Browsing by Author "Huang, Peng"

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    Characterization of Muscle-Associated Cells in Adult Zebrafish
    (2019-12-20) Ruel, Tyler David; Huang, Peng; Childs, Sarah J.; Biernaskie, Jeff A.
    Skeletal muscles make up 40% of body weight in humans. Any compromises in muscle function will cause major consequences to the quality of a person’s life. It is therefore extremely important that this tissue is maintained in a state of homeostasis. To do this, muscle fibres that become damaged must be repaired by tissue-resident muscle-stem cells throughout the life of an animal. Several different kinds of muscle-associated cells have been described, including the two main populations: satellite cells (a population of muscle stem cells) and fibro/adipogenic progenitors (FAPs) (a population of mesenchymal stem cells). Using zebrafish as a model, the importance of muscle-associated cells in maintaining muscle homeostasis is demonstrated. Our lab has previously generated a col1a2¬-based transgenic line that labels collagen-expressing cells in zebrafish. Using a combination of immunohistochemistry and confocal microscopy, I characterize the dynamics and function of col1a2+ muscle-associated cells. A developmental time course shows that col1a2+ intramuscular cells increase in numbers during juvenile stages. In response to muscle injury, col1a2+ muscle-associated cells are expanded and contribute to muscle regeneration. Genetic ablation of col1a2+ cells, results in a compromised regenerative response. Using Cre-mediated lineage tracing, the developmental origin of intramuscular cells is traced to the dermomyotome and sclerotome, two sub-compartments of the embryonic somite. Finally, characterization of a col1a2 mutant line of zebrafish suggests that Type-I collagen is important for maintaining muscle integrity. These observations suggest the importance of col1a2+ muscle-associated cells in maintaining muscle homeostasis and for producing the extracellular matrix (ECM) within the skeletal muscle tissue to prevent degeneration.
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    Cis-regulatory control of the human short stature homeobox gene in the developing limb
    (2022-01) Skuplik, Isabella; Cobb, John; Hansen, David; Kurrasch, Deborah; Huang, Peng; Menke, Douglas
    Disruption of the human short stature homeobox (SHOX) gene causes shortening of the middle limb segment (zeugopod), affecting up to 1:1000 individuals. SHOX deficiencies are caused by coding lesions, or by the deletion of non-coding sequences surrounding the gene. Several deletion intervals downstream of SHOX are predicted to disrupt enhancers that activate transcription during limb development. However, the precise locations and activities of these enhancers remain to be identified. In this work, we investigated the cis-regulatory landscape of the human SHOX gene. We systematically screened a recurrent 47.5 kb short-stature deletion interval downstream of SHOX for the presence of limb enhancers. Human genomic sequences were placed upstream of a lacZ reporter gene and tested for their ability to activate expression in transgenic mice. This revealed the presence of a zeugopodal enhancer downstream of SHOX (the ZED). Using primary cell luciferase assays, we further delineated the minimal active sequence and identified putative HOX9/11 binding sites required for its activity. Next, we developed the domestic cat as an emerging model to characterize enhancer and gene interactions at the endogenous locus. Rodent genomes lack the SHOX gene, while other commonly used laboratory models lack conservation of the ZED and other non-coding sequences at the SHOX locus. We demonstrated that cats are an effective model to identify enhancers. Cat genomes display a synteny of genes and conserved non-coding elements in the pseudoautosomal region 1 where SHOX is located. Using whole-mount in situ hybridization, we validated the expression of SHOX in the zeugopod of cat embryos. Next, we employed the circular chromosome conformation capture (4C) technique to identify SHOX cis-interacting sequences in embryonic limb tissue. Human orthologous sequences were identified through sequence conservation; and human short-stature deletions and enhancer chromatin signatures were used to delineate enhancer candidates. Finally, we confirmed the limb enhancer activity of three human and cat sequences in transgenic mice. Our findings provide a potential explanation for the pathogenicity of certain non-coding deletions downstream of SHOX. This work also uncovers similarities in the regulation of Shox genes (Shox and Shox2) and the development of limbs in cats, humans, and mice.
