Cellular and Molecular NeuroscienceResearch PaperRegulation of POU4F3 gene expression in hair cells by 5′ DNA in mice
Highlights
▶A 5′ genomic DNA fragment from the murine Pou4f3 gene directs gene expression to hair cells. ▶The pattern of expression suggests multiple hair cell enhancers in the Pou4f3 gene. ▶Three regions within this fragment are highly conserved across mammals. ▶Clustered E-boxes are consistent with class II bHLH transcription factor binding. ▶The class II factor ATOH1 binds to, and regulates expression directed by, the 5′ DNA fragment.
Section snippets
Generation of transgenic mice
All procedures were approved by the Animal Subjects Committee of the San Diego VA Medical Center, and were in accordance with the National Institute of Health policies regarding the treatment of animal subjects.
An 8.5 kb genomic DNA fragment immediately 5′ to the ATG of the murine Pou4f3 gene was isolated from a strain 129 genomic library. The fragment was ligated to one of two reporter constructs. The first was a beta galactosidase (β-gal) coding sequence. The second consisted of an enhanced
Expression of β-gal was restricted to HCs within the inner ear of Pou4f3-8.5-β-gal mice
Three lines of Pou4f3-8.5-β-gal mice were generated and characterized in neonatal and adult inner ears. Within the inner ear, X-gal reaction product was restricted to HCs. In neonatal animals, all types of both cochlear and vestibular HCs exhibited β-gal expression. However, as illustrated in Fig 1a for cochlear HCs, the levels observed were highly variable, ranging from undetectable to highly dense. A similar response was observed in vestibular HCs (data not shown). Essentially the same
Discussion
Expression of eGFP under the control of 8.5 kb of 5′ Pou4f3 genomic DNA largely though not completely mimics, in transgenic mice, in vivo expression of Pou4f3 mRNA (Erkman et al., 1996, Artinger et al., 1998, Leonard et al., 2002). That is, expression occurred in most cells that normally produce POU4F3. Variable X-gal reaction product in the HCs of Pou4f3-8.5-β-gal mice seems likely to have resulted from irregular X-gal penetration into HCs, since in the eGFP transgenic, eGFP was uniformly
Acknowledgments
Supported by grants from the Research Service of the Veterans Administration, the NIH/NIDCD (RO1 DC000139), and the National Organization for Hearing Research (NOHR). Eduardo Chavez managed the mouse colony, Julie Lightner formatted the manuscript and Dr. Elizabeth Keithley read the manuscript and provided critical comments. Dr. Matthew Holley generously provided VOT-E36 cells. Their assistance is gratefully acknowledged.
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