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First published online 27 April 2005
doi: 10.1242/dev.01837

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The Drosophila lymph gland as a developmental model of hematopoiesis

Seung-Hye Jung^*, Cory J. Evans^*, Christine Uemura and Utpal Banerjee

Department of Molecular, Cell and Developmental Biology, Department of Biological Chemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA

View larger version (136K): [in a new window]   Fig. 1. Morphological features of the developing lymph gland. (A,B) Lymph glands at embryonic stage 16. Cells of the embryonic lymph gland (shown in whole mount) express the hemocyte marker Srp (A, red) and Odd (odd-lacZ expression in B, green; the bluish-green appearance is due to overlap with nuclear marker To-pro-3 in blue). Pericardial cells (B, arrowhead), also express Odd. Cells between the two symmetric lymph gland clusters represent dorsal vessel (DV) cells (B). Scale bar: 10 µm. (C,D) Second instar larval lymph gland. Expression of Srp (C) and odd-lacZ (D) is maintained in the second instar. By this stage, the lymph gland consists of a pair of primary (1°) lobes and smaller, posterior secondary (2°) lobes separated by pericardial cells (arrowhead in D). Scale bar: 10 µm. Expression of odd-lacZ is slightly higher in the region of the posterior signaling center (PSC) of the primary lobes. (E) Multi-lobed structure of the third instar lymph gland. Srp (red) is ubiquitously expressed. Pericardial cells separate lymph gland lobes as in the second instar. Scale bar: 100 µm. (F,G) Expression pattern of Viking-GFP fusion protein (F, green) reveals chamber-like structures within the lymph gland. Similar structures are seen in lymph glands of the uncharacterized GFP-trap line ZCL2867 (G, green). Scale bar: 10 µm. To-pro-3 (blue) shows nuclei. (H,I) Morphological features of the third instar lymph gland. (H) A DIC image of the third instar lymph gland shows two distinct regions within the primary lobe that we call the cortical zone (CZ) and the medullary zone (MZ) (see text for details). Arrows indicate the boundary between the zones. The secondary (2°) lobe is similar in appearance to the medullary zone. (I) Confocal section through a lymph gland from the GFP-trap line G147. Cells in the medullary zone (MZ, outlined) are compactly arranged, whereas cells in the cortical zone (CZ) exhibit voids and gaps. Scale bar: 10 µm.

View larger version (106K): [in a new window]   Fig. 2. Maturing hemocytes are found in the cortical zone. (A) Cg-gal4/UAS-GFP-expressing cells (green) are found in the periphery of the primary lobe. (B,B') Flattened confocal image of a third instar G147 lymph gland. GFP expression (green) identifies the cortical and medullary zones. Cg-gal4/UAS-lacZ (B', red) expression is restricted to the cortical zone (CZ) and is excluded from the medullary zone (MZ). (C-G) Mature hemocyte markers are restricted to the cortical zone. Hemocyte markers hml-gal4/UAS-GFP (C, green) and Pxn (D, red), crystal cell markers Lz (E; red) and Black cells (Bc, F), and plasmatocyte marker P1 (G, red) are limited to the cortical zone. (H,I) The uncharacterized GFP-trap line ZCL2826 (H, green) and the transcription factor Cut (I, red) are preferentially expressed in the cortical zone. (J,K) Lamellocytes, when present, are found in the cortical zone. Lamellocytes can be distinguished by morphology in G147 background (J,K, green, arrows) and by co-localization with msn-lacZ (K, red, arrow). To-pro-3 (blue) marks nuclei. All lymph glands presented are of the third larval instar.

