch signal (Go et al. 1998). (E) When raised at 29°C, the nonpermissive temperature, βGal is no longer expressed. (F) lz^r15; 12XSu(H)–βGal/+. βGal expression (green) is maintained in lz null mutant background. (G–I) 12XSu(H)–βGal. Identical confocal images of a third instar lymph gland stained for Lz protein (red, G), βGal (green, H), and merged (I). (I) Arrowheads point to cells that coexpress βGal and Lz (see text for details). (J) Schematic representation of third instar larval lymph gland. Four to six pairs of lymph gland lobes along the dorsal vessel (heart) are separated by pericardial cells. Srp is expressed in hemocyte precursors in lymph gland lobes and also in pericardial cells. Lz is expressed in crystal cell precursors in the first lobe and rarely, if at all, in the further posterior lobes. Ser is expressed at the PSC and also inside the first lobes and other posterior lobes. Bars: A–E, 25 μm; F–I, 10 μm.