Development of a genetic multicolor cell labeling approach for neural circuit analysis in Drosophila

The assembly of functional neural circuits during development is pivotal for the ability of the brain to generate complex behaviors. To facilitate the analysis of the underlying molecular mechanisms in Drosophila, we have developed a genetic multicolor cell labeling approach called Flybow (FB), which is based on the vertebrate Brainbow-2 system. FB relies on the stochastic expression of membrane tethered fluorescent proteins (FPs). FP encoding sequences were arranged in pairs within one or two cassettes each flanked by recombination sites. Recombination mediated by an inducible modified Flp/FRT system results in both excisions and inversions of the flanked cassettes providing temporal control of FP expression. Moreover, FB employs the GAL4/UAS system and hence can be used to investigate distinct cell populations in the tissue of interest. We have generated three FB variants. FB 1.0 consists of one cassette driving expression of either mCherry or V5-tagged Cerulean. FB 1.1 contains a second cassette with opposing enhanced green fluorescent protein (EGFP) and mCitrine cDNAs leading to stochastic expression of four FPs. Finally, FB2.0 contains an additional excisable cassette flanked by classical FRT sites to refine transgene expression in specific cell types, in which Gal4 and Flp activities overlap. The FB approach was validated by investigating neural circuit assembly and connectivity in the visual system. FB makes it possible to visualize dendritic and axonal arborizations of different neuron subtypes and the morphology of glial cells with single cell resolution in one sample. Using live and fixed embryonic tissue, we could show that FB is suitable for studies of this early developmental stage. Additionally, we demonstrated that the approach can be used in non-neural tissues. Finally, combining the mosaic analysis with a repressible cell marker (MARCM) and FB approaches, we demonstrate that our technique is compatible with available Drosophila tools for genetic dissection of neural circuit formation

Sex differences in intestinal carbohydrate metabolism promote food intake and sperm maturation

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signalling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this malebiased intestinal metabolic state in controlling food intake and sperm production through gutderived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, and reveals that metabolic communication across organs is physiologically significant. The instructive role of citrate in inter-organ communication may be significant in more biological contexts than previously recognised

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia.

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homoeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease

Enteric neurons increase maternal food intake during reproduction.

Reproduction induces increased food intake across females of many animal species1-4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain

Multiple new site-specific recombinases for use in manipulating animal genomes

Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast—KD, B2, B3, and R—are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice