Over the last two decades zebrafish has emerged as one of the preeminent model organisms. This rise was in large part due to the promise of using forward genetic screens in a vertebrate animal to discover and elucidate the function of genes relevant to human development and health. Numerous genetic screens have now been performed both for general embryonic morpohological phenotypes as well as more specialized functional screens such as for balance and hearing. These screens have been very successful in identifying mutants and there are now over 6000 described chemically induced mutant lines in zebrafish. However, the gene mutated in these lines has only been identified in less than half the cases due to the current difficulty of positional cloning. The high cost and labor currently required for positional cloning prevents a molecular analysis of many current mutants as well as discouraging future genetic screens. Recent advances in next generation sequencing have reduced the cost of sequencing by several orders of magnitude such that it is now possible to routinely resequence entire genomes. These advances have already revolutionized many areas of genomics yet the way people do positional cloning in zebrafish and other model organisms has changed relatively little. Here we propose to apply next generation sequencing technologies to streamline positional cloning of mutants in model organisms focusing on zebrafish inner ear mutants. Our goal is to reduce the labor and cost of positional cloning by one to two orders of magnitude. We will compare two different approaches for cloning-by-sequencing based on linkage and homozygosity mapping by cloning 10 existing mutants in two mutant classes-semicircular canal morphogenesis and deafness.

Public Health Relevance

Defects in hearing and balance represent a major health problem in humans. Models for these defects can be identified in genetic screens in zebrafish but are currently difficult to molecularly characterize. We will develop an approach to streamline the cloning of such mutants using next generation sequencing.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21DC012097-01
Application #
8224539
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Freeman, Nancy
Project Start
2012-01-10
Project End
2013-12-31
Budget Start
2012-01-10
Budget End
2012-12-31
Support Year
1
Fiscal Year
2012
Total Cost
$254,250
Indirect Cost
$104,250
Name
Harvard University
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Xiong, Fengzhu; Tentner, Andrea R; Huang, Peng et al. (2013) Specified neural progenitors sort to form sharp domains after noisy Shh signaling. Cell 153:550-61