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    Csde1, an RNA Binding Protein, Modulates Neuronal Subtype Specification
    (2021-09-20) Harvey, Emily M.; Yang, Guang; Huang, Peng; Kurrasch, Deborah
    Neuronal diversity is the root of complex function in the cerebral cortex. During cortical development, neural stem cells give rise to neurons. Neurons are a diverse cell type and can be grouped into many subtypes, each with distinct functional identities. Neurogenesis and neuronal subtype specification are carefully regulated, both temporally and spatially, by programmed gene expression. One mechanism controlling gene expression is translational regulation, which alters levels of protein synthesis. Translational regulation orchestrated by RNA binding proteins allows dynamic alterations in protein expression. Here I determine that Cold-shock domain containing E1 (Csde1), an RNA binding protein, regulates the specification of neuronal subtypes in the cerebral cortex. In the developing murine cortex, Csde1 is highly expressed in newborn neurons of the cortical plate during development. I show that reduced Csde1 expression alters neuronal subtype in the embryonic and post-natal cortex. Csde1 reduction disrupts the distribution of neuronal subtypes within the cortical plate. Additionally, reduced Csde1 expression increases mixed neuronal subtype identity. Together, this indicates that the specification of neuronal subtypes is subject to translational regulation, and that Csde1 activity is essential for specification of neurons in the cortex.
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    The Derlin protein CUP-2’s role in regulating the Caenorhabditis elegans germline stem cell niche, the Distal Tip Cell
    (2021-08-19) McMillan, Lauren; Hansen, David; Huang, Peng; Cobb, John
    The balance between stem cell proliferation and differentiation is important for growth and development in various multicellular organisms. This balance is highly regulated, and it requires an interaction between the niche and the stem cell pool in order to be upheld. The presence of a stem cell niche is highly conserved; however, depending on the organism and system they can act through a variety of signaling pathways to maintain the stem cell pool. In C. elegans, the somatic distal tip cell (DTC) acts as the germline stem cell niche by providing cues via GLP-1/Notch signalling. We have identified that the Derlin protein DER-1 (CUP-2) plays a role in regulating the DTC. Normally, the DTC is located at the distal end of the gonad. As worms age, it can move several cell diameters away from the distal most end (Kocsisova et al., 2019). However, as cup-2 mutant worms age, the DTC moves significantly further down the gonad arm than what is seen with wild type. In some more extreme and rare cases where the DTC has moved at least 10 cells from the distal end, the stem cell pool moves along with the DTC. The stem cells are still able to proliferate and differentiate at this new location, which suggests the DTC remains functional at the new location. In this thesis, cup-2’s role in holding the DTC in place has been investigated. First, the displacement and two transition zone phenotypes have been characterized further. Additionally, male strains were investigated to determine whether or not the DTC displacement phenotype is sexually dimorphic. Next, I determined where cup-2 is expressed and required such that the DTC will remain in place. Also, I explored cup-2’s known functions, endoplasmic reticulum associated degradation (ERAD) and endocytosis, to determine if they play a role in the placement of the DTC. Finally, I investigated components involved in the surrounding basement membrane to determine if cup-2 may play a novel role in regulating the basement membrane. This investigation of CUP-2 and the DTC may help us understand how the DTC and potentially other niches are held in place.
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    Development of Rasa1 arteriovenous malformations
    (2022-12-22) Greysson-Wong, Jasper; Childs, Sarah; Brook, William; Huang, Peng; Kurek, Kyle; Fish, Jason
    Precise regulation of signalling is critical to ensure proper formation of blood vessel networks during development. Mutations in key genes may disturb this finely tuned process, resulting in malformed vessels. Mutations in RASA1, a Ras GTPase activating protein, leads to the development of arteriovenous malformations (AVMs), where arteries are connected directly to veins without interceding capillary beds. Using zebrafish, I show that rasa1 mutants also develop AVMs in their tail vasculature, which subsume the posterior of the dorsal aorta and caudal venous plexus. The development of rasa1 AVMs does not depend on flow and modulating flow does not prevent or rescue AVM formation. However, blood flow slows in the cavernous AVM, changing flow-responsive signalling reducing expression of flow responsive transcription factor klf2a. Incomplete remodelling of the caudal venous plexus is visualized by an excess of residual intraluminal pillars, an indication of impaired intussusceptive angiogenesis. I show that AVM development is dependant on venous activation of MEK/ERK signalling and that inhibition of MEK signalling can prevent AVM development during an early developmental window, but MEK inhibition cannot rescue an AVM that has already formed. For the first time, I show that Bmp and Vegf signalling both play a role in AVM development, pathways that are important in other vascular malformations but had yet to be explored in RASA1 models.
