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Highlights d Flies steer using four muscle groups that insert on tiny parts of the wing hinge d Each muscle group consists of both steadily and transiently active muscles d A stabilizing visual pathway preferentially recruits the steadily active muscles d A pathway for random turns preferentially recruits the transiently active muscles

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells.

Like the vertebrate spinal cord, the insect ventral nerve cord (VNC) mediates limb sensation and motor control. Here, we applied automated tools for electron microscopy (EM) volume alignment, neuron reconstruction, and synapse prediction to create a draft connectome of theDrosophilaVNC. To interpret the VNC connectome, it is crucial to know its relationship with the rest of the body. We therefore mapped the muscle targets of leg and wing motor neurons in the connectome by comparing their morphology to genetic driver lines, dye fills, and x-ray holographic nano-tomography volumes of the fly leg and wing. Knowing the outputs of the connectome allowed us to identify neural circuits that coordinate the wings with the middle and front legs during escape takeoff. We provide the draft VNC connectome and motor neuron atlas, along with tools for programmatic and interactive access, as community resources.

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless (wg/wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration (rgn), augmenter of liver regeneration (alr) and Matrix metalloproteinase-1 (Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.

When Drosophila imaginal discs regenerate, specific groups of cells can switch disc identity so that, for example, cells determined for leg identity switch to wing. Such switches in cell determination are known as transdetermination. We have developed a system by which individual cells are marked and monitored in vivo as they transdetermine so that their proliferation, cell sizes, and differentiation are accurately traced. Here, we document that when cells transdetermine, they do not convert to a younger cell cycle. Instead, cell cycle changes precede transdetermination and are different from those observed at any time in normal development. We propose that it is not a younger but a unique cell cycle progression and a big cell size that conditions the cells for developmental plasticity.

SUMMARY Imaginal discs of Drosophila have the remarkable ability to regenerate. After fragmentation wound healing occurs, ectopic wg is induced and a blastema is formed. In some, but not all fragments, the blastema will replace missing structures and a few cells can become more plastic and transdetermine to structures of other discs. A series of systematic cuts through the first leg disc revealed that a cut must transect the dorsal-proximal disc area and that the fragment must also include wg-competent cells. Fragments that fail to both transdetermine and regenerate missing structures will do both when provided with exogenous Wg, demonstrating the necessity of Wg in regenerative processes. In intact leg discs ubiquitously expressed low levels of Wg also leads to blastema formation, regeneration and transdetermination. Two days after exogenous wg induction the endogenous gene is activated, leading to elevated levels of Wg in the dorsal aspect of the leg disc. We identified a wg enhancer that regulates ectopic wg expression. Deletion of this enhancer increases transdetermination, but lowers the amount of ectopic Wg. We speculate that this lessens repression of dpp dorsally, and thus creates a permissive condition under which the balance of ectopic Wg and Dpp is favorable for transdetermination.

In Drosophila, widely-used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the Twin Spot Generator technique (TSG) and demonstrate that through mitotic recombination, TSG generates green and red twin spots in internal fly tissues, visible even as single cells. We discuss the wide applications of TSG to lineage and genetic mosaic studies.