Michael Underhill
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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Tissue development and regeneration rely on the cooperation of multiple mesenchymal progenitor (MP) subpopulations. Recently, the gene Hypermethylated in cancer 1 ( Hic1) has been identified as a marker of quiescent MPs in multiple adult tissues. Hic1⁺ MPs have since been implicated in the repair and regeneration of several organs such as skeletal muscle (SkM), skin, heart, and brain. While the roles that Hic1⁺ MPs play in tissue maintenance are currently being elucidated, knowledge surrounding their developmental origin and fate is lacking. Using the developing forelimb as a model system, we describe the embryonic origin of Hic1⁺ MPs and demonstrate that they first appear in the developing limb ~48 hours after limb bud initiation at embryonic day (E) 11.5 and represent a rare subset of the total mesenchymal cell population. Time-resolved single cell-omics analyses coupled with lineage tracing reveal that Hic1⁺ cells generate a unique MP hierarchy, that includes both recently identified adult universal fibroblast populations (Dpt⁺, Pi16⁺ and Dpt⁺, Col15a1⁺) and more specialized mesenchymal derivatives such as, peri- and endo-neural cells, pericytes, bone marrow stromal cells (BMSCs), myotenocytes, tenocytes, and fascia-resident fibroblasts, with limited contributions to chondrocytes and osteocytes within the skeletal elements. We further demonstrate that, in the adult organism, MPs endure within these compartments, continue to express Hic1 and represent a critical reservoir to support post-natal growth and regeneration. Within the appendicular skeleton, Hic1⁺ descendants do not contribute to the primary skeletal anlagen but represent the elusive MP population that generate progeny which connect bone to tendon (entheses) and tendon to muscle (myotendinous junction). Furthermore, deletion of Hic1 leads to skeletal defects reflective of deficient muscle-bone coupling. Collectively, these findings demonstrate that Hic1 identifies a unique MP population that contributes to multiple cell lineages during limb development. Furthermore, Hic1⁺ MPs are crucial for appendicular skeletogenesis as their progeny primarily constitute several forelimb tendon-bone and tendon-muscle attachment regions. As parallels between tissue regeneration and embryonic development are being increasingly made, understanding the origin and fate of heterogenous tissue-resident MPs will support efforts aimed at promoting regeneration in mature tissues.
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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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No abstract available.
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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No abstract available.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Hypermethylated in Cancer 1 (Hic1) is a gene that marks a population of cells known as mesenchymal progenitors (MPs) in humans as well as mice. MPs can be found in almost all adult tissues, where they support repair and regeneration processes after injury. Hic1 regulates MP quiescence, a state where cells are growth arrested, but are poised to enter the cell cycle following activation with various stimuli (i.e., injury). When Hic1 is deleted, MPs exit quiescence and exhibit a mildly activated state, which is associated with an accumulation of MPs and their progeny. These conditions also provide an environment conducive for the development of various cancers and fibrosis. Both diseases are significant contributors to global mortality and morbidity, and treatment options are limited. This project investigated mechanisms for modifying MP activity, in part, through inducing quiescence. Using Hic1 expression as a surrogate for quiescence, an assay was developed to measure Hic1 transcript abundance. The assay relies on the use of cells from a genetically engineered mouse model, in which a nuclear LacZ gene was knocked-in to the Hic1 locus. In this manner, Hic1 expression and by extension quiescence, can be followed by measuring LacZ activity. CPRG is a chromogenic substrate of LacZ and was used to measure the abundance of LacZ after treating Hic1ⁿᴸªᶜᶻ/⁺ mouse embryonic fibroblasts (within the MP umbrella) with a library of drugs that passed at least Phase I of FDA clinical trials. High-throughput techniques such as robotic cell seeding and 96-well parallel drug administration by pinning tool were used to efficiently work through the over ~ 4,000 drugs. The screen identified various retinoids as hits, which was expected due to the direct targeting of Hic1 by retinoic acid (RA) receptors (Rs), and no non-retinoid compounds resulted in significant increases in Hic1 expression. Screen results were further investigated through a high content X-gal and nucleic acid stain screen, as well as through RNA sequencing. Secondary screening indicated that hit compounds were acting through modulating RAR signaling. Collectively, these findings reinforce the concept that MP quiescence is predominantly regulated by RAR-mediated signaling.
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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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In the choroid plexus (CP) and pituitary gland (PG), blood vessels are known to have a permeable phenotype that allows for the free passage of molecules in contrast to the blood brain barrier that is found in the rest of the CNS. While the endothelium and secretory cells (CP epithelium and PG endocrine cells) of these compartments have been studied in detail, fewer studies have examined the mesenchymal progenitors (MPs, also called pericytes, fibroblasts and mural cells) in these tissues. In this study, the Hic1CreERT2/CreERT2; Rosa26LSL-tdTomato/⁺ (Hic1-tdTom) lineage tracing mouse model was used to examine MPs within the CP and PG. Single-cell RNA-sequencing and reporter gene detection identified and validated distinct patterns of gene expression. We found that permeable brain regions possess substantial populations of distinct Hic1⁺ mesenchymal progenitors that make specific contributions to CP, PG and the meninges by expressing extracellular matrix proteins such as collagen 1 and metabolic enzymes such as Indole-n-methyltransferase. Multiple unique markers of mesenchymal cells in the CP and PG were identified, including ALPL and CD34. In sum, this study has identified and characterized novel populations of Hic1⁺ mesenchymal cells specific to permeable neurovasculature and the meninges.
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Medulloblastoma (MB) is the most prevalent malignant childhood brain tumor which arises in the cerebellum. One molecular subgroup of MB is characterized by mutations in genes in the sonic hedgehog (SHH) signaling pathway, including the receptor Patched 1 (PTCH1). Constitutive activation of the SHH pathway in cerebellar granule cell precursors causes proliferation and cell differentiation failure, leading to tumor formation. While the molecular pathways driving tumor formation are well understood in this subgroup, the MB tumor microenvironment (TME) remains understudied. Mesenchymal progenitors (MPs) are a population of stem-like cells that have been identified as a prominent stromal cell type in a range of primary tumors. MPs are recruited to the TME and contribute to tumor growth and progression through various mechanisms. Previous work by our lab has shown that quiescent MPs are marked by the expression of Hypermethylated in cancer 1 (Hic1). To better understand the role of brain MPs in MB, we utilized an inducible Cre recombinase reporter mouse line to study the contribution of Hic1⁺ MPs to the TME in the Ptch1+/- mouse model of MB. Cerebellar and MB tumor-associated Hic1⁺ MPs were characterized by histological analyses, cell proliferation assays and single-cell RNA sequencing. Lineage tracing revealed that Hic1⁺ MPs are a stable cell population in the cerebellum and contribute dynamically to the MB TME. The large majority of cerebellar and tumor-associated MPs were perivascular and expressed pericyte-associated markers, such as Pdgfrb, Rgs5 and Cspg4. The proliferation rate of tumor-associated MPs was markedly increased compared to cerebellar MPs, indicative of their activation in the MB TME. Single-cell RNA sequencing showed that tumor-associated MPs displayed an altered transcriptomic profile, consistent with an activated phenotype. Furthermore, tumor-associated MPs exhibited upregulation of genes involved in angiogenesis, extracellular matrix reorganization, cell growth and immunomodulation. Overall, Hic1⁺ MPs contribute substantially to the MB tumor stroma and likely provide a microenvironment supportive of tumor growth. A deeper understanding of the role of MPs in the TME has relevance for the development of more targeted treatments for MB, as MPs can potentially be manipulated to slow tumor progression and/or metastasis.
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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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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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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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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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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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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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