Abstract
Purpose :
The transcription factor CEBPG (C/EBPγ) plays a key role in the integrated stress response in partnership with ATF4. Mice lacking CEBPG have reduced body size and microphthalmia. We recently observed expression of Cebpg in zebrafish lens, but no previous studies have examined its lenticular role. Therefore, we are using zebrafish as a model system to examine the impact of Cebpg loss on lens development.
Methods :
We used three gRNAs to target Cas9 double stranded cleavage in exons 2 and 3 of the cebpg gene and confirmed site damage using head loop PCR. Lenses of crispant and control-injected larvae were live-imaged using DIC optics at 3 days post fertilization (dpf) and cryosections were stained with DAPI to assess the loss of fiber cell nuclei. We used RT-qPCR to measure the expression of genes either known to be regulated by Cebpg (slc7a11) or involved in oxidative stress response (hmox1a).
Results :
Approximately 75% of cebpg crispant larvae had abnormal lenses at 3 dpf, compared to 10% of control larvae. Defects ranged from subtle separations between fiber cells and small pocketing, to severe disruption throughout the center of the lens. DAPI staining of frontal sections through crispant larvae showed typical removal of fiber cell nuclei, although some lenses retained speckling of DAPI fluorescence within the fiber cell region. cebpg crispant lenses were significantly smaller than controls at 3 dpf, but not smaller as a proportion of body length, which was also reduced in crispants. Preliminary RT-qPCR analysis showed an increase in hmox1a mRNA but reduction in slc7a11 mRNA in crispant larvae, similar to published findings in a Cebpg knockout mouse. Expression of atf4a in crispants was unchanged.
Conclusions :
Our data show that damaging cebpg causes severe defects in the zebrafish lens, although crispant fish do not appear to show the microphthalmia reported in a Cebpg mouse knockout. Similar changes in gene expression suggest that the mechanisms of Cebpg function are conserved between zebrafish and mouse. We plan to use this new model to investigate the role that Cebpg plays in lens stress response.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.