MicroRNAs (miRNAs) have recently been described as an important new class of regulatory molecules controlling aspects of differentiation and patterning in invertebrates. Similar miRNAs have been discovered in vertebrates, but their function in higher organisms remains largely unknown. This proposal is directed towards understanding the role of miRNAs during vertebrate development. We have found that certain miRNAs are differentially expressed during morphogenesis of the limb and heart. We will use microarray and mini-Sage approaches to identify all miRNAs expressed in these developing structures, including the identification of miRNAs expressed in the developing limb buds, including those differentially expressed in the fore- and hind-limb primordia as well as those left-right asymetrically produced in the heart primordium. The spatial distribution of these miRNAs will be determined by Northern blot and by production of transient transgenic mice carrying """"""""sensor"""""""" constructs which are designed to express lacZ everywhere except where a given miRNA is present. Additional sensors will be made to examine the differential expression patterns of all paralogues of a single miRNA family (Iet7);and also of several distinct miRNAs encoded in a cluster in the genome. Based on these expression patterns, the function of miRNAs will be addressed in the chick using retroviral misexpression. Finally, we will study the roles of 1 family of miRNAs, miR196, in regulating Hox gene function in mice. The 3 miR196 loci will be knocked-out individually and the triple mutant will be constructed. These miRs regulate stability of the HoxB8 mRNA and modulate translation of the HoxA7 mRNA. The miR196 target sequence in the HoxB8 3'UTR will be deleted by a knock-in approach to determine the developmental significance of this regulation and the miR196 target sequence in the HoxA7 3'UTR will be replaced with the HoxB8 miR198 target sequence to test whether the mode of regulation (RNA cleavage versus translational control) functionally matters.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD047360-05
Application #
7571720
Study Section
Development - 1 Study Section (DEV)
Program Officer
Javois, Lorette Claire
Project Start
2005-04-01
Project End
2011-01-31
Budget Start
2009-02-01
Budget End
2011-01-31
Support Year
5
Fiscal Year
2009
Total Cost
$307,131
Indirect Cost
Name
Harvard University
Department
Genetics
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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