Every adult fruit fly leg begins as a crumpled pocket of cells inside a larva, yet by the end of pupal development it has become a precisely segmented appendage. How fate-determined cells actually sculpt that final shape has long remained one of developmental biology’s blind spots. A new study, published in Development Genes and Evolution, used days-long live imaging to watch the tarsus, the segmented tip of the Drosophila leg, take shape inside the intact pupa, and what the cameras captured was far stranger than anyone expected.
Shotaro Hiraiwa, Tetsuya Kojima and colleagues at their institution mounted developing pupae under an Olympus FV3000 confocal microscope and filmed the tarsus continuously through the roughly four days of pupal development. Flies were engineered so that cell membranes glowed green, nuclei glowed red, and actin, microtubules and myosin II lit up in their own fluorescent channels. By removing the puparium after 12 hours after puparium formation, the researchers kept the animals alive until the end of the pupal stage while acquiring three-dimensional image stacks at regular intervals, a technical feat that allowed them to trace individual epithelial cells across days rather than reconstructing development from fixed snapshots.
The team divided the shaping process into five stages. In Stage I, from 0 to 7 hours after puparium formation, the leg disc everts. In Stage II, from 7 to 15 hours, the tarsus elongates dramatically: the folded tarsal segments unfold, and by 15 hours the organ has become a bloated tube whose diameter exceeds three times that of the adult tarsus. During this elongation, the epithelial cells lining the tube shift from columnar to cuboidal. Stage III, from 15 to 27 hours, is when the tube narrows dramatically toward its adult diameter. Stage IV, from 27 to 50 hours, is the joint-forming stage, when bristles fully elongate and claws and pulvilli form. Stage V, from 50 to 96 hours, looks externally uneventful, but the epithelial layer quietly continues to thin.
The surprise came in Stage III. Around 18 hours after puparium formation, the epithelial cells transiently assembled a structure the authors named the Parthenon-like structure, after the ancient Greek ruin. Nuclei and most of each cell’s body lined up in a monolayer on the apical, outer side of the tissue. From there, each cell extended one or two pillar-like cytoplasmic processes basally, inward toward the lumen, and then spread out at the base into a thin planar layer. The result was a cellular colonnade: rows of pillars supporting a basal floor, with open cavities between them running through the interior of what had been a simple single-layered epithelium.
Mosaic analysis, in which small patches of cells were labeled with GFP by the flip-out technique, showed that every epithelial cell participated. At 18 hours after puparium formation, all labeled cells extended either one protrusion, in about two-thirds of cells, or two, in about one-third, and neighboring protrusions bundled together so that each pillar was built from processes of multiple adjacent cells. Imaging with GFP-CAAX, which labels cell membranes, confirmed that the cells themselves deform to build the structure rather than merely arranging their cytoskeletons within it, although actin, microtubule and myosin II signals were all concentrated inside the pillars, suggesting that cytoskeletal cables drive the pillar extension and retraction.
The Parthenon-like structure proved to be transient. Once the epithelial layer reached its maximum thickness, the pillars shortened and the cavities collapsed, and the layer thinned again. Crucially, this formation and disappearance coincided with the dramatic reduction of the tarsus’s overall diameter, and the structure dissolved faster in future joint regions than between joints, re-creating the indentations that mark the tarsal segments. The basal surface of the epithelium, and the basement membrane protein Laminin B1 lying against it, initially showed a mesh-like pattern full of holes; within about two hours those holes shrank away, leaving a smooth, taut, membranous sheet. The authors hypothesize that filopodia-like thin protrusions from the pillar roots interconnect to form this basal meshwork.
There were hints of mechanical tension as well. The basal surface was initially winding along the length of the leg but progressively flattened, becoming straight and taut by 19.5 hours after puparium formation. Using the photoconvertible fluorescent protein Kaede, the researchers marked cells at intervals and tracked their movement: the pillars initially tilted with their basal ends pointing distally, then the basal ends were pulled markedly toward the proximal direction, and by 24 hours the tilt had reversed. These observations imply a proximodistal tension pulling on the basal side of the structure, possibly generated by changes in the basement membrane as its meshwork contracts.
Then came a second surprise. Large cells expressing hemocyte-specific drivers, He-GAL4 and srpHemo-GAL4, entered the tarsal lumen from the proximal end during elongation in Stage II and then took up residence inside the cavities of the Parthenon-like structure. By capturing image stacks every 22 seconds, the team filmed a macrophage-like cell extending filopodia and engulfing an epithelial cell whose body had been extruded basally from the apical layer. A caspase activity reporter called Apoliner showed that the engulfed cells were undergoing apoptosis: an epithelial cell would switch on caspase activity, migrate basally, and then be consumed by a macrophage-like cell, which was often seen carrying multiple fluorescent nuclear signals from previously engulfed victims. The macrophage-like cells were also observed passing back out through the basal surface, apparently through holes in the meshwork, suggesting they move in and out of the cavities freely.
The authors propose that the Parthenon-like structure may function partly as a demolition scaffold: by opening cavities inside the epithelium, it gives macrophage-like cells access to surplus cells so that cell number can be trimmed as the organ narrows. This idea gains support from earlier work showing that depleting hemocytes causes severe leg defects, including truncation, though the researchers caution that macrophage-like cells may play additional roles in tarsal morphogenesis beyond clearing apoptotic cells. They also note that the structure is not unique to the tarsus: they observed it in other leg segments, the head and the notum, and similar cell shapes with basal protrusions have long been described in the pupal wing, including in lepidopterans, where hemocytes likewise move between the protrusions. The transient Parthenon-like structure may therefore be a widespread, perhaps essential, feature of final epithelial shaping in insects.
Why it matters: for decades, developmental genetics has explained how cells acquire their identities, but the mechanical and cellular choreography that converts a patterned sheet of cells into a finished organ has been far harder to observe. By filming the entire process in the intact animal, this study transforms the tarsus from a static series of fixed images into a moving picture, revealing that final shape formation involves a transient internal architecture, a contracting basement membrane meshwork, and an immune-cell cleanup crew working inside the tissue. The authors suggest that hydrostatic pressure, possibly carried by the hemolymph flow that accompanies the invading macrophage-like cells, may help drive the Stage II elongation, consistent with classic observations that pressing on the prepupal abdomen can induce tarsal elongation. The work, they write, should provide a foundation for unraveling how cell identities are maintained through such dramatic cell behaviors, and how the mechanisms of final shape formation generate the extraordinary diversity of insect appendages.
Subject of Research: Cell dynamics during final shape formation of the Drosophila adult tarsus observed by live imaging
Article Title: Unveiling the cell dynamics during the final shape formation of the tarsus in Drosophila adult leg by live imaging
Article References: Hiraiwa, S., Takeshita, S., Terano, T., Hayashi, R., Suzuki, K., Tajiri, R., & Kojima, T. (2024). Unveiling the cell dynamics during the final shape formation of the tarsus in Drosophila adult leg by live imaging. Development Genes and Evolution, 234(2), 117-133. https://doi.org/10.1007/s00427-024-00719-z
Image Credits: AI Generated
DOI: 10.1007/s00427-024-00719-z
Keywords: Drosophila melanogaster, tarsus, live imaging, pupal development, epithelial morphogenesis, Parthenon-like structure, basement membrane, macrophage-like cells, apoptosis, cytoskeleton, Development Genes and Evolution, leg development
News Source: Drew Townsend. (October 29, 2026). Live imaging reveals a hidden Parthenon of cells building the fly leg. Scienmag.
