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The underlying molecular mechanisms driving the wound response remain poorly understood. Thus, identifying the proper sequence of events responsible for tissue repair will be essential to clinical applications in regenerative medicine. Transcriptional and biochemical analyses of injured tissues have identified candidate genes and molecular pathways activated minutes to hours after injury. However, the upstream cues leading to genetic and biochemical changes remain elusive. Studies of the wound response in diverse organisms suggest that electrical changes at the site of tissue damage appear as one of the earliest cues. These studies imply that almost immediately after injury, ionic leakage and localized tissue depolarization result in lateral electric fields directed toward the injury site. Changes in endogenous potentials may also act as a first responder enabling tissues to sense the existence of injury. Here, we use the planarian flatworm Schmidtea mediterranea to test the role of tissue depolarization during early events of the wound response and try to identify which ions may be involved in this response. We developed a novel methodology to induce localized epithelial permeabilization without tissue damage. This is accomplished by brief exposure to amphipathic detergents. Our results show detergent-induced tissue permeabilization induces depolarization eliciting wound-related transcriptional changes, reactive oxygen species signaling, and biochemical activity consistent with the earliest stages of injury. Furthermore, we also noticed long-range spatiotemporal cell death and proliferation characteristics of tissue regeneration. Our experimental strategy revealed the possibility of dissociating molecular events of the injury response from physical tissue damage. We have also found that K+ may be the ion responsible for driving this process. We present comprehensive evidence about the activation of the regenerative response by a brief treatment to disrupt physical properties of the plasma membrane. These findings further support and give insight to the idea that it is possible to bypass the injury event to activate downstream components leading to tissue repair.

Small RNAs have been implicated in the intergenerational epigenetic inheritance of paternal environmental effects, with tRNA-derived fragments (tRFs) being one of the most abundant classes of small RNAs in mature mammalian sperm. These fragments are modulated by environmental conditions and can influence offspring phenotypes; however, their biogenesis and functions remain poorly understood. My research focused on uncovering the enrichment and functional roles of tRFs, particularly the 5′ fragment of tRNA-Valine-CAC-2 (tRFValCAC), which is highly abundant in mature mouse sperm. tRFValCAC is enriched during post-testicular maturation in the epididymis and delivered to sperm via extracellular vesicles secreted by epididymal epithelial cells.Mechanistically, I identified that tRFValCAC interacts with the RNA-binding protein heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) in the epididymis, which regulates tRFValCAC levels within these vesicles and further contributes to the enrichment of tRFValCAC in mature sperm. Functional analyses revealed that inhibiting tRFValCAC in preimplantation embryos altered the expression of genes involved in RNA splicing and mRNA processing, leading to delayed preimplantation development. Together, our results reveal a novel function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance.In a different project, I investigated the impact on diet on tRNAs on tissue level. Environmental factors play a crucial role in shaping tRNA levels and modifications, which in turn can influence protein synthesis and enable cells to adapt to various stresses. While much of our knowledge stems from studies in cell culture and yeast, evidence suggests that tRNA dynamics also occur at the tissue level. For instance, the microbiome has been shown to reprogram host tRNAs in a tissue-specific manner. Despite these insights, the connections between diet, tRNA expression, and tRNA modifications across different mammalian tissues remain largely unexplored. To investigate this, I collected tRNA from mouse tissues subjected to 10 weeks of low-protein and high-fat dietary challenges. RNA mass spectrometry analysis revealed tissue-specific changes in tRNA modifications, such as elevated galactosyl-queuosine levels in the epididymis compared to the liver, and demonstrated that diet influences bulk tRNA modifications across tissues. Using a modified-base-sensitive tRNA sequencing technique (OTTRseq), I identified that dietary challenges affect tRNA abundance in all tissues examined, with liver and heart showing more robust changes. Only liver and heart exhibit detectable mismatch rate changes at sites like 58A. This study could pave the way for exploring how tRNA dynamics in response to diet impact gene expression, uncovering tissue-specific RNA regulation and mechanisms of environmental tissue adaptation.

Human stem cell-derived in vitro models of various cell types and tissues act as useful tools for the study of human biology and disease in a genetic editable, scalable setting. Years of studying in vivo pancreatic development has facilitated the development of in vitro protocols. Stem cell-derived islets (SC-islets) provides an alternative source of cells to study β-cell development, function, and dysfunction. This is particularly useful for research regarding the human pancreatic islet, which is difficult to access in vivo and difficult to obtain for experiments ex vivo. Despite successful induction of pancreatic β-cells which produce and secrete insulin in a glucose-responsive manner, stem cell-derived β-cells (SC-β-cells) still fail to recapitulate primary human β-cell gene expression and functional maturity. We developed a genome-wide screening platform using CRISPR-Cas9 technology to identify genes regulating the mitochondrial glucose response, an important aspect of insulin secretion. Our results identified new avenues of study to understand the genetic underpinnings of β-cell functional acquisition. Among these is the folate cycle, an important driver of cellular proliferation. The folate cycle has been implicated in metabolic function and β-cell development in studies of maternal dietary folate. We assayed each stage of development for folate cycle impacts on β-cell development. We discovered the folate cycle primarily influences β-cell development during foregut specification. This novel finding improves our understanding of the in vivo studies on maternal folate supplementation. Furthermore, we find inhibition of the cycle component SHMT2 is necessary to induce a crucial pancreatic transcription factor. These findings, together with literature evidence and qualitative observations, hint at the importance of cell density for pancreatic differentiation, possibly through mechanotransducive signals. In our investigation of the folate cycle impact on mature β-cell function and metabolism, we report antifolate drugs promote oxygen consumption in SC- and primary human islets. Furthermore, potent folate cycle inhibition augments SC-islet insulin, and elevates AICAR, which amplifies insulin secretion through AMPK activation. Together, this paints a picture of folate cycle inhibition amplifying insulin secretion through AICAR accumulation.Altogether, this dissertation details folate cycle regulation of β-cell development and function, identifying a new target for improving SC-islet differentiation protocols.

