{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T07:19:26Z","timestamp":1783408766890,"version":"3.54.6"},"posted":{"date-parts":[[2025,9,22]]},"group-title":"Neuroscience","reference-count":76,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,22]],"date-time":"2025-09-22T00:00:00Z","timestamp":1758499200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"NIH","award":["5R21EY036219"],"award-info":[{"award-number":["5R21EY036219"]}]},{"name":"NIH","award":["U01NS136405"],"award-info":[{"award-number":["U01NS136405"]}]},{"name":"NIH","award":["1F31NS131016"],"award-info":[{"award-number":["1F31NS131016"]}]},{"name":"NSF","award":["CRCNS 2309039"],"award-info":[{"award-number":["CRCNS 2309039"]}]},{"DOI":"10.13039\/100000888","name":"W.M. Keck Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000888","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Glaucoma Research Foundation CFC4"},{"DOI":"10.13039\/100005270","name":"McKnight Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100005270","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000011","name":"Howard Hughes Medical Institute","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000011","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,22]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The mammalian cerebral cortex is composed of neurons whose properties vary in a continuous fashion rather than falling into discrete cell types. In the mouse visual cortex, excitatory neurons in layer 2 and 3 (L2\/3) form such a continuum along cortical depth, patterned by the graded expression of hundreds of genes. Here we sought to understand how this continuum develops and contributes to cortical wiring. Using single-nucleus multiomics (RNA- and ATAC-Seq) and spatial transcriptomics, we show that the L2\/3 continuum is established in two phases. During the first postnatal week, a genetically hardwired program establishes a primitive continuum of cell identities spanning the depth of L2\/3. The second program, promoted by visual experience, is later superimposed upon the preexisting continuum. This second phase is driven by activity-regulated transcription factors that drive the L2\/3 depth-dependent expression of genes linked to synaptic function and plasticity. We show that neurons at different positions along the L2\/3 continuum project preferentially to distinct higher visual areas and that visual deprivation disrupts targeting to some higher visual areas while sparing others. Thus, cortical continua emerge through a stepwise process in which genetic programs and sensory experience specify neuronal identity and sculpt intracortical wiring specificity.<\/jats:p>","DOI":"10.1101\/2025.09.22.677893","type":"posted-content","created":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T04:20:15Z","timestamp":1758601215000},"source":"Crossref","is-referenced-by-count":2,"title":["Establishing a continuum of cell types in the visual cortex"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4600-7094","authenticated-orcid":false,"given":"Juyoun","family":"Yoo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5232-1648","authenticated-orcid":false,"given":"Fangming","family":"Xie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8679-3715","authenticated-orcid":false,"given":"Salwan","family":"Butrus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5364-4112","authenticated-orcid":false,"given":"Runzhe","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2235-4577","authenticated-orcid":false,"given":"Zhiqun","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8320-0416","authenticated-orcid":false,"given":"Ryan","family":"Gorzek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parmis","family":"Mirshahidi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elaine","family":"Tring","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanjana","family":"Suresh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinho","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1879-6521","authenticated-orcid":false,"given":"Greg","family":"Fleishman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liming","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6439-334X","authenticated-orcid":false,"given":"Dario","family":"Ringach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Trachtenberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5828-1533","authenticated-orcid":false,"given":"Xiangmin","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S.","family":"Lawrence