Biology subjects

Silver, D.

Publications and source records attributed to Silver, D..

9 recordsLinked to original sources

bioRxiv2026

Same-section spatial metabolo-transcriptomics using Stereo-meta-seq reveals DHA-driven kidney maturation

A central unresolved question in developmental biology is whether local metabolites merely accompany, or actively instruct, tissue maturation. Addressing this question requires direct spatial coupling of metabolic states with genome-wide transcriptional programs in situ at high spatial resolution, which existing approaches do not readily achieve. Here, we introduce Stereo-meta-seq, a workflow that integrates quantitative MALDI-MSI with Stereo-seq spatial transcriptomics within a single tissue section. A conductive adapter was designed to overcome the electrical incompatibility of non-conductive Stereo-seq chips with vacuum MALDI platforms, improving efficiency of MSI detection that preserves RNA integrity. MALDI laser-ablation marks are retained in downstream Stereo-seq data and serve as intrinsic fiducials for direct co-registration at 10 m or 20 m resolution, enabling fine grained spatial metabolite-transcript integration. Applying Stereo-meta-seq to human kidney development, we uncover selective enrichment of docosahexaenoic acid (DHA) in maturing proximal tubules. Functional studies in human kidney organoids demonstrate that DHA activates PPAR-and HNF4-driven transcriptional programs and promotes proximal tubule maturation in vitro and after transplantation in vivo. These findings identify lipid metabolism as an instructive regulator of human nephrogenesis and establish Stereo-meta-seq as a practical platform for dissecting metabolite-gene coupling and tissue heterogeneity in situ.

Cell Biology↗

bioRxiv2026

Chd8 haploinsufficiency leads to molecular layer heterotopias and age-dependent cortical expansion

Mutations in the chromatin remodeler CHD8 are associated with autism and macrocephaly. While mouse models of Chd8 haploinsufficiency recapitulate brain overgrowth, the specific cellular mechanisms and developmental timing that lead to these anatomical abnormalities remain poorly understood. Here, we conducted 3D imaging of Chd8V986*/+ mouse brains using magnetic resonance imaging followed by tissue clearing and cellular resolution light-sheet microscopy across embryonic and postnatal developmental stages. We found that brain overgrowth occurs postnatally, driven by an increase in non-neuronal cells prior to volumetric expansion. Unexpectedly, we identified prevalent molecular layer heterotopias (MLH) within the frontal cortex of Chd8V986*/+ mice composed of neurons breaking through the pial surface during embryonic development and persisting throughout life. Increased incidence of MLH, previously identified in individuals with idiopathic autism, was replicated across three independent Chd8+/- mouse models and present across multiple genomic backgrounds, establishing aberrant neuronal migration as a core feature of Chd8 haploinsufficiency.

neuroscience↗

bioRxiv2025

Dissecting mammalian cortical circuit development at single-cell resolution using inducible barcoded rabies virus

Highly organized circuits of connected neurons enable diverse brain functions. Improper development of these circuits is associated with neurodevelopmental disorders, and understanding how circuits are formed is crucial for unraveling the mechanisms of these diseases. We currently have an incomplete picture of how specific brain circuits develop and how they are affected in disease, because we lack methods to study them at scale and with single-cell resolution. Monosynaptic rabies tracing is the gold standard method to study circuit architecture. However, it suffers from cellular toxicity, low throughput, lack of control over the timing of labeling, and the inability to access the molecular profiles of individual neurons. To address these issues, we developed an inducible barcoded rabies virus (ibRV) to enable temporal-controlled labeling of synaptic circuits followed by high-throughput single-cell genomics readout. ibRV allows for dissecting neuronal circuit changes over time at single-cell and spatial resolution. We applied ibRV to study the development of specific mouse cortical circuits during late prenatal and postnatal life using single-cell genomics and unbiased spatial transcriptomics as readouts. We characterized and quantified developmental connectivity patterns and molecular cascades that underlie their formation. Additionally, we constructed functional in silico circuit models that enable interrogation of circuit function and dysfunction at specific developmental stages. Our study provides novel tools for circuit analysis and can provide new insights into the mechanisms of mammalian brain development.

