Characterization of a lamellocyte transcriptional enhancer located within the misshapen gene of Drosophila melanogaster

Abstract

Drosophila has emerged as an excellent model system in which to study cellular and genetic aspects of hematopoiesis. Under normal developmental conditions and in wild-type genetic backgrounds, Drosophila possesses two types of blood cells, crystal cells and plasmatocytes. Upon infestation by a parasitic wasp or in certain altered genetic backgrounds, a third hemocyte class called the lamellocyte becomes apparent. Herein we describe the characterization of a novel transcriptional regulatory module, a lamellocyte-active enhancer of the misshapen gene. This transcriptional control sequence appears to be inactive in all cell types of the wild-type larva, including crystal cells and plasmatocytes. However, in lamellocytes induced by wasp infestation or by particular genetic conditions, the enhancer is activated and it directs reporter GFP or DsRed expression exclusively in lamellocytes. The lamellocyte control region was delimited to a 140-bp intronic sequence that contains an essential DNA recognition element for the AP-1 transcription factor. Additionally, mutation of the kayak gene encoding the dFos subunit of AP-1 led to a strong suppression of lamellocyte production in tumorous larvae. As misshapen encodes a protein kinase within the Jun N-terminal kinase signaling pathway that functions to form an active AP-1 complex, the lamellocyte-active enhancer likely serves as a transcriptional target within a genetic auto-regulatory circuit that promotes the production of lamellocytes in immune-challenged or genetically-compromised animals.

Figure 1. Identification and initial characterization of a msn lamellocyte transcriptional enhancer.

A. Genomic organization of the msn gene and location of the 3.0-kb MSNF9 intron-3 DNA that possesses lamellocyte enhancer activity. B. Lack of MSNF9-GFP expression in a wild-type third-instar larva. C. The MSNF9-GFP transgene is active in a hop^Tum-l third-instar larva. The open arrowhead points to MSNF9-GFP-positive lamellocytes that have contributed to the formation of a melanotic tumor in this animal. D. MSNF9-GFP activity is not detected in circulating hemocytes present in hemolymph obtained from a wild-type third-instar larva. E. MSNF9-GFP-positive lamellocytes present in hemolymph obtained from a hop^Tum-l third-instar larva. F. Lack of co-expression of eater-GFP (plasmatocyte marker, arrowhead) and MSNF9-DsRed (lamellocyte marker, open arrowhead) in circulating blood cells obtained from a hop^Tum-l third-instar larva. G. Lack of co-expression of lzGal4>UAS-GFP (crystal cell marker, arrowhead) and MSNF9-DsRed (lamellocyte marker, open arrowhead) in circulating blood cells obtained from a hop^Tum-l third-instar larva.

Figure 2. Temporal activation of the msn lamellocyte transcriptional enhancer.

A. Inactivity of the MSNF9-GFP transgene in lymph glands dissected from a Basc/Y control larva at 72 hr of development. B. Inactivity of the MSNF9-GFP transgene in circulating hemocytes present in hemolymph obtained from a Basc/Y control larva at 72 hr of development. C. MSNF9-GFP-positive hemocytes observed in lymph glands dissected from a hop^Tum-l/Y mutant larva at 72 hr of development. D. MSNF9-GFP-positive lamellocytes present in the hemolymph obtained from a hop^Tum-l/Y mutant larva at 72 hr of development. E. MSNF9-GFP-positive hemocytes observed in lymph glands dissected from a hop^Tum-l/Y mutant larva at 56 hr of development. F. MSNF9-GFP-positive lamellocytes present in the hemolymph obtained from a hop^Tum-l/Y mutant larva at 42 hr of development.

Figure 3. MSNF9-GFP expression in circulating hemocytes obtained from third-instar larvae of different genetic backgrounds that induce lamellocyte production.

A. w¹¹¹⁸; He-Gal4 control. B. He-Gal4>UAS-dTCF^DN. C. He-Gal4>UAS-dRAC1. D. He-Gal4>UAS-hep^CA. E. Cg-Gal4>UAS-col. F. Cg-Gal4>Ush^DN. Hemolymph samples were also stained for DNA (DAPI) and filamentous actin (phalloidin).

Figure 4. MSNF9-GFP expression in larvae post L. boulardi infestation.

A. Lack of MSNF9-GFP activity in lymph glands dissected from a control, non-wasp infested larva. B. MSNF9-GFP expression in lymph glands dissected from a wasp infested larva. C. MSNF9-GFP-positive lamellocyte present in the hemolymph of a wasp infested larva. D. Detection of numerous MSNF9-GFP-positive lamellocytes encapsulating a wasp egg (asterisk).

Figure 5. Fine mapping of the location of the msn lamellocyte enhancer and its essential DNA sequence elements.

A. Schematic of the various truncated and sequence mutated msn intron-3 DNAs tested for enhancer function in lamellocytes of hop^Tum-l larvae as compared to hemocytes of control larvae. B. Sequence of the 590-bp MSNF9e DNA with putative binding elements for the STAT, Srp, and AP-1 transcription factors highlighted.

Figure 6. The AP-1 recognition sequence in the msn lamellocyte enhancer can compete for dFos-dJun DNA binding.

A. Sequence of the hMTII AP-1 DNA probe, MSNF9 AP-1 recognition site, and mutated MSNF9 AP-1 recognition site. B. Electromobility shift competition assay with hMTII wt AP-1, MSNF9 wt AP-1, and MSNF9 mut AP-1 double-stranded oligonucleotide DNAs.

Figure 7. Suppression of lamellocyte production by a mutant allele of kay, which encodes dFos.

A. Supernumerary lamellocytes produced in a hop^Tum-l/+ larva. B. Substantial reduction in the lamellocyte population in a hop^Tum-l/+; kay¹/+ larva. C. Quantification of the number of lamellocytes and non-lamellocytes produced in larva of Canton S control versus mutant larvae. Asterisk (*) denotes a significant difference in values from those observed in hop^Tum-l animals.

Figure 8. Model for the regulation of the msn gene during lamellocyte induction and differentiation.

The MSNF9 lamellocyte-active enhancer is depicted as serving as a transcriptional target within a JNK pathway auto-regulatory circuit that promotes the production of lamellocytes in immune-challenged or genetically compromised animals. The schematic of the JNK pathway is adapted from Xia and Karin (19).