Biology subjects

Levine, M. S.

Publications and source records attributed to Levine, M. S..

14 recordsLinked to original sources

bioRxiv2026

Multiscale spatial analysis implicates chromosomal metaloops in gene patterning across the Drosophila brain

Scores of chromosome-scale loops, or metaloops, arise in the Drosophila brain, but their spatial organization and relationship to neural gene expression patterns remain unclear. Here, we used multiplexed Optical Reconstruction of Chromatin Architecture (ORCA) to examine the multiscale spatial organization of metaloops in cross-sections of 100s of larval and adult Drosophila brains. We find metaloops form preferentially in the central regions of the brain, where they nucleate the formation of metadomains, characterized by the intermingling of distal topologically associating domains (TADs). At the sub-cellular scale, metaloops tend to arise towards the nuclear center, and multiple metaloops in the same cell have a preference to form hubs (3 or more contacts). Each brain nucleus generally harbors only a few loops or hubs. An in-depth analysis of the hub centered on DIP-epsilon, a synaptic wiring gene, identified a three-way metadomain that brings together the DIP-epsilon TAD; a distal TAD carrying a paralog of DIP-epsilon, DIP-zeta; and a putative regulatory TAD, across 3 Mb. This metadomain adopts distinct conformations depending on gene expression; cells expressing DIP-epsilon or DIP-zeta show preferential interactions between the TAD carrying the corresponding gene and the putative regulatory TAD. We posit that the neuron-specific formation of different subsets of metadomains might coordinate the expression of diverse combinations of synaptic wiring genes underlying complex brain architecture.

molecular biology↗

bioRxiv2026

Evolution of compound eye cell types shapes visual behaviors across Heliconius butterflies

The evolution of visual systems is tightly linked to the diversification of visually guided behaviors. Using Heliconius butterflies, renowned for their rapid diversification of wing color patterns and corresponding visual mate preferences, we investigate the evolution of compound eye cell types across a complete speciation continuum. By integrating population genomics, chromatin conformation capture, and single-nucleus multi-omics, we uncover multi-modal mechanisms driving visual system evolution. Within a population polymorphic for mate preference, we identify senseless-2 as a glia-expressing candidate visual preference gene. Across species, we reveal that photoreceptors, particularly the color-sensing R7s, are the fastest-evolving cell types. Furthermore, we molecularly characterize novel R2/5 and R7 photoreceptor subtypes, showing that cellular subfunctionalization arises through the co-option of existing gene regulatory networks. Together, this comparative single-cell analysis of compound eyes bridges microevolutionary transcriptomic divergence with macroevolutionary cellular innovations.

evolutionary biology↗

bioRxiv2026

Sequence-to-function modeling uncovers the context-specific grammar of Drosophila chromatin insulation

Chromatin is organized into self-interacting topologically associating domains partitioned by boundary elements that insulate adjacent domains and restrict regulatory interactions. Yet, how sequence context and combinations of factors dictate boundary strength remains incompletely understood. Here we present Domino, a deep learning framework that maps genomic sequences to quantitative insulation scores defined directly from single-nucleosome resolution Drosophila melanogaster Micro-C data. Unlike traditional transcription factor motif scanning, Domino captures broad sequence context to resolve the functional contributions of individual sequence elements. We validate model predictions through experimental perturbations of insulator sequences. Model interpretation yields insulation-associated motifs genome-wide. Across 7,311 embryonic boundaries, Domino reveals a comprehensive insulation grammar defined by just 24 primary motifs that account for 59% of the boundaries, with an average of only two motifs per motif-containing boundary. Beyond known factors, we identify the zinc-finger proteins Trem, CG4854 and CG17385 as previously unreported insulation factors. We uncover distance- and orientation-dependent motif synergy, including a strict orientation preference of the prominent architectural factor M1BP. Finally, Domino traces tissue-specific shifts in the insulator landscape from the embryo to larval and adult brains, nominating new brain-specific insulation motifs. In sum, Domino provides a generalizable framework for decoding the regulatory logic of 3D genome architecture.