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    Elucidating the role of tcf15 in somite development
    (2024-06-25) Lim, Nicholas Farn Wei; Huang, Peng; Childs, Sarah J.; Cobb, John; Chu, Li-Fang
    Somites are transient embryonic structures that give rise to the axial musculoskeletal system. Eventually, the somite gives rise to three distinct compartments: the dermatome, myotome, and sclerotome. These compartments give rise to the skin, skeletal muscles, and the axial skeleton, respectively. Thus, proper formation of somitic compartment is necessary for a functional body. Despite many early studies elucidating the development of the somite, little is known about the regulator of somite compartment proportion. Here I characterized the basic helix-loop-helix transcription factor, tcf15, as a key regulator of somitic compartments in zebrafish. I showed that tcf15 expression is highly dynamic during somite development, with initial robust expression throughout the presomitic mesoderm, followed by restricted expression in the dermomyotome and ultimately in dermomyotome-derived muscle progenitors and in sclerotome-derived tenocytes along the somite boundary. Through mutant analysis, I found that loss of tcf15 results in a reduction in the pax7a+ dermomyotome and the myoD+ myotome compartment accompanied by an expansion of the nkx3.1+ sclerotome. Interestingly, migration of sclerotome cells and the development of sclerotome-derived cells such as tenocytes and fin mesenchymal cells were unaffected in tcf15 mutants. To manipulate Tcf15 activity, I developed various gain-of-function and loss-of function tools. However, the broad application of these tools was limited by technical challenges. Nonetheless, by combining the expression and mutant analysis, my results suggest that Tcf15 functions as an important regulator of somite compartmentalization in zebrafish, promoting the dermomyotome/myotome fate while repressing the sclerotome fate.
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    Fine-tuning blood vessel development
    (2020-09-11) Watterston, Charlene; Childs, Sarah J; Brook, William J; Huang, Peng; Bonni, Shirin; Yelon, Deborah
    Blood vessel development is typically characterized by stages marking the growth and gradual refinement of vascular networks. Understanding how these stages integrate is essential to our understanding of how the early signals which control vessel growth can influence later stages of vessel stabilization. In this thesis, I use a zebrafish model (Danio rerio) to explore the roles of two negative regulators that modulate key signaling pathways controlling vessel growth. At the early stages, the initial growth of vessels is carefully controlled by distinct gene expression patterns. As a vessel forms, in response to the attractive Vascular endothelial growth factor (Vegf) pathway, its sprouting is often opposed by repulsive Semaphorins (Semas) which limit directional growth. I investigated the role of semaphorin3fb (sema3fb) which I found to be expressed within developing endothelial cells of the zebrafish embryo. I found that sema3fb likely acts through auto-secretory feedback to modulate Vegf responses to promote appropriate vessel growth. At later stages, a supportive layer of vascular smooth muscle cells (vSMCs) is recruited to form the contractile layer of the vessel wall. Bone morphogenic protein (Bmp) signaling is implicated in cellular crosstalk from the underlying endothelium to vSMCs which is critical to the structural integrity of a blood vessel. I investigated the microRNA26a (miR26a), which I found enriched in the endothelial lining of the blood vessel. I identified a non-autonomous role for miR26a in regulating Bmp signaling through its effector Smad1 to control vSMC maturation. Together my work offers mechanistic insight into the cellular communication pathways that regulate blood vessel formation and focuses on how both internal and external signaling pathways communicate to promote vessel formation.
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    Identifying Novel Causes of Human Neuromuscular Disease
    (2016) Smith, Christopher; Parboosingh, Jillian; Innes, A. Micheil; Lamont, Ryan; Huang, Peng
    Neuromuscular diseases (NMDs) are a class of disorders that affect the peripheral nervous system or muscle. These conditions generally result in the loss of voluntary control of movement. They are often genetic, follow Mendelian inheritance, and to date more than 500 causative genes have been identified. Clinical and genetic heterogeneity make correct and specific diagnosis challenging. Identifying novel disease genes will likely improve diagnostic rates, especially as the use of genome scale sequencing techniques in clinical diagnostics increases. Exome sequencing is an extremely powerful tool in Mendelian disease gene discovery. We have leveraged the power of whole exome sequencing in a cohort of 16 individuals with disparate NMDs to: identify novel candidate disease genes (IARS, MSTO1, RAB11FIP2, RNMT), validate previously identified candidate disease genes (MYH14, TAF1, COQ7), propose expansions to the recognized clinical phenotypes of known genes (GLE1, DOK7), and in collaboration with clinical geneticists provide diagnosis to participant patients.
