{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T05:08:45Z","timestamp":1788930525599,"version":"build-2803163510"},"reference-count":53,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T00:00:00Z","timestamp":1788739200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["31970917"],"award-info":[{"award-number":["31970917"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32371020"],"award-info":[{"award-number":["32371020"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["31970917"],"award-info":[{"award-number":["31970917"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"award":["32371020"],"award-info":[{"award-number":["32371020"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Shanghai Municipal Science and Technology Major Project","award":["2018SHZDZX01"],"award-info":[{"award-number":["2018SHZDZX01"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background\/Objectives: Precise genetic access to the zebrafish habenula remains limited by a scarcity of compact, sequence-defined cis-regulatory elements (CREs). Here, we integrated developmental expression mapping, deep-learning predictions on long-range sequences, and in vivo reporter assays to identify compact regulatory sequences driving habenular expression. Methods: Using a transgenic zebrafish line enriched for habenular reporter expression, we isolated GFP-positive cells from larval brains and profiled their transcriptomes via microarray. A subset of candidate genes enriched in this dataset was validated using whole-mount in situ hybridization across two developmental stages. This analysis identified genes with highly reproducible habenular expression, leading to the selection of the gng8 and ano2 loci for subsequent CRE characterization. We developed ZEN-former (Zebrafish EN-former), an Enformer-based sequence-to-function model trained on neuronal subclass chromatin accessibility profiles from the adult mouse brain. Results: The model demonstrated strong correlation between predicted and experimentally measured signals across held-out genomic regions. To prioritize regulatory candidates, we integrated ZEN-former predictions with available zebrafish ATAC-seq data, RepeatMasker annotations, and gene models, identifying two ~600 bp intervals at each gene locus. These selected intervals were combined to generate ~1.2 kb reporter constructs for gng8 and ano2 loci, which were then evaluated using Tol2 transposon mediated transgenesis assays in zebrafish. In transiently injected larvae, both constructs successfully drove reporter expression in the habenular region. Furthermore, the resulting stable transgenic lines displayed highly specific and reproducible habenular expression. Quantitative confocal analysis showed mean habenular labeling completeness values of 84.5% and 96.2% for the gng8- and ano2-derived lines, respectively. Conclusions: Together, these findings provide a proof of concept that sequence features learned from mammalian chromatin accessibility datasets can effectively guide the prioritization of functional regulatory elements across species in zebrafish. The compact regulatory constructs and stable transgenic lines generated here offer robust genetic tools for investigating habenular circuitry.<\/jats:p>","DOI":"10.3390\/genes17091079","type":"journal-article","created":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T16:30:02Z","timestamp":1788798602000},"page":"1079","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Deep Learning-Guided Identification and In Vivo Validation of Compact Cis-Regulatory Elements for the Zebrafish Habenula"],"prefix":"10.3390","volume":"17","author":[{"given":"Zeran","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Brain Function and Disorders, MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Shanshan","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Brain Function and Disorders, MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Quan","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Brain Function and Disorders, MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Cuizhen","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Brain Function and Disorders, MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6625-5426","authenticated-orcid":false,"given":"Gang","family":"Peng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Brain Function and Disorders, MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"conceptualization","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"software","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1098\/rstb.2008.0213","article-title":"The habenular nuclei: A conserved asymmetric relay station in the vertebrate brain","volume":"364","author":"Bianco","year":"2009","journal-title":"Philos. Trans. R. Soc. B Biol. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1016\/j.neubiorev.2007.01.004","article-title":"A conductor hidden in the orchestra? Role of the habenular complex in monoamine transmission and cognition","volume":"31","author":"Lecourtier","year":"2007","journal-title":"Neurosci. Biobehav. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1038\/nrn2866","article-title":"The habenula: From stress evasion to value-based decision-making","volume":"11","author":"Hikosaka","year":"2010","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1016\/j.cub.2014.03.073","article-title":"The right dorsal habenula limits attraction to an odor in zebrafish","volume":"24","author":"Krishnan","year":"2014","journal-title":"Curr. