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Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In Drosophila melanogaster, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. Although it is the most studied pheromone in flies, the mechanisms that determine the complexity of the response, its intensity and final output with respect to social context, sex and prior interaction, are still not well understood. Here we explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. Odorant binding protein 69a (Obp69a) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. We show that activation of cVA-sensing neurons is sufficeint to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies.

The Drosophila melanogaster sex hierarchy controls sexual differentiation of somatic cells via the activities of the terminal genes in the hierarchy, doublesex (dsx) and fruitless (fru). We have targeted an insertion of GAL4 into the dsx gene, allowing us to visualize dsx-expressing cells in both sexes. Developmentally and as adults, we find that both XX and XY individuals are fine mosaics of cells and tissues that express dsx and/or fruitless (fru(M)), and hence have the potential to sexually differentiate, and those that don't. Evolutionary considerations suggest such a mosaic expression of sexuality is likely to be a property of other animal species having two sexes. These results have also led to a major revision of our view of how sex-specific functions are regulated by the sex hierarchy in flies. Rather than there being a single regulatory event that governs the activities of all downstream sex determination regulatory genes-turning on Sex lethal (Sxl) RNA splicing activity in females while leaving it turned off in males-there are, in addition, elaborate temporal and spatial transcriptional controls on the expression of the terminal regulatory genes, dsx and fru. Thus tissue-specific aspects of sexual development are jointly specified by post-transcriptional control by Sxl and by the transcriptional controls of dsx and fru expression.

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells—the progeny of basal stem cells—are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. Here, we investigate how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila . We find they gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity. Galenza et al. show that basal stem cell progeny seamlessly integrate into a physiologically active barrier epithelium by gestating their future apical surface in a sheltering niche created by a transient occluding junction.

Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.

Background/objectivesHeart failure (HF) is a growing clinical and economic burden for patients and health systems. The COVID-19 pandemic has led to avoidance and delay in care, resulting in increased morbidity and mortality among many patients with HF. The increasing burden of HF during the COVID-19 pandemic led us to evaluate the quality and safety of the Hospital at Home (HAH) for patients presenting to their community providers or emergency department (ED) with symptoms of acute on chronic HF (CHF) requiring admission.Design/outcomesA non-randomised prospective case–controlled of patients enrolled in the HAH versus admission to the hospital (usual care, UC). Primary outcomes included length of stay (LOS), adverse events, discharge disposition and patient satisfaction. Secondary outcomes included 30-day readmission rates, 30-day ED usage and ED dwell time.ResultsSixty patients met inclusion/exclusion criteria and were included in the study. Of the 60 patients, 40 were in the HAH and 20 were in the UC group. Primary outcomes demonstrated that HAH patients had slightly longer LOS (6.3 days vs 4.7 days); however, fewer adverse events (12.5% vs 35%) compared with the UC group. Those enrolled in the HAH programme were less likely to be discharged with postacute services (skilled nursing facility or home health). HAH was associated with increased patient satisfaction compared with Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) score in North Carolina. Secondary outcomes of 30-day readmission and ED usage were similar between HAH and UC.ConclusionsThe HAH pilot programme was shown to be a safe and effective alternative to hospitalisation for the appropriately selected patient presenting with acute on CHF.

The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ∼1000 GAL4 lines, using assays for general courtship, male–male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male–male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation.

The Drosophila melanogaster sex hierarchy controls sexual differentiation of somatic cells via the activities of the terminal genes in the hierarchy, doublesex (dsx) and fruitless (fru). We have targeted an insertion of GAL4 into the dsx gene, allowing us to visualize dsx-expressing cells in both sexes. Developmentally and as adults, we find that both XX and XY individuals are fine mosaics of cells and tissues that express dsx and/or fruitless (fruM), and hence have the potential to sexually differentiate, and those that don't. Evolutionary considerations suggest such a mosaic expression of sexuality is likely to be a property of other animal species having two sexes. These results have also led to a major revision of our view of how sex-specific functions are regulated by the sex hierarchy in flies. Rather than there being a single regulatory event that governs the activities of all downstream sex determination regulatory genes--turning on Sex lethal (Sxl) RNA splicing activity in females while leaving it turned off in males--there are, in addition, elaborate temporal and spatial transcriptional controls on the expression of the terminal regulatory genes, dsx and fru. Thus tissue-specific aspects of sexual development are jointly specified by post-transcriptional control by Sxl and by the transcriptional controls of dsx and fru expression.

