Michael Underhill

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Elucidation of the fate and function of mesenchymal progenitors in tissue renewal and regeneration (2019)

Adult tissues contain multiple stem and progenitor cells that are critical for their renewal and regeneration. Tissue resident stem/progenitor populations include mesenchymal progenitors (MPs) whose function and fate are unclear. These studies have been hampered by the lack of suitable solitary markers of these cells which would enable both lineage tracing and functional analyses of MPs. Using a discovery-based approach, the gene Hypermethylated in cancer 1 (Hic1) has been identified as such a marker. Subsequently, two novel knock-in alleles of Hic1 were generated and characterized as part of this project and these analyses showed that Hic1 is restricted to quiescent MPs in muscle and other tissues. Single cell RNA-seq was employed to examine Hic1⁺ cells in muscle and this led to the identification of 3 predominant MP subpopulations with distinct function(s) and lineage potential. Further analysis in muscle injury models revealed that these cells exhibit diverse stage-specific activities, which coordinate multiple aspects of the regenerative process. Following regeneration, Hic1⁺ progeny contribute to several mesenchymal derivatives including Col22a1-expressing cells in the myotendinous junction.In numerous tissues, MPs have been found to play a vital role in stem cell niches. Single cell-seq was employed to characterize MPs across tissues, and these analyses revealed extensive intra and inter-tissue heterogeneity. Within some tissues, unique populations could be identified that appeared based on gene expression to have niche-like properties. This was very evident in the populations characterized from bone marrow (BM), which contribute to the hematopoietic stem cell (HSC) niche. Deletion of Hic1 led to widespread MP hyperplasia, including in the BM. Interestingly, this led to a 2-fold increase in HSC number. Taken together, these results suggest that the Hic1⁺ MPs contribute to the HSC niche and that MP frequency regulates HSC niche capacity. In summary, these findings identify Hic1 as a marker of MPs, and resulting genetic tools have been instrumental in defining MP subpopulations that exhibit transient and enduring roles in regeneration and non-cell autonomous activity in the HSC niche. This provides a solid foundation for understanding MP biology and their utility in cell-based and/or in situ modification to affect health and disease.

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The Molecular Basis of SS18-SSX Action in Synovial Sarcoma (2014)

No abstract available.

The Role of Pannexins, a Novel Gap Junction Family, in Glioma Oncogenicity and Neuronal Differentiation (2010)

Gap junctions are unique intercellular channels assembled from the canonical gap junction family, connexins (Cxs). These channels connect the cytosols of adjacent cells, allowing direct passages of small ions and molecules for intercellular communication and homeostasis within tissues. A novel family of gap junction proteins, pannexins (Panxs), with low sequence similarity to the invertebrate gap junctions, innexins, was recently discovered in chordates. Similar to Cxs, Panxs are also capable of forming functional hemichannels as well as intercellular channels. Aberrations in gap junctions have been associated with abnormal CNS development and diseases including gliomas. The main purpose of this thesis was to determine if Panxs play a functional role under pathological and normal CNS conditions, each of which is represented by gliomas and neuronal differentiation, respectively. A loss of Panx expression was found in the C6 glioma cell line when compared to its normal counterparts, primary astrocytes. Restoring Panx1 and Panx2 expression in C6 glioma cells by stable transfection induced a dramatically flattened morphology, which is similar to the flat and polygonal shape of cultured astrocytes. Both Panx1 and Panx2 also significantly suppressed the neoplastic phenotype of C6 glioma cells, including in vitro monolayer growth, anchorage-independent growth, and in vivo tumorigenesis in immunodeficient mice. Interestingly, while Panx1 reduced cell motility in C6 glioma cells, Panx2 did not elicit a similar effect. Panx1 and Panx2 exhibited a distinct subcellular localization. Panx1 was detected at the plasma membrane and perinuclear regions, whereas Panx2 was only found in membrane-bound compartments within the cytosol, hence suggesting mechanistically different tumor-suppressive pathways employed by the two Panxs. Furthermore, it was determined that Panx1 and Panx3, but not Panx2, increased neurite numbers and further enhanced neurite outgrowth in PC12 cells during nerve growth factor-induced neuronal differentiation. In conclusion, findings from this thesis suggest a functional role of Panxs in normal and pathological conditions of the CNS, and merit critical future investigations to explore their underlying mechanisms and therapeutic implication in diseases.

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Elucidation of the chondrogenic program using a combination of biology and technology (2008)

No abstract available.