neuroscience↗

bioRxiv2025

Intraspecific sequence variation and complete genomes refine the identification of rapidly evolved regions in humans

Summary Humans exhibit significant phenotypic differences from other great apes, yet pinpointing the underlying genetic changes has been limited by incomplete reference genomes and a reliance on single assemblies to represent a species. We aligned 20 telomere-to-telomere (T2T) assemblies spanning great ape divergence and variation to define 1,596 Consensus HAQERs (consensus human ancestor quickly evolved regions), regions that diverged rapidly between the human-chimpanzee ancestor and an ancestral node of modern humans. Unlike prior HAQER sets, Consensus HAQERs incorporate population variation, reducing the likelihood of intraspecies variation appearing as interspecies divergence. Consensus HAQERs exhibit signatures of elevated mutation rates, ancient positive selection, bivalent regulatory function, are enriched in disease-linked loci, and often emerged in previously inaccessible repetitive DNA. Through multiplex, single-cell enhancer assays, we identify HAQERs as active enhancers in the developing brain and cardiomyocytes, highlighting their potential contributions to human-specific gene regulation across multiple tissues. Highlights[bullet] Telomere-to-telomere alignments of diverse human and great ape genomes identify 1,596 Consensus HAQERs, regions of rapid sequence divergence separating human ancestors from other great apes. [bullet]Consensus HAQERs exhibit signatures of both elevated mutation rates and ancient positive selection. [bullet]HAQERs are enriched in bivalent regulatory elements and disease-linked loci. [bullet]Multiplex, single-cell gene regulatory assays identify HAQERs as enhancers in the developing heart and brain.

genomics↗

bioRxiv2025

Subcellular transcriptome of radial glia reveals compartmentalized control of cortical development

RNA localization and local translation mediate spatial and temporal control of polarized cells, including radial glial cells (RGCs) which produce and organize neurons and glia. Within RGCs, RNAs are transported long distances to basal endfeet, where they can undergo local translation. However, the subcellular composition of RGCs and function of local gene regulation remains largely unknown. Here, we discover that basal endfeet harbor a rich transcriptome including a Dynein component critical for subcellular RGC function. By purifying RGC compartments in vivo, we discover [~]3000 endfoot transcripts, including [~]800 highly enriched compared to cell bodies. Many endfoot-enriched transcripts exhibit conserved subcellular localization in neurons and glia and are associated with neurodevelopmental disease. We show that endfoot-enriched Dync1li2 regulates RGC basal morphology and subsequently interneuron organization. Finally, we develop LOCAL-KD, a CRISPR-Cas13 based method for subcellular mRNA knockdown in vivo. Leveraging this, we demonstrate that endfoot-localized Dync1li2 is critical for RGC morphology. Our study establishes experimental paradigms to understand RNA localization in the nervous system. Moreover, we discover RGCs have a vast subcellular transcriptome, revealing foundational insights into how RGCs control cortical development.

neuroscience↗

bioRxiv2025

Species-specific chromatin architecture and neurogenesis mediated by a human enhancer

