Gene regulatory networks controlling hematopoietic progenitor niche cell production and differentiation in the Drosophila lymph gland

Abstract

Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. In this report, we utilize a PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. We assessed the effect of abrogating the function of 820 genes as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, we characterized loss- and gain-of-function phenotypes. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland.

Figure 1. Lymph gland domains, cell markers, and strategy for a PSC-specific gene function knockdown analysis.

(A) Dissected dorsal vessel and associated lymph glands assayed for DAPI (DNA), Antp protein (PSC cells), and eater-GFP transgene activity (plasmatocytes). Abbreviations: CZ, cortical zone; PSC, posterior signaling center. (B) Enlargement of a primary lymph gland lobe stained for Su(H) protein expressed in hematopoietic progenitors of the medullary zone (MZ) and crystal cells of the cortical zone (CZ). Also highlighted are hhF4f-GFP-positive niche cells of the PSC. (C) Focus on a lymph gland PSC niche assayed for nuclear Antp protein and membrane-associated GFP due to expression of the col>UAS-gapGFP combination. The arrow points out a filopodia extending from a niche cell. (D) Strategy for the PSC-specific gene function knockdown analysis undertaken in this study.

Figure 2. Summary of findings of a PSC-specific gene function knockdown analysis of 820 Drosophila loci.

Center: hhF4f-GFP transgene activity (hh in green circle) was the primary marker assessed in the RNAi-based gene function knockdown analysis, while expression of Antp protein (Antp in yellow circle) and filopodia formation based on col>UAS-gapGFP expression were monitored as secondary markers. Periphery: Grouping of genes (based on their encoded protein products), that when functionally altered by RNAi expression in PSC cells, resulted in abnormalities in hh-GFP, Antp, and/or col>UAS-gapGFP expression. Negative (blue circle) and positive (red circle) regulators are indicated. A negative regulator is defined as a gene whose loss-of-function condition leads to increased numbers of hhF4f-GFP-positive cells and/or enhanced transgene expression, while a positive regulator is defined as a gene whose loss-of-function condition leads to decreased numbers of hhF4f-GFP-positive cells and/or decreased transgene expression. Those loss-of-function conditions that resulted in lamellocyte induction (#) or absence of filopodia (*) are also indicated.

Figure 3. Gene function knockdown of BAP chromatin-remodeling complex genes.

(A) hhF4f-GFP transgene activity in a wild-type lymph gland. Arrow points out GFP-positive PSC cells. (B–F) Lack of hhF4f-GFP transgene activity in lymph glands expressing brm, moira, Bap60, Bap111, or osa RNAi constructs, respectively. Arrowheads point out GFP-negative PSC cells in these tissues. (G) Wild-type lymph gland assayed for DAPI (DNA), gapGFP expression (differentiated niche cells), and dome-lacZ expression (hematopoietic progenitors of the medullary zone, MZ). (G’) Wild-type niche cells show normal differentiation as monitored by extension of filopodia (arrow). (H) PSC-specific RNAi knockdown of osa function results in a decreased population of dome-lacZ-positive hematopoietic progenitors and reduced gapGFP-positive PSC cells. (H’) osa-knockdown niche cells fail to differentiate filopodial processes (arrowhead). Scale bar indicates 20 µm.

Figure 4. Genetic interaction between the osa and srp genes.

(A) hhF4f-GFP transgene activity in PSC cells (arrow) of a wild-type lymph gland. (B) Antp expression in PSC cells (arrow) of a wild-type lymph gland. (C) osa³⁰⁸/+ heterozygous lymph glands contain a decreased number of hhF4f-GFP-positive PSC cells (arrowhead). (D) osa³⁰⁸/+ heterozygous lymph glands contain a decreased number of Antp-positive PSC cells (arrowhead). (E) Normal hhF4f-GFP transgene activity in PSC cells (arrow) of a srp⁰¹⁵⁴⁹/+ heterozygous lymph gland. (F) Normal Antp expression in PSC cells (arrow) of a srp⁰¹⁵⁴⁹/+ heterozygous lymph gland. (G) Absence of hhF4f-GFP-positive PSC cells (arrowhead) in a srp⁰¹⁵⁴⁹/osa³⁰⁸ double-heterozygous lymph gland. (H) srp⁰¹⁵⁴⁹/osa³⁰⁸ double-heterozygous lymph glands contain a reduced population of Antp-positive PSC cells (arrowhead).