genomics↗

bioRxiv2025

Metadomain and metaloop genome interactions in mammalian T cells

Recent studies have advanced our understanding of chromosomal organization and its principal role in gene regulation. However, most analyses have focused on short-range interactions (<2 Mb), limiting insight into broader regulatory architecture. In particular, the relationships between topologically associating domains (TADs), sub-TAD loops, long-range cross-TAD interactions, and higher-order chromosomal compartmentalization remain poorly understood. Here, we identify extensive multi-megabase and interchromosomal interactions (metaloops) in T lymphocytes, which organize into larger meta-TAD associations (metadomains). Metaloops bridge distal promoters and regulatory elements of key T cell-specific genes such as Ctla4, Ikzf2, Il2ra, Ets1, Lef1, Runx1, Bach2, Foxo1 and others, and are both shared and cell type-specific across functionally distinct T cell lineages. Reanalysis of published data confirms the reproducibility of these interactions in both mouse and human T cells and their dependence on superenhancers. Genome-wide clustering of metadomains reveals three interchromosomal hubs with distinct epigenomic profiles, including a superenhancer-enriched hub associated with T cell-specific gene activation. By integrating a compendium of new and public T cell epigenomic data, we infer distinct architectural factors associated with short-range loops and long-range metaloops. Altogether, our study reveals new features of T cell-specific 3D genome organization across scales, and our computational framework is broadly applicable to analyses of chromatin architecture across different cell types and experimental systems. Highlights- Ultra-long-range (>2 Mb) chromatin interactions linked to gene regulation in T cells - A new algorithm identifies distal and interchromosomal meta-TADs and metaloops - An interchromosomal T cell-specific active hub emerges from metadomain clustering - Epigenomic compendium implicates TFs associated with long-range interactions

genomics↗

bioRxiv2025

A pair-rule-like transcription network coordinates neural tube closure in a proto-vertebrate

Neural tube closure (NTC) is a conserved morphogenetic process in chordates, during which the neural plate folds and fuses to form a closed neural tube. While the mechanical forces and signaling pathways governing NTC have been characterized in vertebrates, the transcriptional programs coordinating cell behaviors during closure remain less understood. Here, we identify a transcriptional circuit involving Lmx1, Cdkn1b, and Msx that regulates dorsal midline dynamics during NTC in the tunicate Ciona. High-resolution HCR in situ hybridization reveals that Lmx1 is dynamically enriched at the zippering point and advances in a posterior-to-anterior transcription wave, while Msx is downregulated in the same region, marking a transition from early neural patterning to morphogenetic execution. As closure progresses, Lmx1 and Cdkn1b exhibit complementary, alternating expression at the dorsal midline, resembling a pair-rule-like pattern. Misexpression studies show that Lmx1 promotes proliferation and activates its own expression, whereas Cdkn1b limits proliferation and impedes closure. Single-cell RNA-seq reveals transcriptionally distinct dorsal neural populations enriched for Lmx1 or Cdkn1b, supporting spatially organized cell-cycle states. These findings suggest that a transcriptional switch from Msx to Lmx1, followed by spatially alternating Lmx1 and Cdkn1b activity, coordinates proliferation and neural fold fusion during NTC. This mechanism may represent a general strategy for regulating epithelial remodeling in animal embryos.

developmental biology↗

bioRxiv2025

Striatal cell-type specific stability and reorganization underlying agency and habit

Goal-directed behavior is guided by learned action-outcome associations, allowing actions to adapt when outcomes change. With repetition, behavioral control shifts toward habit, enabling more automatic action execution, but reducing flexibility to outcome changes. Here, we used longitudinal, cellular-resolution calcium imaging, chemogenetics, instrumental conditioning, and the outcome devaluation test to ask how dorsomedial striatum D1+ and D2/A2A+ neurons contribute to the formation of goal-directed behavior and habit. DMS D1+ neurons stably encode instrumental actions, carry information about behavioral engagement and organization, and develop and maintain action-reward representational similarity and conjunctive activation with learning. Correspondingly, these neurons are critical for action-outcome learning and goal-directed decision making. In contrast, one ensemble of DMS A2A+ neurons transiently encodes actions during early action-outcome learning and another slowly starts to encode actions as habits form. Similarly, DMS A2A+ neurons transiently mediate action-outcome learning and become dispensable for goal-directed decision making. Thus, action-outcome associations are learned through DMS D1+ and A2A+ neuronal activity but only DMS D1+ neurons maintain support of adaptive, goal-directed decision making. Habit formation is associated with reorganization of A2A+ neuronal encoding. These data reveal a cell-type-specific dissociation between stable v. reorganizing striatal action representations paralleled by persistent v. transient contributions to goal-directed control.

neuroscience↗

bioRxiv2024

Corticostriatal Maldevelopment in the R6/2 Mouse Model of Juvenile Huntington Disease

