UC San Diego

This dissertation is presented in three parts. In the first part, using a combination of simultaneous RNA and protein detection, high-resolution confocal microscopy, and image segmentation, I show that it is possible to resolve and count the number of mRNA transcripts for a given gene within single-cells of fixed Drosophila embryos. I used these methods to study the stochastic nature of transcription at the endogenous locus of the Hox gene Sex combs reduced, and I uncovered evidence for transcriptional bursting as well as divergent modes of transcription. This was the first time such analyses had been carried out in an intact metazoan organism. In the second part, I present evidence that despite the presence of several well conserved putative binding sites in the 3'UTR of the Hox gene Antennapedia, the microRNA miR-iab-4 -5p does not appear to play a large role in the regulation of Antennapedia protein levels during embryogenesis, and at most has very subtle effects. This is important, because despite the fact that Drosophila Hox genes and Hox -cluster encoded microRNAs are very strongly predicted to interact in silico, most investigations of these interactions have uncovered only very subtle (if any) effects on protein levels. This suggests that our ability to predict miRNA target sites in silico is very lacking, or that, in general, microRNAs play very subtle roles during development. In the third part, I investigate the function of the homolog of the Grainy head transcription factor in the fungus Neurospora crassa. In all animal model organisms in which they have been studied, Grainy head transcription factors play a conserved role in epidermal barrier formation and healing. I therefore thought it would be interesting to investigate the function of this transcription factor in fungi, organisms which lack an epidermis. Using microarray and phenotypic analyses I uncovered evidence that the Grainy head homolog in Neurospora plays a role in cell-wall formation, defense, and virulence. This points to an interesting connection between transcriptional control of physical-barrier formation in animals, and physical-barrier formation, defense, and virulence in fungi