The generation of post-gastrulation stem cell-derived mouse embryo models (SEMs) exclusively from naive embryonic stem cells (nESCs) has underscored their ability to give rise to embryonic and extra-embryonic lineages. However, existing protocols for mouse SEMs rely on the separate induction of extra-embryonic lineages and on ectopic expression of transcription factors to induce nESC differentiation into trophectoderm (TE) or primitive endoderm (PrE). Here, we demonstrate that mouse nESCs and naive induced pluripotent stem cells (niPSCs) can be simultaneously co-induced, via signaling pathway modulation, to generate PrE and TE extra-embryonic cells that self-organize into embryonic day (E) 8.5-E8.75 transgene-free (TF) SEMs. We also devised an alternative condition (AC) naive media that in vitro stabilizes TF-SEM-competent OCT4+/NANOG+ nESC colonies that co-express antagonistic CDX2 and/or GATA6 extra-embryonic fate master regulators and self-renew while remaining poised for TE and PrE differentiation, respectively. These findings improve mouse SEM strategies and shed light on amplifying an inherent and dormant extra-embryonic plasticity of mouse naive pluripotent cells in vitro.

Fluorescent proteins have transformed biological imaging, yet their limited photostability and brightness restrict their applications. We used deep learning-based de novo protein design methods to design binders to three bright, stable and cell-permeable dyes spanning the imaging spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). We obtain highly specific dye-binding proteins with low nanomolar affinities for the intended target; a crystal structure of one binder confirms close resemblance to the design model. Simultaneous labeling of mammalian cells expressing three dye-specific binders at different subcellular compartments demonstrates the utility in multiplex imaging. We further expand the functionality of the binder by incorporating an active site that carries out nucleophilic aromatic substitution to form a covalent linkage with the dye, and develop split versions which reconstitute fluorescence at subcellular locations where both halves are present towards monitoring in-cell protein interactions and chemically induced dimerization. Our designed high affinity and specificity dye binders open up new opportunities for multiplexed biological imaging.

Just as genomes revolutionized molecular genetics, connectomes (maps of neurons and synapses) are transforming neuroscience. To date, the only species with complete connectomes are worms and sea squirts (103-104 synapses). By contrast, the fruit fly is more complex (108 synaptic connections), with a brain that supports learning and spatial memory and an intricate ventral nerve cord analogous to the vertebrate spinal cord. Here we report the first adult fly connectome that unites the brain and ventral nerve cord, and we leverage this resource to investigate principles of neural control. We show that effector cells (motor neurons, endocrine cells and efferent neurons targeting the viscera) are primarily influenced by local sensory cells in the same body part, forming local feedback loops. These local loops are linked by long-range circuits involving ascending and descending neurons organized into behavior-centric modules. Single ascending and descending neurons are often positioned to influence the voluntary movements of multiple body parts, together with endocrine cells or visceral organs that support those movements. Brain regions involved in learning and navigation supervise these circuits. These results reveal an architecture that is distributed, parallelized and embodied (tightly connected to effectors), reminiscent of distributed control architectures in engineered systems.

Genetically encoded calcium indicators (GECIs) are vital tools for fluorescence-based visualization of neuronal activity with high spatial and temporal resolution. However, current highest-performance GECIs are predominantly green or red fluorescent, limiting multiplexing options and efficient excitation with fixed-wavelength femtosecond lasers operating at 1030 nm. Here, we introduce OCaMP (also known as O-GECO2), an orange fluorescent GECI engineered from O-GECO1 through targeted substitutions to improve calcium affinity while retaining the favorable photophysical properties of mOrange2. OCaMP exhibits improved two-photon cross-section, responsiveness, photostability, and calcium affinity relative to O-GECO1. In cultured neurons, zebrafish, and mouse cortex, OCaMP outperforms the red GECIs jRCaMP1a and jRGECO1a in sensitivity, kinetics, and signal-to-noise ratio. These properties establish OCaMP as a robust tool for high-fidelity neural imaging optimized for 1030 nm excitation and a compromise-free option within the spectral gap between existing green and red GECIs.

Epithelial polarity is essential for proper tissue organization and function, yet the molecular mechanisms governing apical membrane formation during secretory epithelial development remain incompletely understood. Here, we investigate the role of the small GTPase Cdc42 in salivary gland acinar cell development using a mouse model designed to knock out Cdc42 specifically at the onset of acinar cell formation. Loss of Cdc42 resulted in defective apical membrane formation accompanied by accumulation of vesicles around the apical lumen. These vesicles contained the apical water channel AQP5 and the apical recycling endosome (ARE) marker Rab11a, while the basolateral transporter NKCC1 retained normal localization, indicating an apical-selective trafficking defect. Importantly, Cdc42 deficiency caused a selective 40% reduction in the expression of the SNARE protein VAMP2, while other vesicle trafficking proteins including VAMP8, SNAP23, and EEA1 remained unchanged. Our findings reveal that Cdc42 controls apical membrane formation by maintaining VAMP2 expression, which is essential for the fusion of Rab11a-positive recycling endosomes. The accumulation of fusion-incompetent AREs near the apical surface demonstrates the critical role of the Cdc42-VAMP2 pathway in epithelial development. These results provide new insights into how polarity regulators integrate vesicle trafficking and fusion machinery, and may have implications for understanding glandular diseases involving epithelial polarity defects.