There is a growing consensus that brain development in Huntingtons disease (HD) is abnormal, leading to the idea that HD is not only a neurodegenerative but also a neurodevelopmental disorder. Indeed, structural and functional abnormalities have been observed during brain development in both humans and animal models of HD. However, a concurrent study of cortical and striatal development in a genetic model of HD is still lacking. Here we report significant alterations of corticostriatal development in the R6/2 mouse model of juvenile HD. We examined wildtype (WT) and R6/2 mice at postnatal (P) days 7, 14, and 21. Morphological examination demonstrated early structural and cellular alterations reminiscent of malformations of cortical development, and ex vivo electrophysiological recordings of cortical pyramidal neurons (CPNs) demonstrated significant age- and genotype-dependent changes of intrinsic membrane and synaptic properties. In general, R6/2 CPNs had reduced cell membrane capacitance and increased input resistance (P7 and P14), along with reduced frequency of spontaneous excitatory and inhibitory synaptic events during early development (P7), suggesting delayed cortical maturation. This was confirmed by increased occurrence of GABAA receptor-mediated giant depolarizing potentials at P7. At P14, the rheobase of CPNs was significantly reduced, along with increased excitability. Altered membrane and synaptic properties of R6/2 CPNs recovered progressively, and by P21 they were similar to WT CPNs. In striatal medium-sized spiny neurons (MSNs), a different picture emerged. Intrinsic membrane properties were relatively normal throughout development, except for a transient increase in membrane capacitance at P14. The first alterations in MSNs synaptic activity were observed at P14 and consisted of significant deficits in GABAergic inputs, however, these also were normalized by P21. In contrast, excitatory inputs began to decrease at this age. We conclude that the developing HD brain is capable of compensating for early developmental abnormalities and that cortical alterations precede and are a main contributor of striatal changes. Addressing cortical maldevelopment could help prevent or delay disease manifestations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/618500v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@16db9eborg.highwire.dtl.DTLVardef@c9329borg.highwire.dtl.DTLVardef@1198fc1org.highwire.dtl.DTLVardef@12ce530_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Exact centriole counts are critical for B cell development but not function

Centrioles define centrosome structure and function. Deregulation of centriole numbers can cause developmental defects and foster malignant disease. The p53 tumor suppressor limits the growth of cells lacking or harboring additional centrioles and can be engaged by the "mitotic surveillance" or the "PIDDosome pathway", respectively. Here, we show that early B cell progenitors frequently present extra centrioles that are rapidly lost during maturation. Increasing centriole counts beyond physiological levels by Polo-like kinase 4 (PLK4) overexpression induces apoptosis, suggesting clearance of such cells during development. Remarkably, this apoptotic response is independent of PIDD1 or p53, but can be blocked by excess BCL2. In contrast, loss of centrosomes upon Plk4 deletion arrests B cell development at the pro B cell stage. This defect can be rescued by co-deletion of Usp28, a critical component of the mitotic surveillance pathway that restores cell number and function in the absence of centrioles. In both scenarios, too many and too few centrosomes, mitochondrial apoptosis is engaged to kill B cells with abnormal centriole counts during their development with progenitor B cells being intolerant to centriole loss but permissive to centriole amplification. Unexpectedly, our findings show that centrioles are dispensable for mounting an effective humoral immune response.

cell biology↗

bioRxiv2023

Quantitative proteome dynamics across embryogenesis in a model chordate

The evolution of gene expression programs underlying the development of vertebrates remains poorly characterized. Here, we present a comprehensive proteome atlas of the model chordate Ciona, covering eight developmental stages and [~]7,000 translated genes, accompanied by a multi-omics analysis of co-evolution with the vertebrate Xenopus. Quantitative proteome comparisons argue against the widely held hourglass model, based solely on transcriptomic profiles, whereby peak conservation is observed during mid-developmental stages. Our analysis reveals maximal divergence at these stages, particularly gastrulation and neurulation. Together, our work provides a valuable resource for evaluating conservation and divergence of multi-omics profiles underlying the diversification of vertebrates.

developmental biology↗

bioRxiv2021

ERK signaling dissolves ERF Repression Condensates in Living Embryos

Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor ERF, which has been linked to various human developmental disorders. Using high resolution imaging we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during the cell cycle, providing the first visualization of a nuclear phase separation process that is regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.

