Transcription factors that can convert adult cells of one type to another are usually discovered empirically by testing factors with a known developmental role in the target cell. Here we show that standard genomic methods (RNA-seq and ChIP-seq) can help identify these factors, as most are more strongly Polycomb repressed in the source cell and more highly expressed in the target cell. This criterion is an effective genome-wide screen that significantly enriches for factors that can transdifferentiate several mammalian cell types including neural stem cells, neurons, pancreatic islets, and hepatocytes. These results suggest that barriers between adult cell types, as depicted in Waddington’s "epigenetic landscape", consist in part of differentially Polycomb-repressed transcription factors. This genomic model of cell identity helps rationalize a growing number of transdifferentiation protocols and may help facilitate the engineering of cell identity for regenerative medicine.

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

Long-term memory depends on the control of activity-dependent neuronal gene expression, which is regulated by epigenetic modifications. The epigenetic modification of histones is orchestrated by the opposing activities of two classes of regulatory complexes: permissive co-activators and silencing co-repressors. Much work has focused on co-activator complexes, but little is known about the co-repressor complexes that suppress the expression of plasticity-related genes. Here, we define a critical role for the co-repressor SIN3A in memory and synaptic plasticity, showing that postnatal neuronal deletion of Sin3a enhances hippocampal long-term potentiation and long-term contextual fear memory. SIN3A regulates the expression of genes encoding proteins in the post-synaptic density. Loss of SIN3A increases expression of the synaptic scaffold Homer1, alters the mGluR1α- and mGluR5-dependence of long-term potentiation, and increases activation of extracellular signal regulated kinase (ERK) in the hippocampus after learning. Our studies define a critical role for co-repressors in modulating neural plasticity and memory consolidation and reveal that Homer1/mGluR signaling pathways may be central molecular mechanisms for memory enhancement.

A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast produces a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, we marked and isolated lineage-specific neuroblasts for RNA sequencing. We labeled particular neuroblasts throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single-cell picking. Transcriptome analysis of mushroom body, antennal lobe and type II neuroblasts compared with non-selective neuroblasts, neurons and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. Besides transcription factors that are likely to be pan-neuroblast, many transcription factors exist that are selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates.

In a recent publication, Ma et al [1] claim that a transformer-based cellular segmentation method called Mediar [2] — which won a Neurips challenge — outperforms Cellpose [3] (0.897 vs 0.543 median F1 score). Here we show that this result was obtained by artificially impairing Cellpose in multiple ways. When we removed these impairments, Cellpose outperformed Mediar (0.861 vs 0.826 median F1 score on the updated test set). To further investigate the performance of transformers for cellular segmentation, we replaced the Cellpose backbone with a transformer. The transformer-Cellpose model also did not outperform the standard Cellpose (0.848 median F1 test score). Our results suggest that transformers do not advance the state-of-the-art in cellular segmentation.

Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

In most animals, a relatively small number of descending neurons (DNs) connect higher brain centers in the animal’s head to motor neurons (MNs) in the nerve cord of the animal’s body that effect movement of the limbs. To understand how brain signals generate behavior, it is critical to understand how these descending pathways are organized onto the body MNs. In the fly, Drosophila melanogaster, MNs controlling muscles in the leg, wing, and other motor systems reside in a ventral nerve cord (VNC), analogous to the mammalian spinal cord. In companion papers, we introduced a densely-reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC, Takemura et al., 2023), including cell type and developmental lineage annotation (Marin et al., 2023), which provides complete VNC connectivity at synaptic resolution. Here, we present a first look at the organization of the VNC networks connecting DNs to MNs based on this new connectome information. We proofread and curated all DNs and MNs to ensure accuracy and reliability, then systematically matched DN axon terminals and MN dendrites with light microscopy data to link their VNC morphology with their brain inputs or muscle targets. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify communities of intrinsic neurons linked to control of different motor systems, including putative ventral circuits for walking, dorsal circuits for flight steering and power generation, and intermediate circuits in the lower tectulum for coordinated action of wings and legs. Our analysis generates hypotheses for future functional experiments and, together with the MANC connectome, empowers others to investigate these and other circuits of the Drosophila ventral nerve cord in richer mechanistic detail.

In most animals, a relatively small number of descending neurons (DNs) connect higher brain centers in the animal’s head to circuits and motor neurons (MNs) in the nerve cord of the animal’s body that effect movement of the limbs. To understand how brain signals generate behavior, it is critical to understand how these descending pathways are organized onto the body MNs. In the fly, Drosophila melanogaster, MNs controlling muscles in the leg, wing, and other motor systems reside in a ventral nerve cord (VNC), analogous to the mammalian spinal cord. In companion papers, we introduced a densely-reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC, (Takemura et al., 2024)), including cell type and developmental lineage annotation (Marin et al., 2024), which provides complete VNC connectivity at synaptic resolution. Here, we present a first look at the organization of the VNC networks connecting DNs to MNs based on this new connectome information. We proofread and curated all DNs and MNs to ensure accuracy and reliability, then systematically matched DN axon terminals and MN dendrites with light microscopy data to link their VNC morphology with their brain inputs or muscle targets. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify communities of intrinsic neurons linked to control of different motor systems, including putative ventral circuits for walking, dorsal circuits for flight steering and power generation, and intermediate circuits in the lower tectulum for coordinated action of wings and legs. Our analysis generates hypotheses for future functional experiments and, together with the MANC connectome, empowers others to investigate these and other circuits of the Drosophila ventral nerve cord in richer mechanistic detail.

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.

Perhaps the most valuable single set of resources for genetic studies of Drosophila melanogaster is the collection of multiply-inverted chromosomes commonly known as balancer chromosomes. Balancers prevent the recovery of recombination exchange products within genomic regions included in inversions and allow perpetual maintenance of deleterious alleles in living stocks and the execution of complex genetic crosses. Balancer chromosomes have been generated traditionally by exposing animals to ionizing radiation and screening for altered chromosome structure or for unusual marker segregation patterns. These approaches are tedious and unpredictable, and have failed to produce the desired products in some species. Here I describe transgenic tools that allow targeted chromosome rearrangements in Drosophila species. The key new resources are engineered reporter genes containing introns with yeast recombination sites and enhancers that drive fluorescent reporter genes in multiple body regions. These tools were used to generate a doubly-inverted chromosome 3R in D. simulans that serves as an effective balancer chromosome.