Deep Learning-Guided Identification and In Vivo Validation of Compact Cis-Regulatory Elements for the Zebrafish Habenula.

BACKGROUND/

objectivesPrecise 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.

methodsUsing 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

resultsThe 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

conclusionsTogether, 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.