Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae

Abstract

A GFP expression screen has been conducted on >1000 Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes, with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process.

Keywords: Drosophila hematopoiesis; blood cell-specific gene expression; gene enhancer-Gal4 line screening; larval hemocyte essential genes; wasp-challenged larvae.

Figure 1

Lymph gland structure, cellular domains, blood cell types, and the results of the enhancer-Gal4 line screen. (A) Organization of the lymph glands into primary, secondary, and tertiary lobes. The primary lobe is positive for the plasmatocyte marker eater-GFP, while the arrowheads indicate positive pericardial cells as well. (B) Cellular and functional domains within the primary lobe of a midthird instar larval lymph gland. The primary lobe is composed of three parts: the CZ (blue) marked by NimC1 protein expression, MZ (red) marked by domeMESO expression, and the PSC (green) marked by col > GFP expression. Gray corresponds to DAPI labeling. (C–E) Three mature hemocyte types are found in the CZ and hemolymph. (C) Plasmatocytes (green) are marked by eater > YFP expression. Blue corresponds to DAPI labeling. (D) Crystal cells (blue) are marked by BcF6CFP expression. Red corresponds to phalloidin detection of plasmatocytes. (E) Lamellocytes (red) are labeled by MSNF9mCherry expression. Green indicates phalloidin staining of these large cells. (F and G) Summary of the enhancer-Gal4 > UAS-GFP-expressing lines as to (F) expression in cells of the primary lymph gland lobe and (G) blood cells found in the circulating hemolyph under normal or wasp-challenged growth conditions. Bar, 20 μm in all images. CZ, cortical zone; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; MZ, medullary zone; PSC, posterior signaling center; YFP, yellow fluorescent protein.

Figure 2

PSC-expressed enhancer-Gal4 lines. (A–L) The various lines tested are indicated in the panels with the enhancer location as to genetic locus noted below the strain name. (A–D) Lines expressing the GFP marker (green) with a strong or complete presence in cells expressing the Antp PSC protein marker (red). (E–H) Lines expressing the GFP reporter in a subset of Antp⁺ PSC cells. (I–L) Wasp infestation can induce enhancer activity and reporter gene expression in PSC cells. (I) Under normal growth conditions, the GMR44C05 line shows GFP expression in CZ cells but not PSC cells. (J) Post-wasp infestation, the lola gene enhancer is active in PSC cells. (K) The GMR36G01 line fails to show any expression in cells of the lymph gland. (L) Post-wasp challenge, the cnc enhancer is active in both PSC cells and lamellocytes, the latter marked by the MSNF9mCherry transgene (red). Arrowheads indicate PSC cells expressing GFP and/or Antp. Bar, 20 μm in all images. Antp, Antennapedia; CZ, cortical zone; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; PSC, posterior signaling center.

Figure 3

MZ-expressed enhancer-Gal4 lines. (A–F) The various lines tested are indicated in the panels with the enhancer location as to genetic locus noted below the strain name. GFP expression patterns (green) of (A) GMR47F05, (B) GMR12H06, (C) GMR36B11, and (D) GMR50A12 are consistent with enhancer activity in all or most domeMESO⁺ MZ cells (red). The (E) GMR13B08 and (F) GMR26D06 lines show GFP expression in a subset of domeMESO⁺ cells. Bar, 20 μm in all images. DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; MZ, medullary zone.

Figure 4

CZ and specific blood cell type-expressed enhancer-Gal4 lines. (A–L) The various lines tested are indicated in the panels with the enhancer location as to genetic locus noted below the strain name. (A–F) Representative lines that express the GFP reporter (green) in peripheral CZ cells that do not overlap with domeMESO + MZ cells (red). Lymph glands are also stained with the nuclear marker DAPI (blue). (G–K) Five enhancer-Gal4 lines showing GFP expression in mature crystal cells, marked by the activity of the crystal cell-specific transgene BcF6-mCherry (red). (L) The GMR40F10 line, harboring an enhancer from the bbx gene, fails to show expression in CZ cells of the lymph gland but is solely expressed in circulating plasmatocytes. Bar, 20 μm in all images. CZ, cortical zone; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; MZ, medullary zone.

Figure 5

Lamellocyte-expressed enhancer-Gal4 lines. (A, C, and E). Various lines tested are indicated in the panels with the enhancer location as to genetic locus noted below the strain name. Three lines are presented that failed to show GFP expression (green) in any cell type of the lymph gland. (B, D, and F). Post-wasp infestation, lamellocytes identified by MSNF9mCherry transgene expression (red) are induced in high numbers. Lymph glands are also stained with the nuclear marker DAPI (blue). Bar, 20 μm in all images. DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein.

Figure 6

Phenotype analyses of select PSC, MZ, and lamellocyte-expressed genes. (A–D) PSC-expressed or activated genes, with lymph glands stained with nuclear DAPI (blue), PSC markers hh-GFP (green), Antp (red), or GFP (green), and lamellocyte marker MSNF9mCherry (red). (A and C) Control lymph glands. (B) cut knockdown lymph glands through col-Gal4 > UAS cut RNAi expression. (D) lola gain-of-function lymph glands through col-Gal4 > UAS lola expression. (E–G) Knockdown phenotypes of MZ-expressed genes. Lymph glands were stained for nuclear DAPI (blue) and mCD8::GFP (green). (E) Control lymph glands. (F) h knockdown lymph glands through TepIV-Gal4 > UAS h RNAi expression. (G) Btk29A knockdown lymph glands through TepIV-Gal4 > UAS Btk29A RNAi expression. (H–J) Examples of lamellocyte-inducing genes. Lymph glands were stained with nuclear DAPI (blue), GFP (green), and the lamellocyte marker MSNF9mCherry (red). (H) Control lymph glands. (I) dFOXO gain-of-function lymph glands through Pxn-Gal4 > UAS dFOXO expression. (J) cnc gain-of-function lymph glands through Pxn-Gal4 > UAS cncB expression. Bar, 20 μm in all images. Antp, Antennapedia; CZ, cortical zone; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; MZ, medullary zone; PSC, posterior signaling center; RNAi, RNA interference.

Figure 7

(A) Requirement of the dFOXO, kay, cnc, and dei genes for a full cellular immune response to wasp infestation of larvae. (B–D) Quantification of lamellocyte production in lymph glands of the above tested genotypes in response to wasp parasitization. Three values are assigned. (B) Green indicates no (0) lamellocytes observed in assayed lymph glands. (C) Orange indicates a few (1–9) lamellocytes observed in assayed lymph glands. (D) Red indicates a strong induction (>10–100) of lamellocytes in assayed lymph glands. Bar, 20 μm in all images. DAPI, 4’,6-diamidino-2-phenylindole; RNAi, RNA interference.

Figure 8

Lymph gland phenotypes in E2f1 and Dp gain- and loss-of-function lymph glands from third instar larvae. (A and D) The TepIVGal4 > UASmCD8GFP combination is used to identify a normal population of progenitors (green) in wild-type lymph glands. MSNF9mCherry marks lamellocytes (red, none detected). (B and C) Abrogation of E2f1 or Dp function by RNAi knockdown leads to a complete loss of stem cell-like prohemocytes. (E) Gain-of-function E2f1 leads to a massive overproduction of hematopoietic progenitors (green). (F) Gain-of-function E2f1 and Dp results in a massive expansion of the prohemocyte population (green) and the high-level inducement of lamellocytes (red). Bar, 20 μm in all images. DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; RNAi, RNA interference.