Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the Drosophila lymph gland

Abstract

The Drosophila melanogaster hematopoietic organ, called lymph gland, proliferates and differentiates throughout the larval period. The lymph gland of the late larva is comprised of a large primary lobe and several smaller secondary lobes. Differentiation into two types of hemocytes, plasmatocytes and crystal cells, is confined to the outer layer (cortical zone) of the primary lobe; the center of the primary lobe (medullary zone), as well as the secondary lobes, contain only proliferating prohemocytes. A small cluster of cells located at the posterior tip of the primary lobe serves as a signaling center (PSC) that inhibits precocious differentiation of the medullary zone. The larval lymph gland is stabilized by layers of extracellular matrix (basement membranes) that surround individual hemocytes, groups of hemocytes, as well as the lymph gland as a whole. In this paper, we investigated the events shaping the lymph gland in the early pupa. The lymph gland dissociates and hemocytes disperse during the first 12 h after puparium formation (APF), leaving behind empty husks of basement membrane. Prior to lymph gland dissociation, cells of the medullary zone differentiate, expressing the early differentiation marker Peroxidasin (Pxn), as well as, in part, the late differentiation marker P1. Cells of the PSC spread throughout the pupal lymph gland prior to their dispersal. Cells of the secondary lobes undergo a rapid phase of proliferation that lasts until 8 h APF, followed by expression of Pxn and dispersal. These hemocytes do not express P1, indicating that they disperse prior to full maturation.

Figure 1. Expression of hemocyte differentiation markers in the pupal lymph gland

Panels show Z-projections of confocal sections of central part of primary lymph gland lobe. Expression of GFP driven by Trol-Gal4 in extracellular matrix outlines the lymph gland (green). Panels of left column (A, D, G, J) show labeling with early plasmatocyte marker anti-Peroxidasin (Pxn, red). Second column (B, E, H, K) represents labeling with late differentiation marker P1 (red); in right column (C, F, I), crystal cell marker Propo was used (red). Upper row (A, B, C) represents late third instar to white prepupa (0–2h APF), second row (D–F) 2–4h pupa, third row (G–I) 8h pupa, and fourth row (J, K) 10h pupa. Panel L provides a timeline (in h after puparium formation) of the expression pattern of P1 (red) and Peroxidasin (blue) in different domains of pupal lymph gland. The top portion of the figure represents the cortical zone. Below this is seen the medullary zone and finally the secondary lobes. White arrowheads in A and D indicate medullary zone (mz) which, until about 6hAPF, can be clearly distinguished from cortical zone (cz; white arrows in A and D). Other abbreviations: dv dorsal vessel; sl secondary lobe; pc pericardial cell; tr trachea. Bar: 20μm (A–K)

Figure 2. Ultrastructure of the lymph gland of the third instar larva and 4h pupa

A, B: Low magnification electron micrographs of primary lymph gland lobe of third instar larva (A) and 4h pupa (B). Medial at top; note wall of dorsal vessel (dv) visible in both panels. At larval stage, cells in the medial two thirds of the lymph gland form part of the medullary zone, as indicated to the left of panel A. These cells are more densely packed and exhibit the ultrastructural features of prohemocytes (ph). In the periphery of the larval lymph gland (cortical zone) cells are spaced further apart and are mostly differentiated plasmatocytes (pl). In the early pupa (B), the lymph gland has significantly decreased in diameter, and a clear distinction between medullary zone and cortical zone is no longer visible. Most cells represent differentiated plasmatocytes. C, D: Basement membranes deposited in the cleft between two adjacent prohemocytes (arrowhead in C) and plasmatocytes (arrowhead in D; arrow points at extension of plasmatocyte membrane). E–G: Plasmatocytes in 4h pupal lymph gland. Many plasmatocytes have already moved out of the lymph gland, leaving behind the basement membranes that originally surrounded them (arrowhead; arrow points at extensions of plasmatocyte membrane). Plasmatocyte shown at bottom of G appears to be in the process of leaving, as indicated by the wide gap between the cell and the basement membrane (double arrowhead). Bars: 5μm (A, B); 1μm (E, F, G); 0.1μm (C, D)

Figure 3. Fate of the Posterior Signaling Center (PSC) in the dissociating pupal lymph gland

Expression of PSC marker Antennapedia (Antp). Panels show confocal sections of late larval (A), 8h pupal (B),10 hour pupal (C) and 12 hour pupal (D) lymph gland lobes labeled with anti-Antennapedia (Antp, red). Lymph gland lobes are demarcated with a dashed outline. In pupa, the Antp-positive cells appear to migrate away from the Posterior Signaling Center (PSC) and spread out throughout the lymph gland. Other abbreviations: prl primary lobe; sl secondary lobe; dv dorsal vessel. Bar: 20μm (A–D)

Figure 4. Pupal development of the secondary lymph gland lobes

A–C: confocal sections of secondary lobes (sl) of 4h (A), 8h (B) and 10h pupa (C), labeled with ZCL1973X (Trol; green) and anti-Peroxidasin (Pxn, red). D, E: Labeling of proliferating cells in secondary lobes of 4h (D) and 8h (E) lymph gland with anti-BrdU (green), following 45min incubation of dissected lymph glands in BrdU-containing solution. F: Labeling of 10h secondary lobe with anti-P1 (red). P1-positive cells (arrows) are dispersed hemocytes; cells within the secondary lobes (sl) are P1-negative. Other abbreviations: dv dorsal vessel; pc pericardial cell Bar: 20μm (A–D)

Figure 5. Ultrastructure of the 10h pupal lymph gland

A: Low power electron micrograph showing remnant of lymph gland in the 10h pupa. Few cells, typically phagocytotically active plasmatocytes (pl), are left. Aside from these cells, the lymph gland forms a “husk” of folded basement membranes (ECM) that at earlier stages contained hemocytes. B: Phagocytotically active plasmatocyte (macrophage; pl/mp), containing lysosomes with electron dense cellular debris (arrow). Bars: 5μm (A); 1μm (B)