Master's Student Supervision (2010 - 2020)
Identification and lineage analysis of Hic1+ and Pdgfra+ presumptive mesenchymal progenitors in the adult murine kidney (2020)

The increase in kidney disease incidence and its associated comorbidities has become a global issue. Repeated bouts of acute insults or chronic injury can significantly compromise normal phsyiological functions of the kidney such as glomerular filtration. If left untreated, these issues may progress to end stage renal disease (ESRD) which oftentimes culminates in death. Because the kidney is composed of several hundred thousand functional units called nephrons, it is highly likely that normal physiological wear and tear does not significantly impact kidney function such that it would activate a reparative or regenerative process. However there is evidence which suggests that the kidney may resume normal function following isolated yet severe instances of acute kidney injury. This may involve the activity of kidney-resident mesenchymal progenitors (MPs). Thus far, renal progenitor populations and their coordinated interactions remain largely undefined. Previously, our lab has identified and characterized Hic1 as a possible marker of MPs that can regulate tissue regenerative processes in various tissues. Pdgfra has also been identified as a marker of MPs with similar capacities. Given this information, we were interested in examining the spacial localization of both these markers and comparing their incidence in young and mature adult murine kidney. Using a variety of mouse genetic models and fluorescence microscopy we found that Hic1⁺, Pdgfra⁺, as well as , Hic1⁺Pdgfra⁺ populations, were localized in the mesangial compartment and juxtaglomerular apparatus (JA) of glomeruli. Our quantitative analysis also revealed that there are significantly more Pdgfra⁺ MPs in mature adult mice when compared to young adult mice. We were interested in further parsing out the heterogeneity in the Hic1⁺ compartment. Bioinformatic analysis revealed 5 unique clusters from which we identified highly enriched and unique markers. Markers indicative of some of these different populations such as GATA3 and ADORA1 were used to identify these poptulations in situ. Both of these markers labelled cells within the mesangial compartments and JA.The results from this study yield important insights into the nature of tissue-resident MPs in the kidney, thereby providing a foundation to support their study in kidney renewal and regeneration.

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Generation and characterization of a novel mouse model of synovial sarcoma (2019)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Characterization and identification of hepatic mesenchymal stromal cell populations (2018)

Mesenchymal stromal or stem cells (MSCs) play central roles in numerous physiological processes including tissue development, regeneration and homeostasis. In this study, the homeostatic properties of hepatic MSC’s were characterized by next generation sequencing (NGS) technology at single cell resolution. Hic1 (Hypermethylated in Cancer 1) was found to be a systemic marker of MSCs, and mouse models incorporating a CreERT2 knock-in into this locus were used to genetically trace and characterize labeled hepatic MSCs (HMSCs). Three hepatic mesenchymal cell types were marked by Hic1-expression, the vitamin A storing hepatic stellate cells (HSC), portal fibroblasts (PF) and the hepatic capsule lining mesothelial cell (MC). These HMSCs were known to contribute to regeneration and renewal, and herein data is provided to show that Hic1-expressing cells proliferate in response to liver injury and acquire a pro-regenerative myofibroblastic-like phenotype. Additionally, analysis of Hic1-lineage marked cells from liver with single cell RNA-seq showed that the liver sinusoidal endothelial cell (LSEC) compartment also appeared to be labelled. Hic1-expression within LSECs remains inconclusive and requires further investigation. No marker to date has captured the full spectrum of HMSCs making Hic1 a unique candidate to study the liver stroma.

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Tracing the cellular origins of mesenchymal derivatives in craniofacial development (2018)

Objectives: Numerous genes have been shown to impact craniofacial development. One such gene, Hypermethylated in cancer 1 (Hic1), is a tumour suppressor gene that encodes for the transcription factor HIC1. Hic1 is broadly expressed within embryonic mesenchyme. Hic1 knockout mice demonstrate multiple craniofacial anomalies, including facial clefting, abnormal closure/truncation of the secondary palate, low-set/underdeveloped ears, abnormal eye development, shortened snouts, acrania, and exencephaly, suggesting a role for Hic1 in craniofacial development. Molecular mechanisms underlying the role of Hic1 in craniofacial development remain largely unknown. Our objective was to elucidate the cellular fate of Hic1⁺ cells during craniofacial development. Methods: The fate of Hic1-expressing cells in craniofacial development was studied using lineage tracing analysis. A Hic1CreERT² mouse, in combination with a Cre-dependent reporter mouse, was used to label and follow Hic1-expressing cells. Reporter gene expression was induced at the onset of Hic1 expression (embryonic age (E) of 10 days). Embryos were collected at E11.5, E13.5, E14.5, E16.5 and E18.5. The distribution and fate of reporter-expressing cells was determined using epi-fluorescence imaging. Micromass cultures of cells derived from the mandibular, frontonasal and maxillary prominences at E11.5 were used to study Hic1-expressing cells in cartilage formation. Samples (n=3) were fixed and stained with Alcian blue or COL2A1 to study the relationship of Hic1⁺ cells to cartilaginous nodules. Single-cell RNA sequencing was performed on cells positive and negative for Hic1-expression in the head. Results: Hic1+ cells contribute to the mesenchyme within facial/masticatory muscles, tendon, tongue, meninges, nasal cartilage/ connective tissue, eyes, salivary gland stroma, teeth (dental papilla/pulp), and to populations of perineural and perivascular cells, including those within the brain. Hic1 is not seen within the palatal shelves, or Meckel’s cartilage. In micromass cultures of facial mesenchyme, most Hic1-expressing cells contribute to the area surrounding cartilaginous nodules, while few were incorporated within the nodules, mimicking the pattern of distribution of Hic1-expressing cells in the nasal structures. Single-cell RNA sequencing suggests Hic1-expressing cells are neural crest-like, and represent mesenchymal progenitors. Conclusions: Hic1-expressing cells contribute to various mesenchymal tissues during craniofacial development, and likely represent a population of cranial neural crest-derived cells or similar.