Genomic modifications underlie the evolution of human features, including a larger neocortex and enhanced cognition. Human Accelerated Regions (HARs) are highly-conserved loci containing human-specific variants and can act as neurodevelopmental enhancers. However, the neurodevelopmental functions of HARs and their mechanism of gene regulation are largely unknown. Here, we show that human (Hs) HAR1984 promotes neurogenesis by influencing species-specific transcription and chromatin interactions. Hs-HAR1984 knock-in chimpanzee (Pt) cortical organoids contain more progenitors and neurons, whereas Pt-HAR1984 knock-in human cortical organoids exhibit the opposite phenotype. Hs-HAR1984 knock-in mice recapitulate increased neurogenesis, producing a thicker cortex with folding. Hi-C reveals HAR1984 exhibits chromatin looping with its target genes ETV5 and TRA2B in human fetal brains, notably reduced in chimpanzee, macaque and mouse neural cells. We show that human-specific mutations in HAR1984 directly promote these interactions, favored by nearby structural variants. Further, we discover that human-specific ETV5 binding auto-regulates enhancer activity. This work demonstrates new molecular mechanisms underlying human-specific neurodevelopment, linking HARs to chromatin architecture, cortical cell fate and expansion and folding of brains.

developmental biology↗

bioRxiv2024

mRNA stability fine tunes gene expression in the developing cortex to control neurogenesis

RNA expression levels are controlled by the complementary processes of synthesis and degradation. Although mis-regulation of RNA turnover is linked to neurodevelopmental disorders, how it contributes to cortical development is largely unknown. Here, we profile the RNA stability landscape of the cortex across development and demonstrate that control of stability by the CCR4-NOT complex is essential for corticogenesis in vivo. First, we use SLAM-seq to measure RNA half-lives transcriptome-wide across multiple stages of cortical development. We characterize cis-acting features associated with RNA stability and find that RNAs that are upregulated across development tend to be more stable, while downregulated RNAs are less stable. To probe how disruption of RNA turnover impacts cortical development, we assess developmental requirements of CNOT3, a core component of the CCR4-NOT deadenylase complex. Mutations in CNOT3 are associated with human neurodevelopmental disorders, however its role in cortical development is unknown. Conditional knockout of Cnot3 in neural progenitors and their progeny in the developing mouse cortex leads to severe microcephaly due to reduced neuron production and p53-dependent apoptosis. Collectively, our findings demonstrate that fine-tuned control of RNA turnover is crucial for brain development.

neuroscience↗

bioRxiv2024

A human-specific enhancer fine-tunes radial glia potency and corticogenesis

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications1-3. Human accelerated regions (HARs) are highly conserved genomic sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to human-specific cortical development is largely unknown4,5. HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development6. Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices containing more neurons. By measuring neural dynamics in vivo we show these anatomical features correlate with increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using live imaging, lineage analysis, and single-cell RNA sequencing. This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

neuroscience↗

bioRxiv2020

Subcellular mRNA localization and local translation of Arhgap11a in radial glial cells regulates cortical development

mRNA localization and local translation enable exquisite spatial and temporal control of gene expression, particularly in highly polarized and elongated cells. These features are especially prominent in radial glial cells (RGCs), which serve as neural and glial precursors of the developing cerebral cortex, and scaffolds for migrating neurons. Yet the mechanisms by which distinct sub-cellular compartments of RGCs accomplish their diverse functions are poorly understood. Here, we demonstrate that subcellular RNA localization and translation of the RhoGAP Arhgap11a controls RGC morphology and mediates cortical cytoarchitecture. Arhgap11a mRNA and protein exhibit conserved localization to RGC basal structures in mice and humans, conferred by a 5'UTR cis-element. Proper RGC morphology relies upon active Arhgap11a mRNA transport and localization to basal structures, where ARHGAP11A is locally synthesized. Thus, RhoA activity is spatially and acutely activated via local translation in RGCs to promote neuron positioning and cortical cytoarchitecture. Altogether, our study demonstrates that mRNA localization and local translation mediate compartmentalization of neural progenitor functions to control brain development. HighlightsO_LIArhgap11a in radial glia non-cell autonomously promotes neuronal migration and lamination C_LIO_LIArhgap11a mRNA localizes to radial glial endfeet via a 5 UTR cis element C_LIO_LIARHGAP11A expression in basal process and endfeet depends upon its localized mRNA C_LIO_LILocalized mRNA and RhoA-GAP activity in endfeet control radial glial morphology C_LI

neuroscience↗