Our bodies are composed of many different types of cells that work together to build and maintain normal physiological functions. In the central nervous system, for example, neurons transmit information, while glial cells such as astrocytes regulate the environment surrounding neurons and support their function. Understanding how these cell types influence one another is essential for uncovering how our bodies develop and function.
Understanding the function of each cell type requires tools that can specifically manipulate gene expression in the cell of interest. Researchers often use binary gene expression systems such as the widely used Gal4/UAS system, which utilizes the transcription factor Gal4 and its binding sequence UAS. However, a single binary gene expression system cannot independently control two distinct cell types, making it difficult to study how different cell types work together. To overcome this limitation, an additional gene expression system that can work independently of the conventional gene expression systems is needed.
A research team at the National Institute of Genetics (NIG), Japan, has now developed a new binary gene expression system based on the bacteriophage-derived DNA-binding protein Cro and its target DNA sequence, OR3. The study, led by Kazuhide Asakawa and colleagues at NIG, is reported in "An orthogonal Cro/OR3 system reveals an influence of astrocytes on motor neuron positioning." The study was published in the journal iScience on October XX, 2026.
In the new system, called Cro/OR3, the researchers used Cro fused to the F2 transcriptional activation domain (CroF2). CroF2 induced expression of a target gene placed downstream of the OR3 sequence in both zebrafish and human cultured cells. This suggests that the system has the potential for use across different species.
The researchers then combined the Cro/OR3 system with the conventional Gal4/UAS system in the zebrafish nervous system. By combining the two systems in the same zebrafish, the researchers were able to successfully control gene expression independently in two distinct cell types: neurons and astrocytes. Astrocytes normally help regulate the levels of extracellular glutamate, an important neurotransmitter, through the function of Slc1a3b, an astrocytic glutamate transporter. Using the Cro/OR3 system, the researchers expressed in astrocytes a mutant form of Slc1a3b associated with episodic ataxia type 6. The manipulation altered the positioning of motor neurons labeled by the Gal4/UAS system in the spinal cord. The findings suggest that proper astrocyte function is important for the normal positioning of motor neurons.
This independent control of two cell types is particularly useful for studying interactions between different cell types—for example, between neurons and glial cells. The Cro/OR3 system could serve as a new genetic tool for investigating the roles of specific cell types and how different cell types influence one another, with the potential for broad application across a variety of species. This approach could also be useful for disease modeling, allowing researchers to express a disease-associated protein in one cell type and examine not only its effects on those cells, but also how the affected cells influence other cell types that do not themselves express the disease-associated protein.
The team further generated enhancer-trap zebrafish lines that express CroF2 in specific cell populations by using particular DNA sequences, called "enhancers," that drive gene expression in specific cells (Figure). More than 150 Cro lines have been established and made available through an online resource, providing a growing collection of tools for cell-type-specific manipulation in zebrafish ( https://nig-zebra.nbrp.jp/ ).
"The toolkit that we developed could help broaden the scope of research," said Kazuhide Asakawa. "Having multiple independent gene expression systems gives us a way to observe one cell population while manipulating another, allowing us to examine these interactions directly in living animals."
