The purpose of this competing supplemental application is to map by whole mount in situ hybridization the expression of all microRNAs in the chicken embryo through day 8 of development, and to optimize technologies for detecting mRNAs in vivo using short LNA probes. This will expand the presently funded GEISHA (gallus est in situ hybridization analysis) project (HD044767), the aims of which are to map expression of all protein encoding mRNAs through day 4 of chick embryo development, to link this information to the chicken genome browsers and to present information through a freely accessible user interface (http://geisha.arizona.edu). MicroRNAs posttranscriptionally regulate gene expression by binding to sequences in the 3'UTRs of target mRNAs, and represent a new mechanism of biological regulation with important functions in animal growth, development and disease. Mapping microRNA expression patterns is not presently an objective of the GESIHA project because standard antisense RNA probes used for in situ hybridization detection of mRNAs cannot detect microRNAs, the mature forms of which are only 21-23nt. Although standard DNA oligonucleotides have also not proven useful as in situ hybridization probes, a new class of DNA analogues called locked nucleic acids (LNAs) have recently been developed that exhibit significantly improved hybridization characteristics, thermal stability and specificity. Preliminary studies demonstrate that 21-23 nucleotide LNA probes can readily detect microRNAs in vivo, and through protocol modifications can also detect mRNAs. For this supplement we will use this technology to pursue two specific aims; 1) map the expression of all chicken microRNAs through day 8 of embryo development, using whole mount and section in situ hybridization approaches. 2) Optimize the use of short LNA probes for in situ hybridization detection of protein encoding mRNAs. The ability to use short LNAs to detect mRNAs in vivo will allow probes to be designed directly from sequence information, eliminating the need for a cDNA template for probe production. This new technology could fundamentally change the way in which in situ hybridization analyses are performed. ? ? ?
