Yorkie and Scalloped signaling regulates Notch-dependent lineage specification during Drosophila hematopoiesis

Abstract

Cellular microenvironments established by the spatial and temporal expression of specific signaling molecules are critical for both the maintenance and lineage-specific differentiation of progenitor cells. In Drosophila, a population of hematopoietic progenitors, or prohemocytes, within the larval lymph gland gives rise to three mature cell types: plasmatocytes, lamellocytes, and crystal cells. Removal of the secreted signaling molecules Hedgehog and PVF1 from the posterior signaling center (PSC), which acts as a niche, leads to a loss of progenitors and complete differentiation of the lymph gland. Here, we characterize a novel population of signaling cells within the lymph gland, distinct from the PSC, that are required for lineage-specific differentiation of crystal cells. We provide evidence that Yorkie and Scalloped, the Drosophila homologs of YAP and TEAD, are required in lineage-specifying cells to regulate expression of Serrate, the Notch ligand responsible for the initiation of the crystal cell differentiation program. Genetic manipulation of yorkie and scalloped in the lymph gland specifically alters Serrate expression and crystal cell differentiation. Furthermore, Serrate expression in lineage-specifying cells is eliminated in the lymph gland upon the immune response induced by wasp parasitization to ensure the proper differentiation of lamellocytes at the expense of crystal cells. These findings expand the roles for Yorkie/Scalloped beyond growth to encompass specific cell-fate determination in the context of blood development. Similar regulatory functions may extend to their homologs in vertebrate progenitor cell niches that are required for specifying cell fate.

Figure 1. Scalloped and Yorkie are required for proper crystal cell differentiation

Crystal cell progenitors (CCP) are labeled with Lz (H–P, red). (A) Schematic of the 3^rd Instar lymph gland and hemocyte differentiation. PSC in grey, prohemocytes (PH, green) of the MZ, intermediate progenitors (IP, yellow), plasmatocytes (PL, red) and crystal cells (CC, blue) in the CZ. (B) Yki (red) is expressed in scattered cells of the CZ in a 3^rd instar lymph gland. (C) Yki (red) is observed in CCPs (Lz, blue) amongst differentiating hemocytes (hml, green) of the CZ. (D) Yki (red) is present in mature CCs labeled with Black cells-GFP (green). (E) sd (sd-gal4 > UAS-2xEGFP, green) is expressed in clusters of cells scattered throughout the lymph gland. CZ is demarcated by a dotted line. (F–G) sd (green) is present in a subset of Yki⁺ cells (F, arrows) and mature CCs (G, arrows) and is also seen adjacent to Yki⁺ cells (F, arrowhead) and CCs (G, arrowheads). (G, inset) Lineage traced (sd-gal4, UAS-GFP > UAS-FLP, A5C-FRT-STOP-FRT-LacZ) (red) mature CCs (ProPO, white) do not express sd (green). H–L For each panel, its corresponding pattern of HLT> GFP expression is demonstrated in Fig. S1F–J (H) WT lymph gland (I) Widespread over-expression of yki^WT in the lymph gland increases CCP numbers while (J) depletion of yki or (K) sd blocks CC formation. (L) sd knock-down blocks the increase of CCPs observed upon over-expression of yki^WT. (M) WT lymph gland. (N) Over-expression of yki^WT in sd expressing cells (sd-gal4 >) increases CCP numbers, while (O) depletion of yki or (P) sd strongly inhibits CC differentiation. (Q–R) Quantification of H–P (n=10). * p value <.05, *** p value < .001. Scale bar 10 μm. See also Fig. S1.

Figure 2. Yorkie and Scalloped are required specifically in Serrate-expressing cells for proper Crystal Cell differentiation

Green labels Ser⁺ cells (Ser-gal4, UAS-GFP) (A, E) and red labels ProPO⁺ CCs. (A) Ser expressing cells (arrowhead) observed in the periphery of the Cortical Zone, distinct from the PSC (outlined by a dotted line). (B) WT (C) Knockdown of yki or (D) sd in Ser⁺ cells blocks CC formation. (E) Ser⁺ cells observed in direct contact with CCs in the Cortical Zone. (F) WT (G) Knockdown of yki or (H) sd in the PSC (Antp-gal4 >) has no effect on CC formation. (I) Quantification of yki and sd Knockdown in Ser⁺ cells. (J) Quantification of yki and sd Knockdown in the PSC. (n=10) *** indicates pValue <.001. Scale bar 10 μm. See also Fig. S2.

Figure 3. Yorkie and Scalloped regulate Serrate-expressing lineage specifying cell (LSC) numbers

Red labels Ser⁺ LSCs (SerLacZ, A–E, K–O) and CCs (F–J). (A) Ser expressing cells are located in the CZ, outlined by white hatch marks (asterisk denotes PSC) (B) Ser does not co-localize with the PL marker hml. (C–D) Ser⁺ cells are observed adjacent to lz⁺ CCPs (C, green) and ProPO⁺ mature CCs (D, green) but do not co-localize. (E) sd (green) is co-expressed with Ser (red) (F) WT lymph gland. (G) Depletion or (H) inhibition of Ser function in sd expressing cells (sd-gal4>) blocks CC differentiation. (I) CC differentiation is similarly blocked by inhibition of Ser after over-expression of yki^WT. (J) Loss of CCs observed upon yki depletion is rescued by over-expression of Ser. (K) Ser expression in larvae containing a single copy of SerLacZ is only observed in the PSC (asterisk). (L) Over-expression of yki^WT (HLT >)greatly increases Ser expression in lymph glands containing a single copy of SerLacZ. (M) WT lymph gland. (N) Depletion of yki or (O) sd in sd expressing cells (sd-gal4>) blocks Ser expression outside of the PSC (asterisk). (P–Q) Quantification of F–J and M–O (n=10). *** p value < .001. Scale bar 10 μm. See also Fig. S3.

Figure 4. Serrate expression in LSCs is down-regulated in the lymph gland of immune challenged larvae

(A) Schematic representation of the immune response generated upon wasp infection. Notch signaling, which promotes crystal cell (CC) differentiation in the lymph gland, is blocked by wasp parasitization, allowing intermediate progenitors (IP) to differentiate into lamellocytes [14]. Asterisk denotes PSC (B–C). LSCs (Ser) are red in B–C while CCs (ProPO) are red in E, E", G, G", I, I", K–M. Lamellocytes are labeled by green (L1). (B) Ser expression in WT. (C) Loss of LSC Ser expression upon wasp parasitization. (D) Quantification of Fig.4B–C, (n= 10) (E–E”) WT lymph gland contains CCs (E) and lacks lamellocytes (E'). (F) Quantification of Fig.4E, G (n=10). (G–G”) Wasp parasitization eliminates CCs (G) and promotes lamellocytes (G’). (H) Quantification of Fig.4G, I (n=10) (I–I”) Enforced expression of Ser upon wasp parasitization rescues CC loss (I), and inhibits lamellocyte formation (I'). (J) Quantification of Fig. 4G’, I’ (n= 10) (K) WT lymph gland. (L) Over-expression of Ser (HLT >) rescues CC numbers upon wasp parasitization, but (M) over-expression of yki^WT has no affect compared to WT. (N) Quantification of Fig.4 K–M. (n=10) ** pValue < .01, *** pValue < .001. Scale bar 10 μm. See also Fig. S4.