Single-Cell Transcriptome Analysis of Olfactory Sensory Neurons

Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.

In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Transcriptome and Functional Analysis of Carotid Body Glomus Cells

The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited.

To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response.

Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.