developmental biology↗

bioRxiv2021

A bidirectional corticoamygdala circuit for the encoding and retrieval of detailed reward memories

Adaptive reward-related decision making often requires accurate and detailed representation of potential available rewards. Environmental reward-predictive stimuli can facilitate these representations, allowing one to infer which specific rewards might be available and choose accordingly. This process relies on encoded relationships between the cues and the sensory-specific details of the reward they predict. Here we interrogated the function of the basolateral amygdala (BLA) and its interaction with the lateral orbitofrontal cortex (lOFC) in the ability to learn such stimulus-outcome associations and use these memories to guide decision making. Using optical recording and inhibition approaches, Pavlovian cue-reward conditioning, and the outcome-selective Pavlovian-to-instrumental transfer (PIT) test in male rats, we found that the BLA is robustly activated at the time of stimulus-outcome learning and that this activity is necessary for sensory-specific stimulus-outcome memories to be encoded, so they can subsequently influence reward choices. Direct input from the lOFC was found to support the BLA in this function. Based on prior work, activity in BLA projections back to the lOFC was known to support the use of stimulus-outcome memories to influence decision making. By multiplexing optogenetic and chemogenetic inhibition we performed a serial circuit disconnection and found that the lOFC[->]BLA and BLA[->]lOFC pathways form a functional circuit regulating the encoding (lOFC[->]BLA) and subsequent use (BLA[->]lOFC) of the stimulus-dependent, sensory-specific reward memories that are critical for adaptive, appetitive decision making.

neuroscience↗

bioRxiv2021

Human Neural Stem Cells Differentiate and Integrate, Innervating Implanted zQ175 Huntington's Disease Mouse Striatum

Huntingtons disease (HD), a genetic neurodegenerative disorder, primarily impacts the striatum and cortex with progressive loss of medium-sized spiny neurons (MSNs) and pyramidal neurons, disrupting cortico-striatal circuitry. A promising regenerative therapeutic strategy of transplanting human neural stem cells (hNSCs) is challenged by the need for long-term functional integration. We previously described that hNSCs transplanted into the striatum of HD mouse models differentiated into electrophysiologically active immature neurons, improving behavior and biochemical deficits. Here we show that 8-month implantation of hNSCs into the striatum of zQ175 HD mice ameliorates behavioral deficits, increases brain-derived neurotrophic factor (BDNF) and reduces mutant Huntingtin (mHTT) accumulation. Patch clamp recordings, immunohistochemistry and electron microscopy demonstrates that hNSCs differentiate into diverse neuronal populations, including MSN- and interneuron-like cells. Remarkably, hNSCs receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD.

neuroscience↗

bioRxiv2020

DeepArk: modeling cis-regulatory codes of model species with deep learning

To enable large-scale analyses of regulatory logic in model species, we developed DeepArk (https://DeepArk.princeton.edu), a set of deep learning models of the cis-regulatory codes of four widely-studied species: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, and Mus musculus. DeepArk accurately predicts the presence of thousands of different context-specific regulatory features, including chromatin states, histone marks, and transcription factors. In vivo studies show that DeepArk can predict the regulatory impact of any genomic variant (including rare or not previously observed), and enables the regulatory annotation of understudied model species.

bioinformatics↗

bioRxiv2019

Mutations in CalDAG-GEFI Lead to Striatal Signaling Deficits and Psychomotor Symptoms in Multiple Species Including Human

Syndromes caused by mutations in Ras-MAP kinase signaling molecules are known as RASopathies and share features such as developmental delay, autistic traits, and cancer. Syndromic features of Rap-MAP kinase signaling defects remain undefined. CalDAG-GEFI is a calcium-responsive Rap-GTPase activator that is enriched in the matrix of the sensorimotor striatum and down-regulated in Huntingtons disease. We show here that CalDAG-GEFI mutations, including striatum-specific deletions and spontaneous mutations in the enzymatic domain, are associated with psychomotor phenotypes in humans, dogs and mice. The identification of these neural mutants was guided by the overt bleeding phenotype in CalDAG-GEFI knockout mice, and then in humans and other species with conserved platelet signaling deficits. Knockout mice exhibit loss of striatal long-term potentiation and deficits in dopamine, acetylcholine and glutamate signaling, along with delayed motor learning and drug-induced perseverative behaviors. Thus, loss of CalDAG-GEFI signaling produces an evolutionarily conserved syndrome characterized by bleeding and psychomotor dysfunction.

neuroscience↗