A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors - PubMed (original) (raw)

A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors

Lolitika Mandal et al. Nature. 2007.

Abstract

The Drosophila melanogaster lymph gland is a haematopoietic organ in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work has defined three domains, the medullary zone, the cortical zone and the posterior signalling centre (PSC), within the developing third-instar lymph gland. The medullary zone is populated by a core of undifferentiated, slowly cycling progenitor cells, whereas mature haemocytes comprising plasmatocytes, crystal cells and lamellocytes are peripherally located in the cortical zone. The PSC comprises a third region that was first defined as a small group of cells expressing the Notch ligand Serrate. Here we show that the PSC is specified early in the embryo by the homeotic gene Antennapedia (Antp) and expresses the signalling molecule Hedgehog. In the absence of the PSC or the Hedgehog signal, the precursor population of the medullary zone is lost because cells differentiate prematurely. We conclude that the PSC functions as a haematopoietic niche that is essential for the maintenance of blood cell precursors in Drosophila. Identification of this system allows the opportunity for genetic manipulation and direct in vivo imaging of a haematopoietic niche interacting with blood precursors.

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Figures

Figure 1. Embryonic specification of the PSC by Antp

a, Schematic representation of the development of the Drosophila lymph gland. T1–T3, three thoracic segments; dv, the dorsal vessel; cb, cardioblast; lgprim, lgsec lgtert, primary, secondary and tertiary lobes, respectively, of the lymph gland; and pc, pericardial cells. b–e, Immunohistochemical analysis showed that the lymph gland is formed by the fusion of the three Odd-positive cell groups (T1, T2 and T3). Antp expression is confined to a group of cells at the posterior boundary of T3 (b, e). The cells that will become larval PSC remain confined to the posterior edge of the embryonic lymph gland (e). f, g, Fas III (Fasciclin III, a homophilic cell adhesion molecule) is upregulated in the PSC. h, In the late embryo, the PSC cells incorporate BrdU. i, Antp expression is maintained in the embryonic col mutant background. j, k, Expression of col, detected by in situ hybridization, in the PSC (WT, arrow in j) is eliminated in an Antp mutant background (arrow in k). l, Antp protein is expressed in the PSC, whereas Hth protein is seen in the rest of the lymph gland. m, n, In the hth mutant background (m), the lymph gland is virtually eliminated (arrow). Overexpression of hth (twist-gal4, UAS-hth; n) causes a reduction in the number of cells in the PSC. All colours correspond to the marker label in each panel. All images were acquired using a ×40 objective with additional × 1.3 (b, c, d) or ×2.5 (e–n) confocal magnification.

Figure 2. The larval PSC functions as a haematopoietic niche to maintain blood precursors in the medullary zone

a, In wild-type (WT), the PSC consists of a cell cluster located along the posterior edge of the lymph gland. b, Antp expression in the lymph gland is missing in _col_−(PSC−). The residual expression is in the cardioblasts of the dorsal vessel. c, The PSC is greatly expanded on Antp overexpression (PSCexp). d–k′, In wild type, medullary zone markers (d, g, j) are restricted to the precursor population of cells. These markers are all eliminated in the absence of the PSC (e, h, k). Rarely, a much reduced medullary zone can be seen (example shown in k′). Expansion of the PSC causes expansion of the medullary zone (f, i). l–n, In wild-type (l), BrdU incorporation is limited to the cortical zone. In the absence of PSC, cells in more medial regions incorporate BrdU (m), whereas in an expanded PSC genotype (n), the number of cells in S-phase is greatly reduced. o–q, In wild-type (o), differentiating cells reside in the peripheral cortical zone. In the absence of a PSC (p), differentiated cells are found throughout the lymph gland lobe. On expansion of the PSC (q), these cells are restricted to a thin layer along the distal edge. The colour of molecular markers for each row corresponds to those of the side labels (the label ‘domelessGFP’ corresponds to the expression of GFP under the control of domeless-gal4; ‘SerLacZ’ is expression of β-galactosidase under the control of the Ser9.5 (ref. 4) enhancer). All images were acquired using a 40× objective. The PSC panels (b, e, h, k, k′, m, p) also reflect an additional ×1.5 confocal magnification.

Figure 3. A Hedgehog signal from the PSC is required for the maintenance of the precursor cell population of the medullary zone

a, Wild-type (WT) expression of the cortical zone markers P1 (green) and ProPO (red). b, Third-instar _Antp_-mutant larvae show a lymph gland phenotype similar to that seen in col mutants (c). hhts2 (d) shows an identical phenotype (compare to c). Misexpression of dominant-negative Ci (e, f) phenocopies Antp, col and hh loss-of-function lymph gland phenotypes (b–d). g–i, Hh expression (green) is restricted to the PSC in the second (g) and third instars (h, i). All cells expressing Antp (red in i) also express Hh (yellow in i). A few dispersed cells (arrowhead) in the cortical zone also initiate Hh expression (i). j–l, The PSC (Antp in green) is present in hhts2 mutants (k, l) as in wild-type (j). m, Hh expression marks the PSC and Ptc marks the medullary zone. n–p, Ptc and Ci (red) expression co-localizes with the medullary zone marker domeless-gal4, UAS-GFP (labelled ‘Dome’). Ci forms a gradient with highest staining intensity near the PSC. The asterisk marks the PSC. Haematopoietic markers for each panel are colour-coded. All images were acquired using a ×40 objective. Panels (c, d, k and l) reflect an additional ×1.5 confocal magnification. The confocal magnification used for the close-up of the PSC in panel (i) was ×3.

Figure 4. PSC cells exhibit extensive processes that project into the lymph gland

a, b, GFP expressed exclusively in PSC cells (using Antp-gal4, UAS-mCD8_–_GFP) reveals the presence of numerous thin processes that extend over several cell diameters into the medullary zone. c, d, Analysis of PSC morphology in live animals. Whole-mount live third instar larva showing GFP expression in the PSC. A close-up view (d) of the region indicated by the arrowhead in (c), showing processes extending into the lymph gland. dv, dorsal vessel. e, A schematic representation of the PSC region of the lymph gland as a niche involved in the maintenance of medullary zone progenitors. All images were acquired using a ×40 objective except panel c, which was taken with a ×10 objective. Panels a,b and c and d, reflect additional ×6, ×2.5, and ×8 confocal magnification, respectively.

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A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors - PubMed (original) (raw)