Single cell transcriptomics of the developing zebrafish lens and identification of putative controllers of lens development

Abstract

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos at single cell resolution through five days of development, identifying a number of novel regulators of lens development as potential targets for future functional studies. Our temporospatial expression data address open questions about the function of α-crystallins during lens development and provides the first detailed view of β-and γ-crystallin expression in and outside the lens. We describe subfunctionalization in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, RNA-binding, and transcription factors genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.

Transcriptome-Based Identification of Genes Responding to the Organophosphate Pesticide Phosmet in Danio rerio

“…Crystallins, which constitute a substantial part of the refractive structure of the vertebrate eye lens and play a critical role in embryonic lens development, were downregulated in PTZF [63]. A single-cell transcriptome analysis study of ZF showed that crystallins (cryaa (crystallin, alpha A) and crybgx (crystallin beta-gamma X)) are exclusively expressed in lens fiber cells starting at 2 dpf [64]. In our study, the biological process linked with visual system development and perception was among the top GO terms (Figure 5), indicating that phosmet triggers ocular toxicity.…”

Effects of α-crystallin gene knockout on zebrafish lens development

“…In their more recent CRISPR lines there were no reported general larval defects, further suggesting that the morpholino-induced lens defects were not specifically due to the loss of aB-crystallin proteins. Existing data suggest that neither cryaba nor cryabb are significantly expressed in the zebrafish lens through 5 dpf, based on shotgun proteomics (Greiling, Houck & Clark, 2009) and single cell RNA-Seq (Farnsworth, Posner & Miller, 2021). These previous findings might explain the lack of noticeable early lens phenotypes in our aB-crystallin homozygous null larvae.…”

“…This allowed us to determine if mutation led to a decrease in mRNA levels for the damaged gene or changes in expression of the other α-crystallin genes. Whole larvae were assessed at 7 dpf as earlier work showed low levels of cryaba and cryabb expression through 4 and 5 dpf, respectively (Posner et al, 2017), and almost no expression in the lens (Farnsworth, Posner & Miller, 2021). We found reduced levels of cryaa mRNA in 7 dpf homozygous null mutants with the large deletion removing the proximal promoter and start codon (Fig 7A).…”

“…Existing data suggest that neither cryaba nor cryabb are significantly expressed in the zebrafish lens through 5 dpf, based on shotgun proteomics (Greiling, Houck & Clark, 2009) and single cell RNA-Seq (Farnsworth, Posner & Miller, 2021). These previous findings might explain the lack of noticeable early lens phenotypes in our aB-crystallin homozygous null larvae.…”

“…This result differed from work in mouse, which showed that while the loss of aA-crystallin led to reduced lens size and early cataract development, the loss of aB-crystallin did not produce a lens phenotype, although heart defects were found (Brady et al, 1997(Brady et al, , 2001. Furthermore, our recent analysis of zebrafish gene expression at single cell resolution indicated that neither of the two aBcrystallin genes were expressed in the lens at significant levels through five days of development (Farnsworth, Posner & Miller, 2021). Therefore, there seems to be conflicting reports on what role, if any, aB-crystallins play in early lens development.…”