The functional impacts of olfactory rosette variation in elasmobranchs is unresolved. Our goal was to quantify rosette morphology and shape from 14 species using dissections, phylogenetic comparisons, and microCT imaging. We hypothesized that lamellar count and rosette shape (fineness ratio) would not scale with animal size, but internal rosette size variables must scale positively. We found that fineness ratio and lamellar counts varied significantly among species, and were positively correlated. The first two principal components of the pPCA explained 82% of the variation, with fineness ratio and lamellar count contributing the most. There were no significant differences between rosette structure or volume when comparing dissected values to in situ values obtained using diceCT. Based on our results, we hypothesize that variations in rosette shape and morphology will impact hydrodynamics and optimize odorant detection, and these data can be used to create 3D models for future hydrodynamic studies. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

A functional sense of smell is mediated by Olfactory Receptor proteins (ORs), which reside in olfactory sensory neurons (OSNs) in the epithelium of our nose. Only a singular OR allele out of roughly 2,400 other OR alleles is expressed in every OSN⁽¹˒ ²⁾. Singular expression of the active OR gene occurs in a unique transcriptional hub⁽³⁻⁵⁾. This hub contains one OR promoter and many interchromosomal enhancers that converge upon the hub from far nuclear distances⁽⁵˒ ⁶⁾. Once in the hub, the enhancers work in tandem with each other and with the transcription factors (TFs) Lhx2, EBF, and their cofactor, Ldb1⁽⁵˒ ⁷˒ ⁸⁾ The Greek islands contain a novel “composite” motif containing an Lhx2 and EBF binding site directly next to each other⁽⁸⁾. My work aims to understand how these proteins interact with each other and the composite motif to contribute to the accumulation of many enhancers around only a single promoter in the hub. Our findings illuminate how transcription factor interactions contribute to the hub's unique DNA architecture. To investigate the biochemical foundation of OR hubs, we used 𝑒. 𝑐𝑜𝑙𝑖 to grow and purify full-length and truncated forms of Lhx2, Ebf1, and Ldb1. We used the recombinant proteins with other biochemical methods to characterize the interactions between Lhx2, Ebf1, Ldb1, and different types of DNA found in the OR hub. We used EMSAs to quantify the binding affinity that Lhx2 and Ebf1 have for promoter versus enhancer DNA. Finally, we mixed the purified full-length proteins and used fluorescence microscopy to visualize their behavior in solution. This research combined with in vivo imaging in the Lomvardas lab provides a better understanding as to how molecular interactions 𝑖𝑛 𝑣𝑖𝑡𝑟𝑜 contribute to the hub’s architecture 𝑖𝑛 𝑣𝑖𝑣𝑜, and ultimately, stable OR expression. Our biochemical studies suggest that Lhx2 and Ebf1 can bind at the same time to a single composite motif yet they seem to bind independently of one another. We have used EMSAs to determine that Lhx2 binds much better to OR enhancer DNA compared to Ebf1 and that it might stabilize enhancer contacts. We have also found that Lhx2 and Ebf1 do not cooperatively bind to enhancers- indicating that affinity alone does not explain the accumulation of TFs to the OR hub. Our 𝑖𝑛 𝑣𝑖𝑡𝑟𝑜 imaging shows that Lhx2, Ebf1, and Ldb1 self-assemble into rigid nucleoprotein condensates driven by the composite motif of enhancer DNA. This imaging work also reveals that Lhx2 and Ldb1 are scaffolding proteins with low mobility which drive rigid condensate formation over enhancers. Ebf1 displays more plasticity and turnover into condensates indicating that it plays a more complex role as a recruited factor to these assemblies. We have coupled this data with 𝑖𝑛 𝑣𝑖𝑣𝑜 imaging of endogenous Lhx2, Ebf1, and Ldb1 to find that these factors display similar binding and dynamics 𝑖𝑛 𝑣𝑖𝑣𝑜. This data helps to provide a biophysical model of how OR hubs support multi-enhancer and protein-rich environments that are succinct from their surrounding environment. Our studies suggest that the OR hub forms a rigid phase separated compartment in the nucleus- driven by Lhx2 and Ldb1. This solid-like phase separation may contribute to how singular OR expression is achieved. Such long-range enhancer contacts must stay assembled long-term for continuous OR transcription. Therefore, traditional TF DNA binding dynamics would not explain the longevity of these contacts in the OR hub. This work challenges the traditional model of liquid phase separated nuclear compartments and may provide a broader understanding to how long range and inter-chromosomal compartments are maintained.