Persistent internal states are important for maintaining survival-promoting behaviors, such as aggression. In female Drosophila melanogaster, we have previously shown that individually activating either aIPg or pC1d cell types can induce aggression. Here we investigate further the individual roles of these cholinergic, sexually dimorphic cell types, and the reciprocal connections between them, in generating a persistent aggressive internal state. We find that a brief 30-second optogenetic stimulation of aIPg neurons was sufficient to promote an aggressive internal state lasting at least 10 minutes, whereas similar stimulation of pC1d neurons did not. While we previously showed that stimulation of pC1e alone does not evoke aggression, persistent behavior could be promoted through simultaneous stimulation of pC1d and pC1e, suggesting an unexpected synergy of these cell types in establishing a persistent aggressive state. Neither aIPg nor pC1d show persistent neuronal activity themselves, implying that the persistent internal state is maintained by other mechanisms. Moreover, inactivation of pC1d did not significantly reduce aIPg-evoked persistent aggression, arguing that the aggressive state did not depend on pC1d-aIPg recurrent connectivity. Our results suggest the need for alternative models to explain persistent female aggression.

Preprint: https://www.biorxiv.org/content/10.1101/2023.06.07.543722v2

Fluorescence microscopy, a key driver for progress in the life sciences, faces limitations due to the microscope’s optics, fluorophore chemistry, and photon exposure limits, necessitating trade-offs in imaging speed, resolution, and depth. Here, we introduce MicroSplit, a computational multiplexing technique based on deep learning that allows multiple cellular structures to be imaged in a single fluorescent channel and then unmixed computationally, allowing faster imaging and reduced photon exposure. We show that MicroSplit efficiently separates up to four superimposed noisy structures into distinct denoised fluorescent image channels. Furthermore, using Variational Splitting Encoder-Decoder (VSE) networks, our approach can sample diverse predictions from a trained posterior of solutions. The diversity of these samples scales with the uncertainty in a given input, allowing us to estimate the true prediction errors by computing the variability between posterior samples. We demonstrate the robustness of MicroSplit across various datasets and noise levels and show its utility to image more, image faster, and improve downstream analysis. We provide MicroSplit along with all associated training and evaluation datasets as open resources, enabling life scientists to benefit from the potential of computational multiplexing and accelerate the pace of scientific discovery.

Power-law scaling in coarse-grained data suggests critical dynamics, but the true source of this scaling often remains unclear. Here, we analyze neural activity recorded during spatial navigation, reproducing power-law scaling under a phenomenological renormalization group (PRG) procedure that clusters units by activity similarity. Such scaling was previously linked to criticality. Here, we show that the iterative nature of the procedure itself leads to the emergence of power laws when applied to heterogeneous, non-interacting units obeying spatially structured activity without requiring critical interactions. Furthermore, the scaling exponents produced by heteregeneous non-interacting units match the observed exponents in recorded neural data. A simplified version of the PRG further reveals how heterogeneity smooths transitions across scales, mimicking critical behavior. The resulting exponents depend systematically on system and population size, predictions confirmed by subsampling the data.

Cryo-ultramicrotomy, developed by Bernhard in 1965 [1], has long been regarded as the pinnacle of achievement for electron microscopists. This technique allows biological samples to be sliced into ultrathin sections and examined in a cryo-electron microscope, revealing the most intricate subcellular structures without chemical fixation or staining. The advent of vitrification [2,3] and high-pressure freezing (HPF) technology [4,5] provided reliable methods for preserving cellular structures, and the introduction of diamond knife to cryo-ultramicrotomy [6] offering cryo-ultramicrotomists reassurance in consistency of the quality [7].

Understanding how neural circuits give rise to behavior requires comprehensive knowledge of neuronal morphology, connectivity, and function. Atlas platforms play a critical role in enabling the visualization, exploration, and dissemination of such information. Here, we present FishExplorer, an interactive and expandable community platform designed to integrate and analyze multimodal brain data from larval zebrafish. FishExplorer supports datasets acquired through light microscopy (LM), electron microscopy (EM), and X-ray imaging, all co-registered within a unified spatial coordinate system which enables seamless comparison of neuronal morphologies and synaptic connections. To further assist circuit analysis, FishExplorer includes a suite of tools for querying and visualizing connectivity at the whole-brain scale. By integrating data from recent large-scale EM reconstructions (presented in companion studies), FishExplorer enables researchers to validate circuit models, explore wiring principles, and generate new hypotheses. As a continuously evolving resource, FishExplorer is designed to facilitate collaborative discovery and serve the growing needs of the teleost neuroscience community.