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    Investigating the mechanisms underlying age-related dysfunctions in skin and hair follicle regeneration
    (2020-05-29) Shin, Wisoo; Biernaskie, Jeff A.; Huang, Peng; Cross, James C.; Cobb, John A.
    Age-associated decline in overall skin function and impaired cutaneous wound healing are both direct consequences of the progressively weakening dermis and degeneration of necessary appendages such as glands, nerves and hair follicles (HFs). With a fresh perspective and access to new tools, I revisit aging skin phenotypes including hair loss and deficiency in wound healing from the perspective of the mesenchyme and its progenitors. In Chapter 2, I present a manuscript establishing the transcriptomic identities of bipotent hair follicle mesenchymal stem cells (hfDSCs) and its progeny dermal papilla (DP) via bulk RNA sequencing. Utilizing in vitro cell culture drug treatments, in vivo drug injections, and genetic deletion of Rspondin3 (Rspo3), my work define Rspo3 as an important modulator of epithelial-mesenchymal crosstalk in HF regeneration. In Chapter 3, I ask whether hfDSCs are lost with age and whether their dysfunction contributes to age-associated hair loss. Reporter mice experiments including long-term fate mapping and in vivo clonal analysis characterize the functional deficits of hfDSCs. Analysis of single-cell RNA sequencing (scRNAseq) data reveals that the driver of HF mesenchymal aging is senescence. In Chapter 4, I present on going work that describes the failure of aged mice to undergo wound induced hair neogenesis (WIHN). Using scRNAseq, I determine that mesenchymal fibroblasts in aged mice cannot acquire a regenerative phenotype after injury due to an overabundance of senescent fibroblasts. Senescent fibroblasts persist into late stages of wound healing, contributing to the loss of WIHN. In Chapter 5, I present a co-lead study investigating the impact of genomic instability on progenitor maintenance. I introduce a novel model of accelerated aging with skin deficiencies such as hair loss and hyper pigmentation. Genomic instability significantly impairs the HF regeneration cycle, and as a result, the HF degenerates in a process closely resembling natural HF aging. Characterizing the roles of aging dermal progenitors in skin deficiencies provides new insights into skin aging and aging stem cell research. Additionally, my work on defining functionally diverse fibroblast populations contribute to the growing appreciation for the importance of fibroblast heterogeneity in maintaining overall skin function.
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    Investigating the role of RACK-1 in the C. elegans germ line
    (2021-07-21) Vanden Broek, Kara Dawn; Hansen, Dave; Huang, Peng; Samuel, Marcus
    Stem cells are central to the development of multi-cellular organisms, including C. elegans and humans. Key to their function is their ability to differentiate into more specialized cells or proliferate to maintain their population for future use. Germline stem cells (GSCs) are a class of stem cells that are crucial for an organism’s reproductive success. GSCs proliferate to maintain the stem cell pool (self-renew) and differentiate to produce gametes (sperm/oocytes). A balance between proliferation and differentiation is necessary for proper germline function and the production of offspring throughout an organism’s life. This thesis uses the C. elegans hermaphrodite germline as an in vivo model to investigate the molecular mechanisms that regulate the proliferation/differentiation balance. In this thesis I have characterized RACK-1 as a modulator of the stem cell proliferation/differentiation balance. I found that RACK-1 is required for the proper activity of the conserved STAR family, RNA-binding protein, GLD-1/Quaking. GLD-1 is expressed throughout the cytoplasm in wildtype germlines, whereas in the absence of rack-1, GLD-1 levels are reduced, and GLD-1 becomes mislocalized to perinuclear aggregates (germ granules). This leads to a decrease, but not a complete loss, of GLD-1 activity. Specifically, a loss of rack-1 in combination with a loss of GLD-2 pathway function results in an over-proliferation phenotype. This phenocopies a partial reduction of gld-1 (gld-1(het)), but not a complete loss of gld-1, in the same genetic background. Additionally, loss of rack-1 rescues the proliferation defects in an fbf-1(0) fbf-2(0) mutant background similar to a reduction of gld-1 (gld-1(het)). Loss of rack-1 enhances the defective progression through meiosis phenotype associated with reduced GLD-1 activity. This data supports the model that rack-1 functions to modulate GLD-1 activity through controlling GLD-1’s subcellular localization and levels, thereby maintaining the proper proliferation/differentiation balance. This thesis reveals a novel mechanism that fine-tunes the activity of a key meiotic protein, GLD-1, to provide an additional layer of regulation in the proliferation/differentiation balance in the C. elegans germline
