We developed a method for fluorescence in situ identification of individual mRNA molecules, allowing quantitative and accurate measurement, in single cells, of allele-specific transcripts that differ by only a few nucleotides. By using a combination of allele-specific and non-allele-specific probe libraries, we achieved >95% detection accuracy. We used this technique to investigate the allele-specific stochastic expression of Nanog, which encodes a pluripotency factor, in murine embryonic stem cells. We find that Nanog does not switch between monoallelic and biallelic expression when culture conditions are altered.
We next worked towards adapting our allele-specific single molecule mRNA fluorescent in situ hybridization technique to detect early expression of the immunoglobulin kappa gene in Pre-B cells. Mature B cells only express a single allele of the immunoglobulin kappa gene, and assaying allele-specific expression in single cells will allow the study of the mechanism behind this choice.
We also developed a theoretical model of cell specification in the mammalian inner ear using single-molecule mRNA expression data. During mammalian hair cell development, prosensory cells acquire a spatial pattern of distinct cellular fates. This process is dependent upon the expression of the transcription factor Atoh1, and is mediated by Notch signaling between neighboring cells. We find that both the Notch ligand and transcription factor Atoh1 are expressed in an extended region before turning off in non-hair cells. Our model reveals that this extended pattern creates a system that can suppress extraneous expression over a large region and is robust to movement of prosensory cells as the cochlea extends, especially in the case of a limited time window for specification. Our model can also explain the two types of expression patterns of Atoh1 that are observed.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/13064969 |
| Date | January 2014 |
| Creators | Hansen, Clinton Hugh |
| Contributors | van Oudenaarden, Alexander |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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