Zipursky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4349-6600","authenticated-orcid":false,"given":"Karthik","family":"Shekhar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7442-7670","authenticated-orcid":false,"given":"Saumya","family":"Jain","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025092519251195000_2025.09.22.677893v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2012.06.009"},{"key":"2025092519251195000_2025.09.22.677893v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2020.09.022"},{"key":"2025092519251195000_2025.09.22.677893v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2021.07.053"},{"key":"2025092519251195000_2025.09.22.677893v1.4","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-cellbio-100814-125620"},{"key":"2025092519251195000_2025.09.22.677893v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3917"},{"key":"2025092519251195000_2025.09.22.677893v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41583-023-00675-z"},{"key":"2025092519251195000_2025.09.22.677893v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.12.022"},{"key":"2025092519251195000_2025.09.22.677893v1.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2421022122"},{"key":"2025092519251195000_2025.09.22.677893v1.9","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.cels.2018.12.008","article-title":"Continuum of Gene-Expression Profiles Provides Spatial Division of Labor within a Differentiated Cell Type","volume":"8","year":"2019","journal-title":"Cell Syst"},{"key":"2025092519251195000_2025.09.22.677893v1.10","doi-asserted-by":"crossref","unstructured":"Adler, M. et al. Emergence of division of labor in tissues through cell interactions and spatial cues. Cell Rep. 42, 112412 (2023).","DOI":"10.1016\/j.celrep.2023.112412"},{"key":"2025092519251195000_2025.09.22.677893v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.12.013"},{"key":"2025092519251195000_2025.09.22.677893v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.tins.2018.02.010"},{"key":"2025092519251195000_2025.09.22.677893v1.13","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.2006387"},{"key":"2025092519251195000_2025.09.22.677893v1.14","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-019-0483-3"},{"key":"2025092519251195000_2025.09.22.677893v1.15","first-page":"688","article-title":"Continuous and Discrete Neuron Types of the Adult Murine Striatum","volume":"105","year":"2019","journal-title":"Neuron"},{"key":"2025092519251195000_2025.09.22.677893v1.16","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.59003"},{"key":"2025092519251195000_2025.09.22.677893v1.17","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2504-5"},{"key":"2025092519251195000_2025.09.22.677893v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2021.04.021"},{"key":"2025092519251195000_2025.09.22.677893v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06808-9"},{"key":"2025092519251195000_2025.09.22.677893v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06812-z"},{"key":"2025092519251195000_2025.09.22.677893v1.21","doi-asserted-by":"publisher","DOI":"10.1101\/2023.10.10.561757"},{"key":"2025092519251195000_2025.09.22.677893v1.22","doi-asserted-by":"crossref","unstructured":"Yoshinaga, S. et al. Comprehensive characterization of migration profiles of murine cerebral cortical neurons during development using FlashTag labeling. iScience 24, 102277 (2021).","DOI":"10.1016\/j.isci.2021.102277"},{"key":"2025092519251195000_2025.09.22.677893v1.23","doi-asserted-by":"crossref","unstructured":"Murakami, T. & Ohki, K . Thalamocortical circuits for the formation of hierarchical pathways in the mammalian visual cortex. Front. Neural Circuits 17, 1155195 (2023).","DOI":"10.3389\/fncir.2023.1155195"},{"key":"2025092519251195000_2025.09.22.677893v1.24","doi-asserted-by":"crossref","unstructured":"Wang, X. et al. The Influence of Changes in Microglia Development on the Plasticity of the Developing Visual Cortex Circuit in Juvenile Mice. Investig. Ophthalmol. Vis. Sci. 66, 45 (2025).","DOI":"10.1167\/iovs.66.4.45"},{"key":"2025092519251195000_2025.09.22.677893v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03670-5"},{"key":"2025092519251195000_2025.09.22.677893v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-023-01938-4"},{"key":"2025092519251195000_2025.09.22.677893v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.03.058"},{"key":"2025092519251195000_2025.09.22.677893v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/nature17187"},{"key":"2025092519251195000_2025.09.22.677893v1.