View larger version (101K): [in a new window]   Fig. 3. Expression profile of the medullary zone and the PSC. (A-E) Marker expression in the medullary zone. Medullary zone cells are characterized by their expression of DE-cadherin (A, red), dome-gal4/UAS-GFP (B, green), upd3-gal4/UAS-GFP (C, green), the uncharacterized GFP fusion line ZCL2897 (D, green), and act5C-GFP (E, green). The cortical zone is marked by Lz staining (A, green; C, red) as well as by P1-antigen staining (E, red). (F-J) Markers expressed in the posterior signaling center (PSC) cells. PSC cells are marked by Ser-lacZ (F-I, red) and show high levels of expression of Dorothy-gal4/UAS-GFP (F, green), upd3-gal4/UAS-GFP (G, green), uncharacterized GFP-trap lines ZCL2375 (H, green), ZCL2856 (I, green) and ZCL0611 (J, green). In H and I, insets show only the GFP expression in the PSC regions indicated by the arrows. upd3-gal4/UAS-GFP (G) is expressed in only a subset of PSC cells. PC, pericardial cell. (K-O) Lack of expression of specific markers also defines the PSC. PSC cells are positively marked by Ser-lacZ (red) and characterized by absence of expression of Pvr (K, green; inset shows the PSC region without Ser-lacZ for clarity), dome-gal4/UAS-GFP (L, green), Cg-gal4/UAS-GFP (M, green), U-shaped (N, green) and Hemese (O, green). To-pro-3 (blue) marks nuclei.

View larger version (95K): [in a new window]   Fig. 4. Hemocyte development in the secondary lymph gland lobe of third instar larva. (A) The secondary lobe expresses high levels of DE-cadherin (red). (B-E') Hemocyte formation in larger secondary lobes. Cg-gal4/UAS-GFP (B,E', green), hml-gal4/UAS-GFP (C, green) and Lz (D, green) are seen in patches where DE-cadherin (B-E', red) expression is downregulated. This is particularly apparent in high-magnification images (compare E with E'). To-pro-3 (blue) marks nuclei. (F) Schematic diagram of the 3rd instar lymph gland based on our analysis of morphology and hemocyte markers. The lymph gland flanks the dorsal vessel (DV) and each lobe is separated by pericardial cells (PC). The primary (1°) lobe consists of three distinct zones: (1) the cortical zone (CZ), where maturing hemocytes are found; (2) the medullary zone (MZ), with prohemocytes lacking differentiation markers; and (3) the posterior signaling center (PSC), which is a unique population of specialized hemocytes. Secondary (2°) lobes usually contain immature hemocytes except in random sites of maturation (arrowheads).

View larger version (77K): [in a new window]   Fig. 5. Temporal analysis of hemocyte maturation. (A) Hemese (He, red) is expressed by all hemocytes in the primary lymph gland of the second instar larva. (B,C) Maturation marker expression in second instar lymph glands. (B) Expression of Pxn (red) and hml-gal4/UAS-GFP (green) commence at approximately the same time during development. Although most cells express both markers (yellow), some express only hml-gal4/UAS-GFP (arrowhead), and others express only Pxn (arrow). (C) Cg-gal4/UAS-GFP (green) is expressed in a small subset of cells expressing Pxn (red). (D) P1 antigen (red) is first expressed in the early third instar lymph gland. Fewer cells are seen expressing P1-antigen than Cg-gal4/UAS-GFP (green). (E) Schematic representation of the order of appearance of plasmatocyte maturation markers. (F-G) Maturing hemocytes, prohemocytes and pre-prohemocytes in the second instar lymph gland. (F,F') Maturing hemocytes are marked by Pxn expression (F', red). These cells either express low levels of dome-gal4/UAS-GFP (green, white arrowheads) or no dome-gal4/UAS-GFP (yellow arrowheads). Prohemocytes do not express Pxn, but are marked by dome-gal4/UAS-GFP (F,F', arrows). Finally, pre-prohemocytes are located medially, close to the dorsal vessel (DV) and are characterized by the absence of both dome-gal4/UAS-GFP and Pxn expression (asterisks, blue only). These pre-prohemocytes do express Hemese (G; red, arrows). (H-K') The second instar PSC (arrows) are marked by Ser-lacZ (H,J,K'; red). Expression of upd3-gal4/UAS-GFP (I,J, green; RG, ring gland) initiates in the PSC (I, arrows) and expands later to other cells of the lymph gland (J, green). dome-gal4/UAS-GFP (H, green) and Pvr (K,K'; green) are not expressed in the PSC. (L-O) Gal4-based cell-lineage tracing in the larval lymph gland. (L) Schematic diagram of the cell-lineage marking system. Test stock flies of the genotype UAS-FLP, actin5C-FRT-STOP-FRT-lacZ is crossed to various Gal4-expressing lines (enhancer-gal4, UAS-GFP). Gal4 activates GFP (green) and FLP recombinase expression, which then removes the `FLP-out' cassette such that the constitutive actin5C promoter then drives lacZ expression (red) permanently within all subsequent daughter cells. (M) Cortical zone cells are derived from dome-gal4-expressing cells. The dome-gal4 reporter causes cortical zone (CZ) cells in the third instar lymph gland to be permanently marked with ß-gal expression (red) despite the lack of dome-gal4 expression (as assessed by GFP) in this zone. (N) As a positive control, third instar lymph gland cells were found to be permanently marked by ß-gal expression (red) because of twist-gal4 activation in the embryonic mesoderm, from which the lymph gland is derived. No GFP expression is detectable, indicating that twist-gal4 is no longer expressed in the third larval instar. (O) As a negative control, we show that cells of the PSC do not contribute to cortical zone cells. Lineage tracing using Serrate-gal4 revealed that PSC cells remain few in number and do not give rise to cortical zone cells. The majority of the Serrate-expressing cells appear yellow because of simultaneous expression of GFP (green) and ß-gal (red). To-pro-3 (blue) marks nuclei. Scale bars: 10 µm in A-M,O; 100 µm in N.