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. These replacement cells--the progeny of basal stem cells--are born without apical, barrier-forming structures such as a protective, lumen-facing membrane and occluding junctions. How stem cell progeny acquire these structures to become part of the barrier is unknown. Here we use Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), Correlative Light-Electron Microscopy (CLEM), and volumetric imaging of live and fixed organs to investigate progenitor integration in the intestinal epithelium of adult Drosophila. We find that stem cell daughters gestate their future lumenal-apical membrane beneath a transient, basal niche formed by an umbrella-shaped occluding junction that shelters the growing cell and adheres it to mature neighbor cells. The umbrella junction both targets formation of a deep, microvilli-lined, apical invagination and closes it off from the contents of the gut lumen. When the growing cell is sufficiently mature, the umbrella junction retracts to expose this Pre-Assembled Apical Compartment (PAAC) to the gut lumen, thus incorporating the new cell into the intestinal barrier. When we block umbrella junctions, stem cell daughters grow and attempt to differentiate but fail to integrate; when we block cell growth, no umbrella junctions form, and daughters arrest in early differentiation. Thus, stem cell progeny build new barrier structures in the shelter of a transient niche, where they are protected from lumenal insults until they are prepared to withstand them. By coordinating this dynamic junctional niche with progenitor cell differentiation, a physiologically active epithelial organ incorporates new cells while upholding integrity of its barrier. Competing Interest Statement The authors have declared no competing interest.

For my graduate work, I have chosen to investigate sex-specific patterning and function of the olfactory system in the fruit fly, Drosophila melanogaster, by addressing three questions: 1) What is the developmental genetic regulation of the sexual dimorphism in numbers of olfactory sensilla? 2) Do olfactory receptor neurons (ORNs) expressing Or47b (Or47b-ORNs) stimulate courtship in males and females? 3) Which genes are regulated by male-specific fruitless isoforms (FRUM) in olfactory and other peripheral sensory neurons?In Drosophila, terminal sexual differentiation is orchestrated by the activity of the transcription factors fruitless (fru) and doublesex (dsx). I have shown that the action of dsx alone is necessary and sufficient to specify sex-appropriate numbers of olfactory sensilla (the peripheral sensory unit). The dsx locus encodes sex-specific isoforms that differentially organize the developing antenna to specify sex-appropriate numbers of different types of olfactory sensilla. The female-specific DSX isoform promotes female-appropriate numbers of basiconic sensilla and depresses the number of trichoid sensilla on the antenna. Male-specific DSX promotes male-appropriate numbers of trichoid sensilla but appears to have little effect on determining numbers of basiconic sensilla. Inferred from the expression pattern of dsx, the DSX proteins do not show significant overlap with olfactory sensilla precursors. These data and other lines of evidence suggest that it is likely that dsx controls numbers of olfactory sensilla cell non-autonomously.One subtype of sexually dimorphic trichoid sensilla is innervated by ORNs that express the receptor Or47b and respond to a pheromone produced by males and females. I asked whether these ORNs promote male courtship and female receptivity.I have shown that Or47b-ORNs may promote courtship in males, but they appear to play no detectable role in stimulating female receptivity. Finally, in an effort to elucidate how sexual dimorphism is built into the developing olfactory system, I have collaborated with David Mellert (a former graduate student) to identify genes that are regulated by male-specific fru isoforms in peripheral sensory neurons of the olfactory and gustatory systems, as well as in mechanosensory neurons of the genitalia. We used laser capture microdissection to collect fru-expressing neurons in the antennae, prothoracic forelegs, and genitalia from fru+ and fru-null males. By comparing the whole-genome expression profiles of these samples, we were able to identify candidate targets of FRUM regulation in these tissues. For the antenna, this yielded a promising list of ~100 candidates that are in the process of being validated. Moreover, we found essentially no overlap in the candidate lists for different tissues, suggesting that FRUM regulates different genes in different tissues.

Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in specific sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D. melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D. melanogaster, D. simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship in both species, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D. simulans males. However, and similar to its function in D. melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D. simulans. Taken together, we have identified shared as well as distinct evolutionary solutions to chemosensory processing of tastants as well as cues that inhibit or promote courtship in two closely related Drosophila species.