Biology subjects

Romanowski, A. J.

Publications and source records attributed to Romanowski, A. J..

6 recordsLinked to original sources

bioRxiv2026

Widespread Corticothalamic Connectivity Identifies the Inferior Pulvinar as a Central Node in Visual Network Architecture

The medial subdivision of the inferior pulvinar (PIm) has been implicated in motion processing, visuomotor integration, and residual visual function, yet a comprehensive account of its cortical inputs remains unresolved. Previous studies often relied on indirect cortical injections or tracer deposits spanning multiple pulvinar subdivisions, limiting anatomical specificity. Here, we used MRI-guided, cytoarchitectonically restricted retrograde tracer injections to selectively target PI in the common marmoset (Callithrix jacchus) and systematically map its cortical afferents. Across four cases, retrogradely labeled neurons were widely distributed throughout occipital, temporal, parietal, and cingulate cortices, with a strong predominance in layer V, consistent with driver-like corticothalamic projections. Early and middle-tier visual areas (V1, V2, V3, V3A, V4, V6/DM) contributed substantial input, with labeling patterns corresponding to peripheral visual field representations. The middle temporal complex (MT, MTc, MST, FST) represented one of the densest sources of cortical projections. Prominent inputs also arose from posterior parietal regions, including LIP, MIP, VIP, AIP, and inferior parietal areas (e.g., PFG, OPt), linking PIm to visuospatial and action-related networks. Semi-quantitative analyses indicated that occipital cortex and the MT complex together accounted for approximately 60% of total cortical input, while parietal cortex contributed roughly 20%. Additional projections from retrosplenial and posterior cingulate cortices were observed. These findings identify PIm as a central integrative node embedded within distributed visual and visuomotor networks. Rather than functioning as a restricted visual relay, PIm appears positioned to coordinate motion, spatial, and action-relevant signals within cortico-thalamocortical circuits supporting adaptive visually-guided behavior.

neuroscience↗

bioRxiv2024

Developmental transformations of Purkinje cells tracked by DNA electrokinetic mobility

Brain development relies on orchestrated placement and timing of neurogenesis in progenitor zones to produce the expansive cellular diversity of the brain. We took advantage of bioelectric interactions between DNA and embryonic tissue to perform "stereo-tracking", a developmental targeting strategy that differentially labels cells positioned at different depths within intact progenitor zones. This three-dimensional labeling was achieved by delivery of plasmids with distinct electrokinetic mobilities into neural progenitor zones in utero. We applied stereo-tracking with light sheet imaging in the cerebellum and identified that Purkinje cells follow embryonically committed developmental trajectories linking distinct progenitor zone fields to the topography of the mature cerebellar cortex. In the process of stereo-tracking, we identified a previously unreported subcellular structure on the axon initial segment of Purkinje cells. These structures, we termed "axon bubbles", are developmentally timed and differentially labeled by lipid-modified proteins. Our findings demonstrate key rules that orchestrate the stereotyped transformations from fetal progenitors into mature networks of neuronal circuits, and demonstrate the potential of progenitor zone stereo-tracking to reveal new biology within intact developing systems.

neuroscience↗

bioRxiv2024

Symmetry in levels of axon-axon homophilic adhesion establishes topography in the corpus callosum and development of connectivity between brain hemispheres

Specific and highly diverse connectivity between functionally specialized regions of the nervous system is controlled at multiple scales, from anatomically organized connectivity following macroscopic axon tracts to individual axon target-finding and synapse formation. Identifying mechanisms that enable entire subpopulations of related neurons to project their axons with regional specificity within stereotyped tracts to form appropriate long-range connectivity is key to understanding brain development, organization, and function. Here, we investigate how axons of the cerebral cortex form precise connections between the two cortical hemispheres via the corpus callosum. We identify topographic principles of the developing trans-hemispheric callosal tract that emerge through intrinsic guidance executed by growing axons in the corpus callosum within the first postnatal week in mice. Using micro-transplantation of regionally distinct neurons, subtype-specific growth cone purification, subcellular proteomics, and in utero gene manipulation, we investigate guidance mechanisms of transhemispheric axons. We find that adhesion molecule levels instruct tract topography and target field guidance. We propose a model in which transcallosal axons in the developing brain perform a "handshake" that is guided through co-fasciculation with symmetric contralateral axons, resulting in the stereotyped homotopic connectivity between the brains hemispheres.

neuroscience↗

bioRxiv2023

Modeling an ultra-rare epilepsy variant in wildtype mice with in utero prime editing

Generating animal models that mirror a patients seizures within clinically-useful timeframes is an important step toward advancing precision medicine for genetic epilepsies. Here we report a somatic cell genome editing approach that rapidly incorporated a patients genomic variant into mice, which developed seizures recapitulating elements of the patients pathology. This approach offers a versatile in vivo platform for clinical, preclinical, and basic research applications, including tailoring pharmacotherapy, assessing variants of uncertain significance, and screening compounds to develop drugs for rare epilepsies. As proof-of-principle, we modeled an epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A using prime editing in utero directly in the developing brain of wild-type mice. This methodology achieved high-fidelity genome editing in vivo sufficient to induce frequent spontaneous seizures without necessitating germline modification or extensive breeding. Leveraging the speed and versatility of this approach, we propose a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. This advance holds promise for providing a cost-effective, expedient in vivo testing platform that reduces barriers to access for precision medicine, and accelerates drug development for rare and neglected neurological conditions.

neuroscience↗

bioRxiv2022

Phosphorylation Determines Whether Neuroligin-3 is at Excitatory or Inhibitory Synapses in Different Regions of the Brain

Neuroligin-3 is a postsynaptic adhesion molecule involved in development, function, and pathologies of synapses in the brain. It is a genetic cause of autism and a potent component of the tumor microenvironment in gliomas. There are four Neuroligins that operate at distinct synapse types, selectively interacting with presynaptic adhesion and postsynaptic scaffold proteins. We investigated the subcellular localization and scaffold specificities of synaptic Neuroligin-3 and demonstrate an unexpected pattern of localization to excitatory synapses in cortical areas, and inhibitory synapses in subcortical areas. Using phosphoproteomics, we identify Neuroligin-3-specific serine phosphorylation in cortex and hippocampus that obstructs a key binding site for inhibitory synapse scaffolds. Using in utero CRISPR/Cas9 knockout and replacement with phosphomimetic mutants, we demonstrate that phosphorylation at this site determines excitatory versus inhibitory synapse localization of Neuroligin-3 in vivo. Our data reveal a mechanism that differentially regulates the balance of Neuroligin-3 between excitatory and inhibitory synapses, adding to our emerging understanding of their role in the development of brain connectivity and associated pathologies.

neuroscience↗

bioRxiv2020

Cas9 fusions for precision in vivo editing

Cas9 targets genomic loci with high specificity. When used for knockin, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. Here we demonstrate a high-throughput workflow to ratiometrically assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations with orders-of-magnitude increases in knockin precision. Cas9-RC, a novel Cas9 fusion to eRad18 and CtIP, increased knockin performance over 3-fold in vitro and in vivo in the developing mouse brain. Continued comparative assessment of existing and novel editing agents with this ratiometric framework of efficiency and precision will further the development of direct in vivo knockin and future genetic therapies.

synthetic biology↗