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Analysis of Hic1-expressing cells in the murine pancreas and their role in tissue regulation and regeneration (2014)

Mesenchymal stromal cells (MSC) play fundamental roles in tissue development, homeostasis and regeneration and are associated with many pathological conditions. Previous studies analyzing MSCs in skeletal development identified a gene (Hypermethylated in Cancer 1, Hic1) that appears to be solely expressed within embryonic mesenchyme. Analysis of a novel Hic1nLacZ/⁺ mouse line revealed that Hic1 is restricted to interstitial stromal and perivascular cells. In pancreas, Hic1⁺ cells overlap extensively with Pdgfrα and Sca1, which together identify putative MSCs in various tissues. These cells also co-express several markers of Pancreatic Stellate cells (PSC), including vimentin, desmin, nestin and neural/glial antigen 2 (NG2). PSCs are considered effector cells in many pancreatic diseases including fibrosis and cancer, and become activated under these conditions. Using a novel fate-tracking Hic1CreERT² knock-in mouse, we have seen that under homeostatic conditions these cells are capable of some turnover, the labeled cells doubling in number over a 3-month period following induction of the reporter gene in adult 8-week old mice. After caerulein-induced pancreatic damage, the cells become activated, attaining smooth muscle actin α (αSMA) and Collagen-GFP expression, and incorporating EdU, with cells amassing in the damaged areas. Consistent with a potential role for Hic1 in regulating MSC quiescence, conditional deletion of Hic1 in the adult pancreas leads to significantly increased numbers of perilipin⁺ adipocytes in comparison to controls, and an increased number of nLacZ⁺ cells. CD45- CD31- Sca1⁺ cells proliferate at a higher rate after Hic1-deletion. Mouse weight and blood glucose are unaffected. Functional studies into the role of Hic1 have found that Hic1-deletion resulted in a significantly less damage after caerulein-induced damage. This work has identified Hic1 as a novel marker of PSCs and set the stage for a greater understanding of the role of these mesenchymal stromal cells (MSCs) in pancreatic diseases.

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A chemical genetics strategy exposes novel modulators of chondrogenesis that act by blocking a potassium channel, Kcnd2, & reveals a potential role for potassium channels in limb development (2013)

Much of the vertebrate skeleton is formed through endochondral ossification. In this process, a chondrogenic template is laid down, which is subsequently replaced by bone. The first step involves condensation of mesenchymal cells and their differentiation into chondroblasts that initiate elaboration of the chondrogenic template. At later stages, chondrocytes undergo hypertrophy, and produce a matrix for bone formation. To enhance our understanding of molecular programs regulating this process a chemical genetics approach was employed. Our strategies involved the development of screens using primary cultures of murine limb bud-derived mesenchymal (PLM) cells. Chondroblast differentiation is associated with increased SOX5, 6 and 9 activity; while hypertrophic differentiation is associated with reduced SOX5, 6 and 9 activity. Therefore, a SOX5/6/9-responsive reporter gene was used to follow expression of the chondroblast phenotype. Compound libraries representing more than 1400 compounds were screened; 28 compounds were found to increase reporter gene activity greater than 2.5 fold. In secondary screens, 7 of 28 positive compounds stimulated cartilage formation, as assessed by alcian blue staining. Two compounds identified, Butamben (butyl 4-aminobenzoate; BAB) and Phenazopyridine hydrochloride (PHCl), exhibited strong pro-chondrogenic activity and morphologically similar alcian blue staining. BAB is a member of the benzocaine family of analgesics and functions by inhibiting sodium channel activity. However, BAB has also been shown to have potassium channel-blocking activity. Specifically, BAB inhibits the activity of Kcnd2; which through transcriptional profiling was also found to be down-regulated by bone morphogenetic protein-4 (BMP4). We speculated BAB and PHCl may be able to modulate chondrogenesis by acting on potassium channels. To confirm this idea we examined molecular activities of PLM cultures treated with BAB and PHCl at two stages of chondrogenesis: 1. pre-chondrocyte to chondroblast and 2. chondrocyte to hypertrophic chondrocyte. Results confirm, BAB and PHCl increase expression of chondrogenic markers and reduce expression of hypertrophic markers. In addition patch clamp analysis revealed both BAB and PHCl are able to block, at least partially, KCND2 channel activity. We confirmed the dynamic expression pattern of Kcnd2 by qPCR and radioactive section in situ hybridization. Together, these results reveal an unanticipated and novel role for Kcnd2 in chondrogenesis.

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