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    Notch signalling in spinal cord patterning: Crosstalk and fate decisions
    (2021-07-19) Jacobs, Craig Timothy; Huang, Peng; Hansen, David; Brook, William
    The spinal cord is a highly complex structure. During development, initially equivalent neural progenitors divide and give rise to a specific pattern of differentiated neurons. Understanding how this pattern arises would have far reaching impact, not just in developmental biology but also in the understanding of developmental disorders. Building on the decades of work from other groups, this thesis focuses on the extrinsic cues that guide pattern formation in the zebrafish spinal cord, with particular focus on the highly conserved Notch signalling pathway. The major, and most well studied, role of Notch signalling during neural development is progenitor maintenance. This thesis explores the additional roles of Notch signalling in signalling crosstalk and cell fate decisions. The action of Hedgehog signalling during spinal cord development is well characterised, providing the spatial information to drive the patterning of the ventral neural progenitors. In chapter two, I reveal a novel mechanism by which Notch signalling maintains Hedgehog response in spinal cord progenitors. This occurs at the level of the Gli family of downstream transcription factors, not at the primary cilia as previous reports suggest. This raises the interesting question of whether the Notch mediated control of Hh signalling is providing the instructive cues that guide fate determination in the ventral spinal cord. In chapter three, I analyse this through examining the Notch signalling dynamics of the lateral floor plate domain. This reveals that different cell types in the lateral floor plate display discrete durations of Notch signalling. It is the duration of Notch signalling that instructs cell fate determination, as prolonged exposure is required for the later-born cell fates. How the neighbouring progenitors in the lateral floor plate temporally restrict their Notch response remains an open question, though it is likely mediated through differential ligand-receptor interactions. Collectively, this thesis highlights the pleiotropic nature of Notch signalling during neural development. Alongside the classical involvement in progenitor maintenance, Notch signalling also functions to maintain cellular response to key developmental signals and directly guide cell fate decisions.
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    Origin and diversification of tissue-resident fibroblasts
    (2021-03-12) Ma, Roger; Huang, Peng; Grewal, Savraj; Mains, Paul
    Every cell within a multicellular organism contains the same genome and ultimately originates from a single cell. Despite this, a vast array of cell types is created during development. Understanding how a single progenitor can differentiate into distinct cell types is one of the fundamental questions in developmental biology. For example, while it is known that the somites generate much of the axial body, the specific mechanisms of how this occurs remains poorly understood. In this thesis, I use the zebrafish sclerotome as a model to study how different cell types can be generated from a common pool of progenitor cells. During development, the sclerotome is subdivided from the somite and gives rise to the axial skeleton, cartilage and tendons. In Chapter 2, I characterize sclerotome development in zebrafish and identify a novel dorsal sclerotome domain unique in zebrafish. Active hedgehog signaling is required for the migration of and maintenance of sclerotome-derived cells. Lineage analysis reveals that the sclerotome progenitors give rise to tenocytes (tendon fibroblasts) in a stereotypical manner. In Chapter 3, I ask how sclerotome progenitors diversify into multiple fibroblast subtypes. Using single cell lineage analysis, reveals that the sclerotome is multipotent and generates a variety of fibroblasts in the zebrafish trunk. BMP signaling is required for the generation and maintenance of a subset of sclerotome-derived fibroblasts, the fin mesenchymal cells. Together, my work shows that the zebrafish sclerotome is the embryonic origin of a diverse population of tissue-resident fibroblasts and can be a good model to study cell type diversification.
