Deciphering the genetic interactions between Pou4f3, Gfi1 and Rbm24 in maintaining cochlear hair cell survival

Reviewer #1 (Public Review):

Wang and colleagues recently demonstrated the essential role of RBM24 (RNA-binding motif protein 24a) in the development of mouse hair cells (source: https://doi.org/10.1002/jcp.31003). In this study, they further expand on their findings by revealing that Rbm24 expression is absent in Pou4f3 mutant mice but not in Gfi1 mutant mice. This observation suggests that POU4F3 acts as an upstream regulator of Rbm24. The researchers effectively demonstrate that POU4F3 can bind to and regulate Rbm24 through three distant enhancers, which are located in open chromatin regions and are bound by POU4F3. Lastly, Wang and colleagues discovered that ectopic expression of Rbm24 was unable to prevent the degeneration of POU4F3 null hair cells.

The findings in this manuscript hold great significance as they provide additional insights into the transcriptional cascades crucial for hair cell development. The discovery of enhancers capable of driving transgene expression specifically in hair cells holds promising therapeutic implications. The figures presented in the study are of excellent quality, the employed techniques are state-of-the-art, the data are accurately represented without exaggeration, and the study demonstrates a high level of rigor.

Reviewer #2 (Public Review):

Previous studies have shown that two hair cell transcription factors, Pou4f3 and Gfi1, are both necessary for the survival of cochlear hair cells, and that Gfi1 is regulated by Pou4f3. The authors have previously also shown that mosaic inactivation of the RNA-binding protein RBM24 leads to outer hair cell death.

In the present study, the authors show that hair cells die in Pou4f3 and Gfi1 mutant mice. They show that Gfi1 is regulated by Pou4f3. Both these observations have been published before. They then show that RBM24 is absent in Pou4f3 knockouts, but not Gfi1 knockouts. They ectopically activate RMB24 in the hair cells of Pou4f3 knockouts, but this does not rescue the hair cell death. Finally, the authors validate three RMB24 enhancers that are active in young hair cells and which have been previously shown to bind Pou4f3.

The experiments are well-executed and the data are clear. The results support the conclusions of the paper.

Much of the work in the paper has been reported before. The result that hair cell transcription factors operate in a network, with some transcription factors activating only a subset of hair cell genes, is an expected result. Since RMB24 is only one of many genes regulated directly by Pou4f3, it is not surprising that it cannot rescue the Pou4f3 knockout hair cell degeneration.

The identification of new hair cell enhancers may be of use to investigators wishing to express genes in hair cells.

In sum, this work, although carefully performed, does not shed significant new light on our understanding of hair cell development or survival.