Single-Cell RNA Sequencing Characterizes the Molecular Heterogeneity of the Larval Zebrafish Optic Tectum

Abstract

The optic tectum (OT) is a multilaminated midbrain structure that acts as the primary retinorecipient in the zebrafish brain. Homologous to the mammalian superior colliculus, the OT is responsible for the reception and integration of stimuli, followed by elicitation of salient behavioral responses. While the OT has been the focus of functional experiments for decades, less is known concerning specific cell types, microcircuitry, and their individual functions within the OT. Recent efforts have contributed substantially to the knowledge of tectal cell types; however, a comprehensive cell catalog is incomplete. Here we contribute to this growing effort by applying single-cell RNA Sequencing (scRNA-seq) to characterize the transcriptomic profiles of tectal cells labeled by the transgenic enhancer trap line y304Et(cfos:Gal4;UAS:Kaede). We sequenced 13,320 cells, a 4X cellular coverage, and identified 25 putative OT cell populations. Within those cells, we identified several mature and developing neuronal populations, as well as non-neuronal cell types including oligodendrocytes and microglia. Although most mature neurons demonstrate GABAergic activity, several glutamatergic populations are present, as well as one glycinergic population. We also conducted Gene Ontology analysis to identify enriched biological processes, and computed RNA velocity to infer current and future transcriptional cell states. Finally, we conducted in situ hybridization to validate our bioinformatic analyses and spatially map select clusters. In conclusion, the larval zebrafish OT is a complex structure containing at least 25 transcriptionally distinct cell populations. To our knowledge, this is the first time scRNA-seq has been applied to explore the OT alone and in depth.

Valproic Acid Affects Neuronal Specification and Differentiation During Early Optic Tectum Development of Zebrafish

“…NeuroD1 has been shown to be involved in neuronal differentiation, specification, and migration within the developing mouse cortex 38 , making it a good indicator of neurogenesis. Additionally, single-cell RNA sequencing (scRNA-seq) data previously collected in our lab 39 confirmed the presence of NeuroD1 in the zebrafish OT and showed that its expression is sequestered to specific populations by 7dpf (Suppl. To measure redox states after VPA exposure, embryos were treated with 250µM VPA beginning at 18hpf, and media was replaced every 24 hours until 5dpf.…”

“…One study 50 of zebrafish embryos treated with VPA identified upregulation of several miRNAs, including those predicted to regulate cell cycle genes. Among those cell cycle regulating genes, cdkn1a, tp53 are broadly expressed throughout the different neuronal subclasses at 7dpf in our scRNA-seq data 39 , while wee1, cdk2 and chek1 are specifically expressed in one developing neuronal population 39 (Suppl. Fig 2B).…”

“…(B) Expression of various cell cycle genes reveal that cdkn1a and tp5d are found throughout the OT at 7dpf. In contrast, wee1, cdk2, and chek1, are largely found in OT10, a cluster identified as developing neuronal39 . (C) Expression profiles show that both HDAC1 and HDAC4 are present in the OT at 7dpf.…”

The “Primitive Brain Dysfunction” Theory of Autism: The Superior Colliculus Role

“…It was found to be differentially expressed within specific populations in the larval tectum. This strongly suggests a potential genetic link between the SC/tectum and ASD (Martin et al, 2022 ).…”

“…Autism spectrum disorder coherence is not explained by etiologic factors because several prenatal or postnatal genetic or non-genetic conditions could result in ASD (Jure, 2019 ; Martin et al, 2022 ). Similarly, several autism theories have been proposed, from dysfunctions of sensory, motor, autonomic, attentional, or emotional basic processing (for example abnormal social motivation) to exclusive higher-order compromise (theory of mind, social cognition, executive functions abnormalities, etc.)…”