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    Perivascular fibroblasts in tissue development, maintenance, and repair
    (2023-10-30) Rajan, Arsheen; Huang, Peng; Childs, Sarah; Grewal, Savraj; Mcfarlane, Sarah; Mosimann, Christian
    At the microscopic scale, all tissues within multicellular organisms are comprised of diverse cell types embedded in a web of extracellular proteins. How each of these components evolve and are maintained as the tissue grows, and how they are replaced upon tissue injury are fundamental questions at the heart of developmental and regenerative biology. One cell type that has emerged in recent years as an essential modulator of tissue growth and repair is the fibroblast. Fibroblasts are multifunctional, tissue-resident mesenchymal cells that are known to regulate the extracellular environment through matrix production and remodeling, coordinate cell differentiation by releasing paracrine signaling factors, and act as multipotent progenitors themselves. Yet, little is known about how different fibroblast subtypes arise and diversify. Therefore, this thesis focuses on exploring the origin, behavior and function of a poorly characterized fibroblast subtype, the perivascular fibroblast, so named due to its localization around blood vessels. In Chapter 1, we summarize current knowledge on blood vessel associated fibroblasts from numerous murine organs. In Chapter 2, we show that in zebrafish, perivascular fibroblasts, arise from the sclerotome region of the somite early in development. We find these perivascular fibroblasts play dual roles in stabilizing the immature vasculature by depositing extracellular structural collagens and giving rise to blood vessel support cells called pericytes. In Chapter 3, I further clarify that perivascular fibroblasts are distinct from other sclerotome-derived fibroblasts in their transcriptional signature, plasticity, and regenerative potential. We show that in response to tendon injury, perivascular fibroblasts actively migrate, proliferate, and differentiate into specialized tendon fibroblasts, tenocytes, to facilitate tissue regeneration. Finally, in Chapter 4, I outline outstanding questions on perivascular fibroblast biology that can be explored in future studies. Together, my work highlights the functional relevance of fibroblast heterogeneity and provides insights into the potential characteristics of homologous perivascular fibroblast-like populations identified in murine and human tissues.
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    The Role of the RNA-Induced Silencing Complex (RISC) Component VIG-1 in Caenorhabditis elegans Germline Stem Cell Regulation
    (2021-04-26) Zhang, Dan Jia Run; Hansen, David; Huang, Peng; Chua, Gordon
    Stem cells are undifferentiated cells that can give rise to a wide variety of specialized cell types. In development and tissue maintenance, these cells play crucial roles in maintaining homeostasis and reproductive fitness. The C. elegans germline is a powerful model in which the nature of stem cell regulation can be studied. The germline is set up in an assembly line-like format, in which mitotically dividing proliferative cells reside in a distal niche. As cells move away from this niche, they gradually undergo meiosis until becoming fully differentiated sperm or oocytes. Notch signaling in the distal end of the germline provides the proliferative signal, while downstream post-transcriptional regulatory pathways promote differentiation. Although this main pathway has been well characterized, various components serve to fine-tune this balance in subtle but important ways. Previous work has identified small RNA molecules, such as miRNAs, as having a role in germline stem cell regulation. In this thesis, the role of vig-1, a gene involved in the RNA-induced silencing complex (RISC) pathway, in C. elegans germline stem cell regulation is investigated. Although vig-1 performs multiple functions in a variety of tissues, it was initially implicated in the germline through its physical protein interaction with another RISC pathway component, teg-1, that was known to modulate germline stem cell balance. Here, I show that vig-1 functions to repress the activity of Notch signaling. In worms lacking vig-1 activity, Notch signaling is seen to be enhanced, manifesting through ectopic over-proliferation of stem cells. Furthermore, vig-1 can partially suppress Notch loss-of-function mutant phenotypes. Using immunofluorescent microscopy, I show that vig-1 may act directly on Notch signaling through increasing the expression of its direct transcriptional targets. I also show that vig-1 does not work with downstream differentiation-promoting pathways, further strengthening the idea that vig-1 functions at the level of Notch signaling. Finally, I propose a potential miRNA mechanism through which vig-1 exerts its function on the germline proliferation versus differentiation decision. This work provides deeper insight into the complex and interwoven nature of regulatory signaling pathways and lays the foundation for further research and eventual therapeutic or biotechnological applications.
