Hemocytes are essential for wing maturation in Drosophila melanogaster

Abstract

Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, we present studies of two mutant phenotypes. First, the batone mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map. Second, ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic "tumors." Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. We propose that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. We propose that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing.

Figure 1

Morphological observations. (A) Wild-type wing of a GAL4–30A fly. (B) Wing of a bae mutant fly. (C and D) Wings of GAL4–30A/+ ; UAS-PKAcF 5.2/+ flies. (E) Head of a GAL4–30A/+ ; UAS-PKAcF 5.2/+ fly; the arrow points to a small melanotic tumor. (F) Wing of a GAL4–30A, UAS-GFP/UAS-Ricin A fly. Failure of dorsal and ventral surfaces to bond is apparent along the posterior edge of the wing and from the precipitated debris in the distal portion of the wing blade. (G) Wing of a GAL4–30A, UAS-PKAc 15.3/UAS-pan 24 fly, sibling to that shown in H. (H) Wing of a GAL4–30A, UAS-PKAc 15.3/Sp fly, sibling to that shown in G. (I) Wing of a GAL4–30A/+ ; UAS-panΔN5/+ fly.

Figure 2

Blastoderm fate map of the bae mutant focus. The bae mutant focus is positioned on the blastoderm fate map relative to markers for the head, wing, and anterior leg. Sturt distances between cuticular markers are shown with solid lines, and the distances between the bae focus and cuticular markers are shown with dotted lines. Distances were generated from the data in Table 1 by using the Hotta and Benzer (9) procedure.

Figure 3

Observations of GFP fluorescence. (A) Unopened wing of a GAL4–30A, UAS-GFP fly. (B) Newly opened wing of a GAL4–30A, UAS-GFP fly. (C) Newly opened wing of a GAL4–30A, UAS-GFP/+ ; UAS-PKAcF 5.2/+ fly; note the large aggregations of hemocytes, most of which appear to be lamellocytes. Arrows mark some of the few round cells that can be identified. (D–G) Newly opened wings of GAL4–30A, UAS-GFP/UAS-Ricin A flies. In D, the coverslip was moved to show that dorsal and ventral surfaces can be displaced from their normal relationship to one another, creating the wrinkles in the wing blade. In E, partial ablation of hemocytes prevents bonding of the wing surfaces of the posterior wing blade (left), where hemocytes are out of focus, whereas bonding occurs in the central and anterior wing blade where hemocytes are abundant and in focus (right). In F, the wing is fully expanded and filled with hemolymph and some hemocytes; at higher magnification (G), many of the hemocytes appear to be lamellocytes. Note the separation of dorsal and ventral portions of the vein (arrows) in G. (H) Newly opened wing of a GAL4–30A, UAS-GFP/+ ; UAS-panΔN5/+ fly. The dorsal wing blade and veins are in focus, whereas most of the hemocytes are not, because of the separation of the dorsal and ventral surfaces. In D–H, the low level of fluorescence required long exposures, causing some reflected background room light to illuminate portions of the wing blade and veins (F–H). (I) Unopened wing of a bae/Y; GAL4–30A, UAS-GFP/+ fly. [Bar = 0.2 mm.]