Work

An orthogonal Cro/OR3 system reveals an influence of astrocytes on motor neuron positioning

Year: 2026

Type: article

Abstract: Precise spatiotemporal control of gene expression is essential for dissecting complex biological systems. Here, we introduce a phage-derived Cro/O R 3 binary system for transcriptional activation that functions robustly in zebrafish and human cells. A Cro repressor fused to the VP16 activation domain efficiently drives expression of O R 3 operator-linked genes, and its activity can be targeted to specific cell types using enhancer-trap and promoter-controlled zebrafish lines. As a proof of concept, we manipulate glutamate transporter Glast/Slc1a3b-expressing cells in the central nervous system by Cro/O R 3-mediated expression of a human ataxia-associated, uptake-deficient Glast/Slc1a3b variant, causing spastic, uncoordinated movement and axial deformation. Concurrent Gal4/UAS-mediated visualization reveals substantially disrupted positioning of motor neurons, highlighting their selective sensitivity to extracellular glutamate homeostasis during spinal circuit assembly. Together, these results establish the Cro/O R 3 system as an orthogonal platform for targeted gene expression, enabling precise mechanistic dissection of how specific cell types and their interactions underlie diverse biological processes.

Source: iScience

DOI: 10.1016/j.isci.2026.117967