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    The role of TOR kinase signaling in responses to bacterial infection
    (2021-08-10) Deshpande, Rujuta Shailesh; Grewal, Savraj; Brook, William; Huang, Peng; McCafferty, Donna-Marie; Jean, Steve
    Animals in their natural ecology are often exposed to environmental stressors (e.g., starvation, extreme temperature, hypoxia, pathogens) that can affect their physiology, development, and lifespan. An important question in biology is how animals sense these stresses and, in response, adapt their metabolism to maintain homeostasis and survival. In some cases, specific tissues function as stress sensors to control whole body adaptive responses. One well-studied example is the Drosophila intestine. Along with performing absorptive, digestive, and endocrine functions, the intestine also functions as both a stress sensor and signaling hub, to regulate systemic metabolic changes. Upon encountering enteric pathogenic bacteria, Drosophila adults mount organism-wide immune and physiological responses in order to provide infection resistance and promote tolerance. The Drosophila intestine controls both local and systemic anti-bacterial immune responses. Recent work shows that the gut also signals to other tissues to control whole-body metabolic changes to promote infection tolerance. However, the mechanisms underlying how these stress sensing tissues link the stressors such as infection to metabolic adaptations is not well understood. In my thesis, I show that one way by which the fly intestine mediates these adaptive metabolic responses is via induction of target-of-rapamycin (TOR) kinase signaling. TOR is a well-established regulator of metabolism that has classically been shown to be activated by growth cues and suppressed by stress conditions. Interestingly however, I found a rapid increase in TOR activity in the fly gut in response to enteric gram-negative bacterial infection stress, independent of the classic innate immune response. Furthermore, I showed that blocking this TOR induction reduced survival upon infection. My data suggest that these protective effects of gut TOR signaling on organismal survival may be mediated through altered whole-body lipid metabolism. Lipid stores are an important metabolic fuel source. They can be synthesized and stored in specific tissues and then mobilized, transported to other tissues, and used to fuel metabolism, particularly in stress conditions. Infection leads to transient loss of lipids which is perhaps needed to fuel the immune response. This transient loss and restoration of lipids was further exacerbated by TOR inhibition. Infection also induced TOR dependent systemic expression of transcription factors and enzymes that promote de novo lipid biogenesis, indicating one way by which TOR inhibits excess lipid loss is by promoting de novo lipid synthesis upon infection. Moreover, genetic upregulation of intestinal TOR was sufficient to induce the expression of some of these lipid synthesis genes. In addition to systemic effects, enteric infection also induced TOR dependent local intestinal lipolysis and beta oxidation genes, and endocrine signaling peptides which have previously been implicated in whole body lipid homeostasis. I propose a model in which induction of intestinal TOR signaling is an infection stress sensor that leads to local intestinal changes such as lipolysis and secretion of signaling peptides, which perhaps non autonomously signal to the rest of the animal to upregulate lipid synthesis upon infection. TOR upregulation represents a host adaptive response to counteract infection mediated loss of whole-body lipid stores in order to promote survival. While only a handful of studies have investigated a role for TOR signaling upon infection with varying results, my thesis supports the idea of TOR activity being beneficial for the host to survive enteric infection. I propose TOR signaling as a link between infection and metabolic adaptations which contributes to infection tolerance.
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    Roles of muscle-associated cells during muscle regeneration.
    (2018-11-27) Yang, Lucy; Ruel, Tyler; Kocha, Katrinka; Huang, Peng
    Skeletal muscles control many essential functions that we constantly perform including walking, eating, and breathing. Any diseases that compromise muscle function, such as muscular dystrophy, will have a noticeable impact on the quality of a person’s life. Understanding molecular and cellular mechanisms underlying muscle regeneration will help design new therapeutic approaches to promote muscle injury repair and ameliorate different muscular disorders. Current research mostly focus on known muscle stem cells (satellite cells), while little is known about other types of muscle-associated cells and how they contribute to muscle regeneration. For example, some studies have found that fibro/adipogenic progenitors do not generate muscle fibers but create microenvironments that promote muscle stem cell activity during regeneration instead. My research project aims to determine the functions and responses of different muscle-associated cells during muscle injury repair using zebrafish as a model system. We hypothesize that each type of muscle-associated cell has a distinct regulatory function that aids the muscle regeneration process. To address this topic, I first optimized two complementary techniques to section adult muscle tissues — I used vibratome sectioning to generate thicker sections to maintain 3-dimensional architecture and cryosectioning to prepare thin sections for histological staining. Combining these techniques with different muscle injury models, I have performed detailed time course experiments to determine how muscle repair progresses with respect to either glycerol or cardiotoxin-induced injury. Preliminary results have shown that muscle injury repair in adult zebrafish is dependent on the type of injury. Thus far, glycerol-injected fish seem to suffer catastrophic muscle damage that is still evident 5 days post-injury. Next, I will determine how different types of muscle-associated cells contribute to muscle regeneration under different injury conditions.
