Poster abstracts

Poster number 47 submitted by Jennifer Bosse

Enhancing Rx: Keeping an Eye on Retinal Stem Cell Identity

Jennifer Bosse (MCDB and Nationwide Childrens Hospital)

Abstract:
The retinal homeobox gene, Rx/RAX, is a conserved transcription factor vital for normal vertebrate eye development. Loss of Rx gene function causes anophthalmia in many vertebrates including humans (Bailey 2004). Rx is also thought to be necessary for maintaining retinal stem cell identity. Unlike humans, frogs maintain a stable population of retinal stem cells (RSCs) and retinal progenitor cells (RPCs) throughout their lifetime for use in retinal growth and maintenance.
The purpose of this study is to understand how an enhancer of Rx, known as ultra conserved element 2 (UCE2), drives Rx mRNA expression in RSCs. It was previously shown that fragment 1 of UCE2 comprising the first 50 bp of UCE2, in combination with an Rx promoter, is sufficient to drive reporter mRNA expression in RSCs (Pan et al., 2010). To further understand which sequences are necessary for driving Rx mRNA expression, mutational analysis of UCE2 fragment 1 was performed. Specifically, deletions were made within UCE2 fragment 1, dividing the fragment into five 10 bp parts termed A-E (UCE2 fragment 1A-E). Our hypothesis is that UCE2 fragment 1D is essential for Rx mRNA expression since it contains a strongly predicted transcription factor binding site.
To study the effect of these mutations, the UCE2 fragment 1 deletion mutants were combined with a Rx promoter driving expression of GFP and then constructed into Xenopus transgenes. Transgenic Xenopus embryos were analyzed by in situ hybridization against GFP mRNA. Rx expression regions were examined for GFP mRNA. Transgenic tadpoles that fail to display GFP mRNA expression comparable to the control, transgenic tadpoles that have intact UCE2, indicate the deleted sequence in the UCE2 fragment 1 is necessary for Rx expression. Thus far, our data indicate that UCE2 fragment 1A is unnecessary for Rx expression while UCE2 fragments 1C and 1D are necessary for RX expression.
Identifying the necessary sequences in UCE2 fragment 1 will lead to a better understanding of how retinal stem cell identity is established and maintained in Xenopus. Importantly, this knowledge could also be applied to the development of retinal stem cells therapies for the treatment of retinal diseases in human.

References:
Bailey T.J, El-Hodiri H, Zhang L, Shah R, Mathers P.H, Jamrich M. Regulation of vertebrate eye development by Rx genes. Int. J. Dev. Biol. 2004

Pan Y, Martinez-De Luna RI, Lou CH, Nekkalapudi S, Kelly LE, Sater AK, El-Hodiri HM. Regulation of photoreceptor gene expression by the retinal homeobox (Rx) gene product. Dev Biol. 2010

Keywords: Retinal Stem Cells, Retinal Homeobox , Retinal development