Date Thesis Awarded

5-2018

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Margaret S. Saha

Committee Members

Randolph A. Coleman

Shantá D. Hinton

Joshua R. Puzey

Abstract

Embryonic development is a robust process during which embryos must respond and compensate for changes in order to achieve consistent patterning; however, there are still questions about the limits and mechanisms of this robustness. Using tetraploid Xenopus laevis as a model, we have previously shown that embryos respond to perturbations of the highly-conserved Notch signaling pathway in a compensatory manner. We have now demonstrated that this response involves changes in the proliferative status of neural progenitors and differentiated neurons over time. Subsequent RNA-seq analysis of Notch perturbed X. laevis embryos revealed that homeologs (duplicated genes originating from whole-genome duplication) respond differentially to this perturbation, suggesting that the polyploidy of X. laevis may contribute to the compensatory abilities. To address this question, we have perturbed Notch signaling in X. borealis, a tetraploid species that is closely related to X. laevis, and characterized the response over time. Similarly to X. laevis, a compensatory response is seen in X. borealis over time based on gene expression in the developing nervous system, but embryos appear morphologically deformed throughout development. This suggests that X. borealis embryos may be more severely affected by this perturbation and do not compensate as well as X. laevis. RNA-seq was performed on Notch perturbed X. borealis embryos to quantitatively and globally assess the transcriptional response over time. Given that there was previously no reference genome or transcriptome for X. borealis, a de novo assembly of the X. borealis transcriptome was constructed to allow for further analysis. Using the X. laevis genome as a reference has allowed for comparative analysis of the changes in homeolog expression in X. laevis and X. borealis embryos following Notch perturbation. These data have revealed differences in the response to Notch perturbation between X. borealis and X. laevis, with X. borealis generally having more differentially expressed genes when compared to X. laevis under the same condition, again suggesting that X. borealis is more severely perturbed and does not compensate as well as X. laevis. To validate and compliment these RNA-seq results, it would be ideal to visualize homeolog expression in situ; however, given the high degree of sequence homology between homeologs, detection of specific homeolog transcripts in situ has presented a challenge for traditional methods. Using two new in situ hybridization technologies (Molecular Instruments Hybridization Chain Reaction v3.0 and Advanced Cell Diagnostics BaseScopeTM assay), we have been able to visualize X. laevis homeolog expression in situ with extreme specificity, which will enable spatial analysis of homeolog expression and a tool to validate RNA-seq findings. In the future, it will be interesting compare the transcriptional response to Notch signaling perturbation across ploidy levels, in addition to within multiple tetraploid species as we have already done. To enable this type of comparative experiment, we have sequenced and assembled the transcriptome of Xenopus andrei, an octoploid frog, representing, to the best of our knowledge, the first publicly available assembled transcriptome of an octoploid vertebrate.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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