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    Semaphorin3f as a Spatial Regulator of Embryogenesis
    (2019-01-22) Halabi, Rami; McFarlane, Sarah; Childs, Sarah J.; Huang, Peng
    During embryogenesis, cells integrate both spatial and temporal information from their surroundings to influence proliferation, migration, differentiation and physiological functions. Understanding the molecular mechanisms which confer spatial identity is essential to our understanding of tissue development and human disease. In this thesis I explore multiple roles for the secreted chemotactic ligand Semaphorin3f (Sema3f) in different biological contexts. Using zebrafish (Danio rerio) as a model I take advantage of the duplicated genome to study loss of function of both orthologs, Sema3fa and Sema3fb, in discrete contexts due to their differential expression. First, I show that in the eye Sema3fa produced by progenitors is necessary for the generation of amacrine cells within the temporal retina and the spatially-organized transcriptome of stem cells in the ciliary marginal zone (CMZ). Second, I define an endogenous role of Sema3fa to maintain the avascularity of the neural retina and refine the branch pattern of intraocular vessels. Loss of Sema3fa results in the pathologic angiogenesis of leaky blood vessels into the neural retina. Last, I unveil a role for Sema3fb produced by cardiomyocyte progenitors in the differentiation of the ventricle of the developing heart. Overall, my work provides the first evidence of a Sema3 involved in retinal progenitor cell and cardiomyocyte differentiation, and elucidates the endogenous role of Sema3fa as a negative regulator of retinal blood vessels in the embryo and adult. My data exemplifies the necessity of spatial information conferred by a single chemotactic molecule, Sema3f, to impact differentiation and cellular biology.
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    To define the interactome of neuronal primary cilia
    (2021-09-17) Whitmore, Brandon Alexander; Guo, Jiami; Dufour, Antoine; Huang, Peng; Schriemer, David; Mains, Paul
    Primary cilia are projections of the plasma membrane on almost all mammalian cells in the body, including neurons. The purpose of primary cilia is to act as a cellular antenna due to the unique membrane composition of the primary cilia which is abundant in different receptors, like GPCRs and RTKs, and ion channels. Dysfunction of cilia signaling gives rise to a class of diseases known as ciliopathies. Ciliopathies present with brain structural and functional deficits and have been implicated in intellectual disabilities, Autism Spectrum Disorder, and Schizophrenia. While a number of ciliopathies have been determined, there is a gap in the knowledge of the mechanisms of some ciliopathies due to an incomplete understanding of the primary cilia proteome, especially within the brain. Understanding the primary cilia proteome may determine potential gene candidates that have been implicated in ciliopathies. The gap in the knowledge of the neuronal ciliary proteome arises from the difficulty in purifying mammalian primary cilia. Different proteomics techniques have been used to begin to study the primary cilia composition within kidney cells; however no techniques have been applied to neurons to study the proteome of neuronal primary cilia. The brain and kidneys respond to different extracellular cues and have different environments, indicating that neuronal primary cilia should have a unique membrane composition and respond to distinct extracellular cues compared to primary cilia of other cells. To identify the unique membrane composition of neuronal primary cilia I developed an in vitro proximity labeling method to isolate ciliary proteins from primary neuron cultures. From this work, I was able to create the first list of potential neuronal primary cilia proteins. As an alternative method to understand the membrane composition, I identified a number of signaling molecules that affect primary cilia morphology and impact signaling within cilia. These results will be used to begin constructing an interactome of neuronal primary cilia signaling.
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    Ubiquitin Signaling Regulates P-Body Assembly
    (2021-06-21) Kedia, Shreeya; Yang, Guang; Huang, Peng; Mains, Paul Elliott; Corcoran, Jennifer
    During the development of the central nervous system, neural stem cells give rise to different cell populations including neurons and glia. To en¬sure the genesis of the correct cell populations in the developing brain, there exists and intricate system of gene expression regulation. One such mechanism of gene expression regulation is the presence of membrane-less ribonucleoprotein (RNP) granules in the cell such as Processing bodies (PBs). These dynamic organelles are sites of RNA metabolism that can temporarily sequester mRNAs resulting in translational repression and/or decay. Therefore, to understand the molecular mechanism by which PBs regulate stem cell homeostasis, it is critical to delineate the signaling regulating PB dynamics. To this end, my thesis explores a novel non-proteolytic monoubiquitination-based signaling mechanism, where monoubiquitination of a core PB protein called 4E-T drives PB assembly. Mechanistically, PB dynamics are fine-tuned by a deubiquitinase called Otud4, which deubiquitinates 4E-T to disassemble PBs. This dynamic ubiquitination signaling therefore, functions as an essential molecular switch to coordinate PB dynamics in neural stem cells.