Our aim is to develop Xenopus tropicalis as a robust, relevant and rapid vertebrate model system for the analysis of the regulatory functions conferred by gene-specific conserved non-coding elements (CNEs). The hypothesis underlying this proposal is that many developmental processes, including embryonic patterning and organogenesis, are highly conserved among vertebrates, and that the genetic machinery regulating these processes is also likely to be conserved. The Xenopus tropicalis draft genome sequence provides a powerful resource for comparative analysis of vertebrate gene regulation, because the evolutionary distance from frog to mammals permits one to readily identify CNEs, which in many cases act as regulatory elements for nearby genes. Discovery of complex long-range cis-regulation of gene expression, particularly for developmental control genes, is a major biological spin-off of comparative genomics. This collaborative project, aimed at increasing our understanding of both normal organogenesis and human genetic diseases, will use the highly efficient and rapid transgenesis system in Xenopus to screen putative regulatory elements in a system that allows many manipulations of gene activity and complementary assays of biological function. Development of this methodology is highly significant because it will enhance the ability of biologists to rapidly identify and examine regulatory elements in genes, even large sets of genes, involved in many facets of cell and developmental biology. This project serves the Xenopus community at large by developing and integrating genomic and transgenic approaches that strengthen the utility of Xenopus as a model organism. We propose to apply this approach to the problem of eye formation and function, the subject of study in both the Grainger laboratory and the collaborating van Heyningen group, albeit from differing, complementary perspectives. The Grainger lab, and several others, have been involved in identification of regulatory genes involved in embryonic eye formation. The ability to rapidly identify and study the regulatory elements of genes involved in eye formation has begun to provide, and will continue to reveal significant new insights regarding the gene networks controlling eye formation. A very different perspective is the insight that these studies can provide for understanding human genetic diseases, as studied in the van Heyningen laboratory.

Public Health Relevance

Increasingly there is evidence that gene regulatory elements, some quite distant from the coding sequence, are essential for correct gene function, and, when altered, cause disease phenotypes. There has been no systematic way to identify enhancer elements controlling gene expression, however, and therefore the database of putative enhancers that might be affected by mutations is limited to a small group of serendipitously discovered elements. The approaches described here permit one to develop a large database of regulatory elements, their roles in gene expression, and the phenotypes associated with mutations in them, which will be a highly valuable resource for identifying genetic lesions in human patients. Increasingly there is evidence that gene regulatory elements, some quite distant from the coding sequence, are essential for correct gene function, and, when altered, cause disease phenotypes. There has been no systematic way to identify enhancer elements controlling gene expression, however, and therefore the database of putative enhancers that might be affected by mutations is limited to a small group of serendipitously discovered elements. The approaches described here permit one to develop a large database of regulatory elements, their roles in gene expression, and the phenotypes associated with mutations in them, which will be a highly valuable resource for identifying genetic lesions in human patients.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY018000-04
Application #
8212076
Study Section
Special Emphasis Panel (ZRG1-GGG-T (52))
Program Officer
Araj, Houmam H
Project Start
2009-01-01
Project End
2012-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$325,864
Indirect Cost
$88,264
Name
University of Virginia
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Bhatia, Shipra; Monahan, Jack; Ravi, Vydianathan et al. (2014) A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos. Dev Biol 387:214-28
Fish, Margaret B; Nakayama, Takuya; Fisher, Marilyn et al. (2014) Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character. Dev Biol 395:317-30
Nakayama, Takuya; Blitz, Ira L; Fish, Margaret B et al. (2014) Cas9-based genome editing in Xenopus tropicalis. Methods Enzymol 546:355-75
Ravi, Vydianathan; Bhatia, Shipra; Gautier, Philippe et al. (2013) Sequencing of Pax6 loci from the elephant shark reveals a family of Pax6 genes in vertebrate genomes, forged by ancient duplications and divergences. PLoS Genet 9:e1003177
Zeng, Mei; Kuzirian, Marissa S; Harper, Lamia et al. (2013) Organic small hairpin RNAs (OshR): a do-it-yourself platform for transgene-based gene silencing. Methods 63:101-9
Hellsten, Uffe; Harland, Richard M; Gilchrist, Michael J et al. (2010) The genome of the Western clawed frog Xenopus tropicalis. Science 328:633-6