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.conb.2018.11.001"},{"key":"2025092519251195000_2025.09.22.677893v1.30","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03705-x"},{"key":"2025092519251195000_2025.09.22.677893v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2907-3"},{"key":"2025092519251195000_2025.09.22.677893v1.32","doi-asserted-by":"crossref","unstructured":"Jin, S. et al. Inference and analysis of cell-cell communication using CellChat. Nat. Commun. 12, 1088 (2021).","DOI":"10.1038\/s41467-021-21246-9"},{"key":"2025092519251195000_2025.09.22.677893v1.33","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.1124-14.2014"},{"key":"2025092519251195000_2025.09.22.677893v1.34","first-page":"1","article-title":"Higher-Order Areas of the Mouse Visual Cortex","volume":"3","year":"2016","journal-title":"Annu. Rev. Vis. Sci"},{"key":"2025092519251195000_2025.09.22.677893v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.11.024"},{"key":"2025092519251195000_2025.09.22.677893v1.36","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.18372"},{"key":"2025092519251195000_2025.09.22.677893v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2020.04.018"},{"key":"2025092519251195000_2025.09.22.677893v1.38","doi-asserted-by":"crossref","unstructured":"Heo, S. , Kang, T. , Bygrave, A. M. , Larsen, M. R. & Huganir, R. L . Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome. Mol. Cell. Proteom. 22, 100661 (2023).","DOI":"10.1016\/j.mcpro.2023.100661"},{"key":"2025092519251195000_2025.09.22.677893v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/S41583-019-0125-5"},{"key":"2025092519251195000_2025.09.22.677893v1.40","doi-asserted-by":"publisher","DOI":"10.1101\/2024.10.02.616246"},{"key":"2025092519251195000_2025.09.22.677893v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/nature20794"},{"key":"2025092519251195000_2025.09.22.677893v1.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2024.04.021"},{"key":"2025092519251195000_2025.09.22.677893v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/J.NEURON.2018.10.013"},{"key":"2025092519251195000_2025.09.22.677893v1.44","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3808"},{"key":"2025092519251195000_2025.09.22.677893v1.45","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2017.11.026"},{"key":"2025092519251195000_2025.09.22.677893v1.46","doi-asserted-by":"publisher","DOI":"10.1126\/science.3749894"},{"key":"2025092519251195000_2025.09.22.677893v1.47","doi-asserted-by":"publisher","DOI":"10.1016\/0896-6273(90)90106-P"},{"key":"2025092519251195000_2025.09.22.677893v1.48","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-017-0029-5"},{"key":"2025092519251195000_2025.09.22.677893v1.49","doi-asserted-by":"crossref","unstructured":"Kushinsky, D. et al. Daily light-induced transcription in visual cortex neurons drives downward firing rate homeostasis and stabilizes sensory processing. Cell Rep. 43, 114701 (2024).","DOI":"10.1016\/j.celrep.2024.114701"},{"key":"2025092519251195000_2025.09.22.677893v1.50","first-page":"3715","article-title":"Functional and structural features of L2\/3 pyramidal cells continuously covary with pial depth in mouse visual cortex","volume":"33","year":"2022","journal-title":"Cereb. Cortex"},{"key":"2025092519251195000_2025.09.22.677893v1.51","doi-asserted-by":"publisher","DOI":"10.1038\/nature26159"},{"key":"2025092519251195000_2025.09.22.677893v1.52","doi-asserted-by":"crossref","first-page":"5671","DOI":"10.1016\/j.cub.2024.10.048","article-title":"Higher-order cortical and thalamic pathways shape visual processing streams in the mouse cortex","volume":"34","year":"2024","journal-title":"Curr. Biol"},{"key":"2025092519251195000_2025.09.22.677893v1.53","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.04.008"},{"key":"2025092519251195000_2025.09.22.677893v1.54","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2025.05.030"},{"key":"2025092519251195000_2025.09.22.677893v1.55","doi-asserted-by":"crossref","first-page":"e3003176","DOI":"10.1371\/journal.pbio.3003176","article-title":"Molecular