Kicking it up a Notch for the best in show: Scalloped leads Yorkie into the haematopoietic arena

Abstract

Maintenance and differentiation of progenitor cells is essential for proper organ development and adaptation to environmental stress and injury. In Drosophila melanogaster, the haematopietic system serves as an ideal model for interrogating the function of signaling pathways required for progenitor maintenance and cell fate determination. Here we focus on the role of the Hippo pathway effectors Yorkie and Scalloped in mediating and facilitating Notch signaling-mediated lineage specification in the lymph gland, the primary center for haematopoiesis within the developing larva. We discuss the regulatory mechanisms which promote Notch activity during normal haematopoiesis and its modulation during immune challenge conditions. We provide additional evidence establishing the hierarchy of signaling events during crystal cell formation, highlighting the relationship between Yorkie, Scalloped and Lozenge, while expanding on the role of Yorkie in promoting hemocyte survival and the developmental regulation of Notch and its ligand, Serrate, within the lymph gland. Finally, we propose additional areas of exploration that may provide mechanistic insight into the environmental and non-cell autonomous regulation of cell fate in the blood system.

Keywords: CZ, Cortical Zone; LSC, Lineage Specifying Cell; Lz, Lozenge; MZ, Medullary Zone; PSC, Posterior Signaling Center; ProPO, Prophenoloxidase; Sd, Scalloped; WT, wild-type; Yki, Yorkie; Yorkie; crystal cell; haematopoiesis; lymph lland; notch; scalloped; serrate.

Figure 1.

Schematic representation of lymph gland development. (A) The lymph gland is comprised of several lobes paired on either side of the dorsal vessel, separated by pericardial cells. The primary lobes are the largest and most anterior in the larva with progenitors labeled in Green and differentiated hemocytes in Red, while smaller secondary and tertiary lobes consist of mostly progenitor cells and are located posterior to the primary lobes. (B) The early lymph gland (first-second instar) is comprised of undifferentiated prohemocytes (Green) and a small number of PSC cells (Gray). The first differentiating cells are observed at the periphery of the organ at mid-second instar. By the early third instar, fully differentiated Plasmatocytes (Red) and Crystal Cells (Blue) are observed in the CZ. The PSC secretes Hedgehog (Hh, Black Arrow) to maintain Prohemocytes of the MZ (Green). Prohemocytes differentiate through an Intermediate Progenitor (Yellow) state before reaching mature hemocyte lineages found in the CZ of the lymph gland. Plasmatocytes comprise the majority of mature hemocytes, while Crystal Cell Progenitors (Light Blue) and fully mature Crystal Cells (Blue) are also present. PVF1 (White Arrow) secreted from the PSC signals through PVR expressed in differentiating cells of the CZ to maintain levels of ADGF required for the Equilibrium Signal.

Figure 2.

Notch expression in the third instar lymph gland. (A) Notch expression (Red) in the early third instar lymph gland is expressed at low levels in prohemocytes of the MZ (dome, Green). (B) A few Notch⁺ (Red) cells are observed in dome⁺ (Green) cells of the MZ, but high levels of Notch are restricted to the dome^- cells of the CZ. (C) High magnification image of the MZ-CZ boundary reveals the disparity between abundant Notch expression observed in dome^- cells and minimal Notch in dome⁺ cells. (D) Low levels of Notch (Red) expression are observed in prohemocytes of the early third instar lymph gland, while higher expression is present in hml⁺ hemocytes of the CZ. (E) Notch (Red) is highly expressed among hml⁺ (Green) cells of the CZ in the late third instar lymph gland, while hml⁻ cells display low levels of Notch. (F) High magnification of hml⁺ (Green) cells in the CZ demonstrates high levels of Notch (Red) expression. (G) The Notch signaling reporter Enhancer of Split (E(Sp), Red) is active in scattered cells of the CZ. (H) Crystal cells (ProPO, Red) are traced in the CZ of a WT lymph gland with clonal GFP expression under control of HLT (Green). (I) Overexpression of Active Notch (N^ACT) with the HLT driver induces non-autonomous differentiation of crystal cells (ProPO, Red, not green), and inhibits expansion of HLT (Green) traced cells. Scale bar 10 μm.

Figure 3.

