Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis.

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Studying gene function in mammalian neural circuit formation is challenging due to limitations in traditional genetic screening methods.
• Synapse development in the mammalian retina, specifically the outer plexiform layer (OPL), is crucial for visual processing.

Purpose of the Study:

• To overcome limitations in germline mutagenesis for gene screening in neural development.
• To investigate the roles of specific genes in the formation of the outer plexiform layer (OPL) in the mouse retina.

Main Methods:

• Combined RNA sequencing (RNA-seq) with somatic CRISPR mutagenesis in the mouse retina.
• Utilized cell-specific promoters for CRISPR-Cas9 electroporation to assess gene function.
• Identified selectively expressed genes encoding cell surface and secreted proteins.

Main Results:

• Identified Wnt5a and Wnt5b as key genes involved in OPL development.
• Demonstrated that Wnt5a and Wnt5b are produced by rod bipolar cells.
• Showed that Wnt5a and Wnt5b activate a non-canonical signaling pathway in rods, regulating early OPL patterning.

Conclusions:

• The developed RNA-seq and somatic CRISPR mutagenesis approach is effective for studying neural circuit development.
• Wnt5a and Wnt5b play critical roles in the early patterning of the outer plexiform layer.
• This methodology can be extended to investigate gene function in other brain regions.