states underlying neuronal cell type development and plasticity in the postnatal whisker cortex","volume":"23","year":"2025","journal-title":"PLOS Biol"},{"key":"2025092519251195000_2025.09.22.677893v1.56","doi-asserted-by":"crossref","unstructured":"Traag, V. A. , Waltman, L. & Eck, N. J. Van . From Louvain to Leiden: guaranteeing well-connected communities. Sci. Rep. 9, 5233 (2019).","DOI":"10.1038\/s41598-019-41695-z"},{"key":"2025092519251195000_2025.09.22.677893v1.57","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0654-5"},{"key":"2025092519251195000_2025.09.22.677893v1.58","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-023-02103-7"},{"key":"2025092519251195000_2025.09.22.677893v1.59","doi-asserted-by":"crossref","unstructured":"Zhang, Y. et al. Model-based Analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).","DOI":"10.1186\/gb-2008-9-9-r137"},{"key":"2025092519251195000_2025.09.22.677893v1.60","unstructured":"Li, Y. E. et al. A comparative atlas of single-cell chromatin accessibility in the human brain. Science 382, eadf7044 (2023)."},{"key":"2025092519251195000_2025.09.22.677893v1.61","doi-asserted-by":"crossref","unstructured":"Amemiya, H. M. , Kundaje, A. & Boyle, A. P . The ENCODE Blacklist: Identification of Problematic Regions of the Genome. Sci. Rep. 9, 9354 (2019).","DOI":"10.1038\/s41598-019-45839-z"},{"key":"2025092519251195000_2025.09.22.677893v1.62","doi-asserted-by":"publisher","DOI":"10.1016\/j.neucom.2011.06.033"},{"key":"2025092519251195000_2025.09.22.677893v1.63","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1754"},{"key":"2025092519251195000_2025.09.22.677893v1.64","doi-asserted-by":"crossref","unstructured":"McLeay, R. C. & Bailey, T. L . Motif Enrichment Analysis: a unified framework and an evaluation on ChIP data. BMC Bioinform. 11, 165 (2010).","DOI":"10.1186\/1471-2105-11-165"},{"key":"2025092519251195000_2025.09.22.677893v1.65","first-page":"D174","article-title":"20th anniversary of the open-access database of transcription factor binding profiles","volume":"52","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2025092519251195000_2025.09.22.677893v1.66","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0619-0"},{"key":"2025092519251195000_2025.09.22.677893v1.67","doi-asserted-by":"publisher","DOI":"10.1152\/jn.01056.2015"},{"key":"2025092519251195000_2025.09.22.677893v1.68","doi-asserted-by":"publisher","DOI":"10.1126\/science.274.5293.1678"},{"key":"2025092519251195000_2025.09.22.677893v1.69","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-021-00527-z"},{"key":"2025092519251195000_2025.09.22.677893v1.70","doi-asserted-by":"crossref","unstructured":"Kalatsky, V. A. & Stryker, M. P . New paradigm for optical imaging: temporally encoded maps of intrinsic signal. Neuron (2003).","DOI":"10.1016\/S0896-6273(03)00286-1"},{"key":"2025092519251195000_2025.09.22.677893v1.71","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0501-0"},{"key":"2025092519251195000_2025.09.22.677893v1.72","doi-asserted-by":"crossref","unstructured":"Peng, T. et al. A BaSiC tool for background and shading correction of optical microscopy images. Nat. Commun. 8, 14836 (2017).","DOI":"10.1038\/ncomms14836"},{"key":"2025092519251195000_2025.09.22.677893v1.73","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-022-01669-y"},{"key":"2025092519251195000_2025.09.22.677893v1.74","doi-asserted-by":"publisher","DOI":"10.5281\/zenodo.3555620"},{"key":"2025092519251195000_2025.09.22.677893v1.75","doi-asserted-by":"publisher","DOI":"10.1101\/061507"},{"key":"2025092519251195000_2025.09.22.677893v1.76","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-01018-x"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.22.677893","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:38:48Z","timestamp":1768469928000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.22.677893"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,22]]},"references-count":76,"URL":"https:\/\/doi.org\/10.1101\/2025.09.22.677893","relation":{},"subject":[],"published":{"date-parts":[[2025,9,22]]},"subtype":"preprint"}}