Hierarchy of events in crystal cell differentiation. (A) A Notch⁺ (Red outline)/Sd+(Blue) Intermediate Progenitor is activated by a Serrate-expressing LSC (Green outline). Suppressor of Hairless (Yellow) is activated downstream of Notch. Yorkie (Green) expression is then turned on by Notch signaling and it interacts with Scalloped to transcriptionally activate Lozenge (Orange), specifying a crystal cell progenitor. The Lozenge-expressing crystal cell progenitor turns on Prophenoloxidase becoming a mature crystal cell (Filled Red). (B-B”’) Nuclear marker in Blue. (B) High magnification image of crystal cells (ProPO, Red) in the WT lymph gland. (B’) Crystal cells are absent in lz^R15 mutant lymph glands. (B”) Yki (Red) and Lz (Green) in the WT lymph gland. (B”’) Yki (Red) is still present in scattered cells of the CZ in lz^R15 mutant lymph glands. (C) Yki (Red) is observed at low levels throughout the lymph gland. (C’) Yki (Red) is highly co-expressed specifically with some but not all Su(H)⁺ (Green)cells. (C”) Yki (Red) is also co-expressed in most lozenge⁺ (lz, White) cells. (C”’) Expression overlap of all 3 crystal cell progenitor markers, Yki (Red), Su(H) (Green), and lozenge (White). Scale bar 10 μm.

Figure 4.

Role of Yorkie, Scalloped, and Hippo Pathway in crystal cell development. TUNEL labels apoptotic cells (White, A-C) and crystal cells are labeled with ProPO (Red, D-E, G-L). (A) WT (B) yki and (C) sd depletion in crystal cell progenitors increases apoptosis, that is rescued by over-expression of (D) Diap1 and (E) p35. (F) Diap1 (Green) is expressed in the CZ of 3^rd instar lymph glands, and is specifically co-expressed with Lz (Red) and ProPO (Blue). (G) WT LG. (H) depletion of wts, (I) hippo, or (J) expanded does not have a significant effect on crystal cell numbers. (K) Overexpression of hipK does not affect crystal cell numbers, while (L) depletion of hipK leads to a significant loss of mature crystal cells. Scale bar 10 μm.

Figure 5.

Restriction of Serrate expression in the lymph gland. (A-B”) Crystal cell progenitors labeled with lozenge (lz, Green) and mature crystal cells labeled with ProPO (Red). (A) Crystal cell progenitors and (A’) mature crystal cells in the WT lymph gland. (A”) Overlap of crystal cell progenitors and mature cells. (B) Overexpression of Serrate in crystal cell progenitors induces a large increase in the numbers of lz⁺ progenitors and (B’) mature crystal cells. (B”) Merge. (C) Crystal cell progenitors are labeled with lz (Green) and proliferative cells are labeled with phosphorylated Histone H3 (pH3, Red) in a WT early third instar lymph gland. (D) Proliferative cells in an early third instar lymph gland after overexpression of sd in crystal cell progenitors. (D’) Increased numbers of crystal cell progenitors are observed in early 3^rd instar lymph glands upon overexpression of sd. (E-G) Serrate LacZ (Red) expression in the lymph gland. The PSC is identified with an asterisk. (E) Serrate is first observed in the early second instar lymph gland. (F) By the mid second instar, Serrate is observed in the PSC and in the first LSCs. (G) In the early third instar lymph gland, 2 distinct populations of Serrate-expressing cells are observed: the PSC and more scattered LSCs in the CZ. Scale bar 10 μm.

Figure 6.

Schematic representation of the Cellular Immune Response in the lymph gland. The PSC is depicted in Gray. Prohemocytes of the MZ are shown in Green and Intermediate Progenitors are Yellow. Plasmatocytes (Red), Crystal Cell Progenitors (Light Blue) and Crystal Cells (Blue) comprise the CZ. Scattered LSCs (Light Green) are also shown in the CZ. Crystal Cell differentiation occurs when a Serrate-expressing LSC interacts with a Notch⁺ hemocyte. Upon immune challenge, depicted here as a parasitic wasp, the size of the MZ decreases while there is a strong up-regulation of Lamellocytes (Large Gray Cells) and loss of LSCs and Crystal Cells.