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.1016\/j.neuron.2014.10.035","article-title":"The habenulo-raphe serotonergic circuit encodes an aversive expectation value essential for adaptive active avoidance of danger","volume":"84","author":"Amo","year":"2014","journal-title":"Neuron"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1038\/s41583-020-0292-4","article-title":"Circuits and functions of the lateral habenula in health and in disease","volume":"21","author":"Hu","year":"2020","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1038\/nature11527","article-title":"Input-specific control of reward and aversion in the ventral tegmental area","volume":"491","author":"Lammel","year":"2012","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4051","DOI":"10.1002\/cne.23167","article-title":"Molecular characterization of the subnuclei in rat habenula","volume":"520","author":"Aizawa","year":"2012","journal-title":"J. Comp. Neurol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Beretta, C.A., Dross, N., Guiterrez-Triana, J.A., Ryu, S., and Carl, M. (2012). Habenula circuit development: Past, present, and future. Front. Neurosci., 6.","DOI":"10.3389\/fnins.2012.00051"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1016\/j.neuron.2020.03.011","article-title":"Transcriptional and spatial resolution of cell types in the mammalian habenula","volume":"106","author":"Hashikawa","year":"2020","journal-title":"Neuron"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e51271","DOI":"10.7554\/eLife.51271","article-title":"Anatomical and single-cell transcriptional profiling of the murine habenular complex","volume":"9","author":"Wallace","year":"2020","journal-title":"eLife"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1038\/s41398-024-03199-x","article-title":"The disappointment centre of the brain gets exciting: A systematic review of habenula dysfunction in depression","volume":"14","author":"Cameron","year":"2024","journal-title":"Transl. Psychiatry"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1002\/cne.20907","article-title":"Expression of pcp4a in subpopulations of CNS neurons in zebrafish","volume":"495","author":"Mione","year":"2006","journal-title":"J. Comp. Neurol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Parker, M.O., Brock, A.J., Walton, R.T., and Brennan, C.H. (2013). The role of zebrafish (Danio rerio) in dissecting the genetics and neural circuits of executive function. Front. Neural Circuits, 7.","DOI":"10.3389\/fncir.2013.00063"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.pnpbp.2014.03.011","article-title":"The utility of zebrafish to study the mechanisms by which ethanol affects social behavior and anxiety during early brain development","volume":"55","author":"Parker","year":"2014","journal-title":"Prog. Neuro-Psychopharmacol. Biol. Psychiatry"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"21171","DOI":"10.1073\/pnas.1319566110","article-title":"Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway","volume":"110","author":"Hong","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"deCarvalho, T.N., Akitake, C.M., Thisse, C., Thisse, B., and Halpern, M.E. (2013). Aversive cues fail to activate fos expression in the asymmetric olfactory-habenula pathway of zebrafish. Front. Neural Circuits, 7.","DOI":"10.3389\/fncir.2013.00098"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"636","DOI":"10.1002\/dvg.22785","article-title":"Neurotransmitter map of the asymmetric dorsal habenular nuclei of zebrafish","volume":"52","author":"deCarvalho","year":"2014","journal-title":"Genesis"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1449","DOI":"10.1016\/j.neuron.2021.03.011","article-title":"Enhancer viruses for combinatorial cell-subclass-specific labeling","volume":"109","author":"Graybuck","year":"2021","journal-title":"Neuron"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"108754","DOI":"10.1016\/j.celrep.2021.108754","article-title":"Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex","volume":"34","author":"Mich","year":"2021","journal-title":"Cell Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1507","DOI":"10.1016\/j.neuron.2025.04.035","article-title":"Enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits","volume":"113","author":"Hunker","year":"2025","journal-title":"Neuron"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"115730","DOI":"10.1016\/j.celrep.2025.115730","article-title":"Enhancer AAVs for targeting spinal motor neurons and descending motor pathways in rodents and macaque","volume":"44","author":"Kussick","year":"2025","journal-title":"Cell Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1525","DOI":"10.1016\/j.neuron.2025.05.002","article-title":"An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes","volume":"113","author":"Furlanis","year":"2025","journal-title":"Neuron"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1548","DOI":"10.1016\/j.neuron.2025.04.030","article-title":"Machine learning identification of enhancers in the rhesus macaque genome","volume":"113","author":"He","year":"2025","journal-title":"Neuron"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1562","DOI":"10.1016\/j.neuron.2025.03.031","article-title":"Specific targeting of brain endothelial cells using enhancer AAV vectors","volume":"113","author":"Dey","year":"2025","journal-title":"Neuron"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3045","DOI":"10.1016\/j.cell.2025.05.002","article-title":"A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types","volume":"188","author":"Hooper","year":"2025","journal-title":"Cell"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"990","DOI":"10.1101\/gr.200535.115","article-title":"Basset: Learning the regulatory code of the accessible genome with deep convolutional neural networks","volume":"26","author":"Kelley","year":"2016","journal-title":"Genome Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1196","DOI":"10.1038\/s41592-021-01252-x","article-title":"Effective gene expression prediction from sequence by integrating long-range interactions","volume":"18","author":"Avsec","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1038\/s41586-023-06936-2","article-title":"Cell-type-directed design of synthetic enhancers","volume":"626","author":"Taskiran","year":"2024","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1211","DOI":"10.1038\/s41586-024-08070-z","article-title":"Machine-guided design of cell-type-targeting cis-regulatory elements","volume":"634","author":"Gosai","year":"2024","journal-title":"Nature"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1038\/s41588-022-01048-5","article-title":"DeepSTARR predicts enhancer activity from DNA sequence and enables the de novo design of synthetic enhancers","volume":"54","author":"Reiter","year":"2022","journal-title":"Nat. Genet."