View larger version (60K): [in a new window]   Fig. 6. Role of Pvr in plasmatocyte differentiation. (A-E') Pvr-/Pvr- mutant clones marked by the absence of GFP (A-E, green) expression. To-pro-3 marks all nuclei (blue). Representative clones are outlined and analyzed for marker expression in A'-E'. P1 antigen (A,A', red) and Pxn (B,B', red) are missing in Pvr-/Pvr- clones, but Hemese (C,C', red) and Lz (D,D', red) expression remain unchanged. TUNEL (E,E', red) staining shows that cell death is not enhanced in the mutant clones.

View larger version (53K): [in a new window]   Fig. 7. Proliferation profile of the lymph gland. (A) Second instar primary lobe stained for BrdU incorporation. A single confocal section from the middle of the lymph gland shows BrdU (red) incorporation in randomly positioned cells. (B,B') Third instar primary lobe stained for BrdU incorporation. A single confocal section from the middle of the lymph gland shows that BrdU+ cells (red) are restricted to the cortical zone marked by Pxn expression (B', green). (C) Cells incorporating BrdU (red) are excluded from the medullary zone marked by dome-gal4/UAS-GFP (green). (D) Quantitation of data represented in C. Twelve third instar lymph gland lobes were counted. The y-axis represents the percentage of total BrdU-positive cells. (E) In secondary lobes of the third instar lymph gland, BrdU+ cells (red) are randomly distributed. To-pro-3 (blue) marks nuclei. Scale bars: 10 µm. (F) Three phases of proliferation in lymph gland development. Phase 1: growth phase. Lymph gland cells expand in number from the embryonic stage through the second instar (left panel). Cells in the secondary lobes of the third instar also belong to this phase. Phase 2: quiescence phase. This comprises the prohemocyte population of the third instar medullary zone. Cells of the PSC also rarely divide. Phase 3: expansion phase. Maturing and mature hemocytes divide to increase their numbers.

View larger version (27K): [in a new window]   Fig. 8. Schematic diagram of hemocyte maturation in the lymph gland. (A) The earliest lymph gland cells, the hemocyte precursors (HP), express Srp (S+) and Odd (O+). As these cells transition into pre-prohemocyte (PPH) fate, they initiate the expression of Hemese (H+) and Pvr (Pv+). Prohemocytes (PH) initiate dome-gal4 (dg4+) expression. Maturation to the various hemocyte (H) fates requires downregulation of dome-gal4 (dg4-), upregulation of different maturation markers (M+) and the involvement of the indicated signaling pathways. (B) Spatial and temporal sequence of hemocyte development in the lymph gland (see text for details). The PSC cells are marked by Collier (C+) and Serrate (Ser+) expression. Ubiquitous cell markers such as Srp, Odd, Pvr and Hemese are expressed in all the lymph gland cells marked by the nuclear marker To-pro-3, thus they are expressed in all the cell types and the zones. Mature hemocytes markers such as Pxn, P1 and Lz were compared with dome-gal4 expression. Lamellocyte marker msn-lacZ was compared with G147. All the markers used in the diagram except Collier were directly compared with Ser-lacZ. PM, plasmatocyte; CC, crystal cell; LM, lamellocyte; CZ, cortical zone; MZ, medullary zone; PSC, posterior signaling center.