Figure 5. Alteration of insulin-like growth factor signaling pathway gene functions.

Expression of the PSC cell-specific markers hhF4f-GFP and Antp was assessed in lymph glands of the following loss- or gain-of-function genotypes: (A) wild-type, (B) col>dAkt1 RNAi, (C) col>dAkt1 cDNA, (D) col>PDK1 RNAi, (E) col>InR^DN cDNA, (F) col>InR cDNA, (G) col>Pi3K92E RNAi, (H) col>Pi3K92E^DN cDNA, and (I) col>Pi3K92E^CA cDNA. Arrows in panels C, F, and I point out expanded populations of PSC niche cells, with an abnormal niche organization caused by PSC cell-specific expression of the InR cDNA. Arrowheads in panels B, D, E, G, and H point out reduced populations of PSC niche cells. Scale bar indicates 20 µm.

Figure 6. Quantification of PSC cell number.

Figure 7. Alteration of Pten and dFOXO gene functions.

Expression of the PSC cell-specific markers hhF4f-GFP and Antp was assessed in lymph glands of the following loss- or gain-of-function genotypes: (A) wild-type, (B) Pten¹⁰⁰/Pten¹¹⁷, (C) P85col>Pten cDNA, (G) dFOXO²¹/dFOXO²⁵, (H) col>dFOXO cDNA, and (I) Pten¹¹⁷/+;dFOXO²¹/+. Arrows in panels B, G, and I point out expanded populations and abnormal organization of niche cells. Arrowheads in panels C and H point out severely reduced populations of PSC niche cells. (D) Wild-type lymph gland assayed for DAPI (DNA), P1 antigen (plasmatocytes), and dome-lacZ expression (hematopoietic progenitors). (E) Pten¹⁰⁰/Pten¹¹⁷ lymph gland assayed for DAPI, P1 antigen, and dome-lacZ expression. The arrow points out the substantial increase in plasmatocyte number. (F) Pten¹⁰⁰/Pten¹¹⁷ lymph gland assayed for MSNF9mCherry transgene activity (lamellocytes). The arrow points out the de novo production of lamellocytes. Scale bar indicates 20 µm.

Figure 8. Alteration of TOR signaling pathway gene functions.

Expression of the PSC cell-specific markers hhF4f-GFP and Antp was assessed in lymph glands of the following loss-of-function genotypes: (A) col>Tor RNAi, (B) col>raptor RNAi, (C) col>rictor RNAi, (D) col>TSC1 RNAi, (G) col>TSC2 RNAi, (J) S6K^L-1/S6K^L-1 (Antp only), and (K) Thor⁰⁶²⁷⁰/Thor⁰⁶²⁷⁰. Arrows in panels G and K point out expanded populations and abnormal organization of PSC niche cells. Arrowheads in panels A, B, C, and J point out severely reduced populations of PSC niche cells. (E) Expanded expression of the hhF4f-GFP transgene in TSC1^f01910/TSC1^f01910 lymph glands. (F) Increase in plasmatocyte number (detected by P1 antibody) and supernumerary lamellocyte production (detected by MSNF9mCherry activity) in TSC1^f01910/TSC1^f01910 lymph glands. (H) Expanded expression of the hhF4f-GFP transgene in TSC2¹⁰⁹/TSC2¹⁰⁹ lymph glands. (I) Increase in plasmatocyte number and supernumerary lamellocyte production in TSC2¹⁰⁹/TSC2¹⁰⁹ lymph glands. Scale bar indicates 20 µm.

Figure 9. Quantification of hemolymph lamellocyte numbers.

Figure 10. Quantification of lymph gland size and PSC cell number in fed or starved larvae.

(A) Lymph gland cell number. (B) PSC cell number.