},{"key":"ref_32","unstructured":"Westerfield, M. (2007). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio), University of Oregon Press. [5th ed.]."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e80865","DOI":"10.7554\/eLife.80865","article-title":"Kiaa1024L\/Minar2 is essential for hearing by regulating cholesterol distribution in hair bundles","volume":"11","author":"Gao","year":"2022","journal-title":"eLife"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2120","DOI":"10.1016\/S0006-3495(01)76185-1","article-title":"Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components","volume":"80","author":"Pyenta","year":"2001","journal-title":"Biophys. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11403","DOI":"10.1073\/pnas.97.21.11403","article-title":"Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage","volume":"97","author":"Kawakami","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1534\/genetics.106.060244","article-title":"Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition","volume":"174","author":"Urasaki","year":"2006","journal-title":"Genetics"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Zhang, H., Zhang, Q., Gao, G., Wang, X., Wang, T., Kong, Z., Wang, G., Zhang, C., Wang, Y., and Peng, G. (2018). UBTOR\/KIAA1024 regulates neurite outgrowth and neoplasia through mTOR signaling. PLoS Genet., 14.","DOI":"10.1371\/journal.pgen.1007583"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Gao, J., Zhang, C., Yang, B., Sun, L., Zhang, C., Westerfield, M., and Peng, G. (2012). Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0036516"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"12589","DOI":"10.1523\/JNEUROSCI.6518-11.2012","article-title":"Robo2-Slit and Dcc-Netrin1 Coordinate Neuron Axonal Pathfinding within the Embryonic Axon Tracts","volume":"32","author":"Zhang","year":"2012","journal-title":"J. Neurosci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.mce.2016.06.005","article-title":"nr0b1 (DAX1) mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation","volume":"433","author":"Chen","year":"2016","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1038\/s41586-023-06824-9","article-title":"Single-cell analysis of chromatin accessibility in the adult mouse brain","volume":"624","author":"Zu","year":"2023","journal-title":"Nature"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1038\/s41586-023-06812-z","article-title":"A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain","volume":"624","author":"Yao","year":"2023","journal-title":"Nature"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1101\/gr.227819.117","article-title":"Sequential regulatory activity prediction across chromosomes with convolutional neural networks","volume":"28","author":"Kelley","year":"2018","journal-title":"Genome Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1038\/s41556-024-01449-0","article-title":"Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq","volume":"26","author":"Sun","year":"2024","journal-title":"Nat. Cell Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"100083","DOI":"10.1016\/j.xgen.2021.100083","article-title":"Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos","volume":"2","author":"McGarvey","year":"2022","journal-title":"Cell Genom."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1126\/science.1088328","article-title":"Scanning human gene deserts for long-range enhancers","volume":"302","author":"Nobrega","year":"2003","journal-title":"Science"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1038\/ng.650","article-title":"ChIP-Seq identification of weakly conserved heart enhancers","volume":"42","author":"Blow","year":"2010","journal-title":"Nat. Genet."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Lindhorst, D., and Halfon, M.S. (2023). Reporter gene assays and chromatin-level assays define substantially non-overlapping sets of enhancer sequences. BMC Genom., 24.","DOI":"10.1186\/s12864-023-09123-9"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1126\/science.ade6970","article-title":"Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain","volume":"384","author":"Powell","year":"2024","journal-title":"Science"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2409","DOI":"10.1002\/dvdy.22051","article-title":"Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish","volume":"238","author":"Bessa","year":"2009","journal-title":"Dev. Dyn."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Bhatia, S., Gordon, C.T., Foster, R.G., Melin, L., Abadie, V., Baujat, G., Vazquez, M.-P., Amiel, J., Lyonnet, S., and van Heyningen, V. (2015). Functional assessment of disease-associated regulatory variants in vivo using a versatile dual colour transgenesis strategy in zebrafish. PLoS Genet., 11.","DOI":"10.1371\/journal.pgen.1005193"},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Kelley, D.R. (2020). Cross-species regulatory sequence activity prediction. PLoS Comput. Biol., 16.","DOI":"10.1371\/journal.pcbi.1008050"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.ygeno.2015.06.005","article-title":"Decoding enhancers using massively parallel reporter assays","volume":"106","author":"Inoue","year":"2015","journal-title":"Genomics"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/9\/1079\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T04:42:00Z","timestamp":1788928920000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/9\/1079"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,7]]},"references-count":53,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["genes17091079"],"URL":"https:\/\/doi.org\/10.3390\/genes17091079","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,7]]}}}