Deepthi Mahishi @deeMahishi@qoto.org

Vision is critical for the regulation of mating behaviors in many species. Here, we discovered that the Drosophila ortholog of human GABAA-receptor-associated protein (GABARAP) is required to fine-tune male courtship by modulating the activity of visual feedback neurons, lamina tangential cells (Lat). GABARAP is a ubiquitin-like protein that regulates cell-surface levels of GABAA receptors. Knocking down GABARAP or GABAA receptors in Lat neurons or hyperactivating them induces male courtship toward other males. Inhibiting Lat neurons, on the other hand, delays copulation by impairing the ability of males to follow females. Remarkably, the human ortholog of Drosophila GABARAP restores function in Lat neurons. Using in vivo two-photon imaging and optogenetics, we show that Lat neurons are functionally connected to neural circuits that mediate visually-guided courtship pursuits in males. Our work reveals a novel physiological role for GABARAP in fine-tuning the activity of a visual circuit that tracks a mating partner during courtship. ### Competing Interest Statement The authors have declared no competing interest.

Neurons produce and release neuropeptides to communicate with one another. Despite their profound impact on critical brain functions, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects the neuropeptides release presynaptically, and a genetically encoded silencer that specifically degrades neuropeptides inside the LDCV. Monitoring and silencing peptidergic and glutamatergic transmissions from presynaptic terminals using our newly developed tools and existing genetic tools, respectively, reveal that neuropeptides, not glutamate, are the primary transmitter in encoding unconditioned stimulus during Pavlovian threat learning. These results show that our sensor and silencer for peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake behaving animals. ### Competing Interest Statement The authors have declared no competing interest.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model. ### Competing Interest Statement The California Institute of Technology has filed patent applications for the work described in this manuscript, with X.C. and V.G. listed as inventors. V.G. is a co-founder and board member of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company.

Intestinal smooth muscles are the workhorse of the digestive system. Inside the millions of finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic vessel, called the lacteal, sending fats into blood circulation for energy production. Despite this vital function, how villus smooth muscles form, how they assemble alongside lacteals, and how they repair throughout life remain unknown. Here we combine single-cell RNA sequencing of the mouse intestine with quantitative lineage tracing to reveal the mechanisms of formation and differentiation of villus smooth muscle cells. Within the highly regenerative villus, we uncover a local hierarchy of subepithelial fibroblast progenitors that progress to become mature smooth muscle fibers, via an intermediate contractile myofibroblast-like phenotype, a long-studied hallmark of wound healing. This continuum persists in the adult intestine as the major source of smooth muscle reservoir capable of continuous self-renewal throughout life. We further discover that the NOTCH3-DLL4 signaling axis governs the assembly of villus smooth muscles alongside their adjacent lacteal, which is necessary for gut absorptive function. Overall, our data shed light on the genesis of a poorly defined class of intestinal smooth muscle and pave the way for new opportunities to accelerate recovery of digestive function by stimulating muscle repair. ### Competing Interest Statement The authors have declared no competing interest.