A single-cell resolution gene expression atlas of the larval zebrafish brain

Abstract

The advent of multimodal brain atlases promises to accelerate discoveries in neuroscience by offering in silico queries of cell types, connectivity and gene expression in regions of interest. We employed multiplexed fluorescent in situ RNA hybridization chain reaction (HCR) to generate expression maps for an initial set of 200 marker genes across the larval zebrafish brain. The data were registered to the Max Planck Zebrafish Brain (mapzebrain) atlas, thus allowing co-visualization of gene expression patterns, single-neuron tracings, transgenic lines and anatomical segmentations. Additionally, brain activity maps of freely swimming larvae were generated at single-cell resolution using HCR labeling of the immediate-early gene cfos and integrated into the atlas. As a proof of concept, we identified a novel class of cerebellar eurydendroid cells that express calb2a, project to the hypothalamus and are activated in animals that have recently ingested food. Thus, a cellular-resolution gene expression atlas will not only help with rapid identification of marker genes for neuronal populations of interest, but also bridge the molecular and circuit levels by anchoring genetic information to functional activity maps and synaptic connectivity.

Visual recognition of social signals by a tectothalamic neural circuit

“…2j). Registration of the larval data to the Max Planck Zebrafish Brain Atlas 30,31 (https://mapzebrain.org) confirmed localization of the DT-BPN cluster to the vglut2a-positive DT area, ventrally touching the gad1b-positive VT 32 . The larval DT-BPN cluster was molecularly defined by the expression of cort, as seen in juveniles, and pth2 (Extended Data Fig.…”

“…In larvae, s1026tEt drives strong expression in the DT, dorsal VT, ventral pretectum and ventral telencephalon, and additional background expression in the heart and trunk musculature 31,48 . We found that MTZ mediated ablation of s1026tEt, UAS-E1B:NTR-mCherry(c264) double transgenic fish was lethal in 100% of animals.…”

Functional and pharmacological analyses of visual habituation learning in larval zebrafish

“… C) Normalized summed intensity projections comparing the spatial arrangements of i) , and ii) with atlas markers found within the the purple cluster iii) satb1b HCR ( Shainer et al, 2022 ), mapZebrain Atlas iv) nns25 , aka TgBAC(gad1b:GFP) ( Satou et al, 2013 ), mapZebrain Atlas v) nns26 , aka TgBAC(gad1b:LOXP-RFP-LOXP-GFP) ( Satou et al, 2013 ), mapZebrain Atlas vi) TgBAC(gad1b:GFP) ( Satou et al, 2013 ), Z-Brain Atlas…”

“…Virtual co-localization analyses with 3D atlases can be used to identify candidate molecular markers for functionally identified neurons, provided sufficient stereotypy exists in neuronal positioning, and the relevant marker exists in the atlas Dunn et al ( 2016 ); Randlett et al ( 2015 ). Therefore, we analyzed the spatial correlations for markers contained in the Z-Brain Randlett et al ( 2015 ), Zebrafish Brain Browser Gupta et al ( 2018 ); Marquart et al ( 2017 ); Tabor et al ( 2018 ), and mapZebrain atlases Kunst et al ( 2018 ); Shainer et al ( 2022 ). We identified markers showing the highest spatial correlations with any of our functional clusters (corr.…”

“…The subset of the hierarchy containing the gad1b-reporters is coloured in purple. C) Normalized summed intensity projections comparing the spatial arrangements of i) 10 𝑛𝑜𝐴 𝑆 , and ii) 11 𝑤𝑒𝑎𝑘𝐷 𝑆with atlas markers found within the the purple cluster iii) satb1b HCR(Shainer et al, 2022), mapZebrain Atlas iv) nns25, aka TgBAC(gad1b:GFP)(Satou et al, 2013), mapZebrain Atlas v) nns26, aka TgBAC(gad1b:LOXP-RFP-LOXP-GFP)(Satou et al, 2013), mapZebrain Atlas vi) TgBAC(gad1b:GFP)(Satou et al, 2013), Z-Brain Atlas D) 2-photon imaging of Tg(Gad1b:DsRed);Tg(elavl3:H2B-GCaMP6s) larvae depicting the raw data for each channel (top), and the ratio of Gad1b/GCaMP6s fluorescence in each ROI functionally identified using suite2p. E) ROIs imaged in double transgenic larvae are assigned a cluster identity based on their correlation to the cluster mean trace, and classified as Gad1b-positive based on a DsRed/GCaMP6s ratio of greater than 0.25.…”