Defects in telomerase and telomere length maintenance have been implicated in difficult to treat genetic disorders, premature senescence underlying aging disorders, and chromosomal instability contributing to increased risk of cancer development. It has relatively recently been noted that patients with congenitally short telomeres and telomerase dysfunction diseases present with microcephaly and associated neurological symptoms. However, few studies have explored the role of telomere dysfunction on neurodevelopment.This proposal aims to study the role of shortened telomeres and telomerase dysfunction using human induced pluripotent stem cells (hiPSCs) to model neurodevelopmental defects. In previous studies, hiPSCs with measurably shortened telomere length, as well as hiPSCs that have been CRISPR engineered with defects in the telomerase reverse transcriptase (TERT) component of the telomerase complex that is important for end replication function, have elucidated that bone marrow failure associated with the disease can be attributed to cellular dysfunction that underlies failure of hematopoietic stem cell development. Using hiPSCs that have similar defects in telomere length and in TERT (the enzymatic component of telomerase), this proposal will utilize a directed differentiation approach to study early neurodevelopment and neuronal maturation to measure critical points of cellular development that potentially give rise to the reduced neuronal volume that characterizes microcephaly. Furthermore, this proposal will utilize a previously published three-dimensional cellular protocol for studying the microcephaly phenotype using cortical organoid culture, which will allow for visualization and measurement of neuronal development related to spatial organization. Finally, the cellular basis of associated neurodevelopmental aberrations will be measured via bioinformatics analysis and functional activity assays, to elucidate the underlying contribution of prematurely shortened telomeres to neuronal activity and maturation.

Recent reports of bovine embryonic stem cell (bESC) establishment and in vitro gametogenesis (IVG) in the mouse open the door to recapitulate oogenesis from bESCs in vitro, however little progress has been made toward this feat. Critical gaps remain in our understanding of bovine germ cell development, which will be necessary to establish a system that supports efficient in vitro germline formation as well as to assess the resulting cells once obtained. Additionally, progress in other models has shown the importance of pluripotent state on cellular capacity for in vitro germline induction. Therefore, the goal of this dissertation was to shed light on the requirements of livestock pluripotency and germline development to set the basis to establish reliable bovine in vitro oogenesis systems.We first examined the pluripotent state of bESC lines and explored the potential to stabilize these cells in intermediate states of pluripotency, due to the limitations of primed ESCs for germline induction. We found that regardless of the initial blastocyst stage that bESCs were derived from, cell lines exhibited similar transcriptional and functional characteristics of primed pluripotency. However, the demonstration that bESCs could be efficiently established from early blastocysts increases the pool of embryos that may be targeted for bESC derivation approaches. We also investigated alternative formative media for bESC establishment, but found that conditions that supported this state in other mammalian species did not translate to cattle. This highlights the unique signaling requirements of bovine pluripotency and the necessity of further studies to establish alternative conditions for bESCs with high germline competence.Specification of primordial germ cells (PGCs), the precursors of oocytes and sperm, occurs during early embryonic development. The first step of IVG is induction of ESCs into PGC-like cells. Despite wide interest in replicating this event in vitro, little was known about PGC specification in the cow. We identified founder PGCs in the posterior epiblast of bovine embryos at day 16, by unique co-expression of conserved early germline proteins SOX17, PRDM1, and TFAP2C, with absent SOX2. We found that PGCs had initiated migration by day 20, at which point they were distributed through the allantois and hindgut endoderm, but were not yet highly proliferative. Single-cell transcriptome analysis of bovine embryos at day 20-22 allowed us to capture a small population of bovine PGCs, and gave insights into early germline development, including acquisition of the germ cell program, repression of somatic marks, and initiation of epigenetic remodeling. Further, we identified candidate cell surface markers for sorting PGCs – or in vitro-induced counterparts – for future experiments. This work provides the earliest observation of PGCs in cattle and demonstrates a conserved program in bovine germline emergence with other species that form as an embryonic disc.We continued from studying PGC specification and migration onset in the embryo through the advanced development of these cells into oocytes in the fetal ovary. Expanding upon a report from day 50, we traced the bovine germline by single-cell RNA-sequencing at days 70, 90, and 120 of fetal ovarian development. This revealed the transcriptional profile of bovine germ cells from gonadal PGCs through germline commitment into oogonia, initiation of meiosis, arrest at prophase I, and development of primordial oocytes within follicles, as well as cell surface marker candidates for each of these stages. Further, we explored the molecular profile of the somatic niche of the ovary and the interactions of these cells with the germline, which gives evidence for signals that could support germ cell development in a culture dish.In conclusion, this dissertation provides detailed and novel insights into bovine pluripotency and germline establishment. The knowledge gained from these studies may help in the development of assisted reproductive technologies of livestock, humans, and endangered ungulates. Moreover, these studies highlight the cow as a valuable model of human reproductive development and offer important context of embryonic/fetal events that cannot be directly studied in humans.

Faithful cell division is crucial for all eukaryotic cells to ensure that daughter cells receive thecorrect number of chromosomes. Errors in chromosome segregation during meiosis in germcells can result in aneuploid gametes, potentially leading to infertility, genetic disorders likeDown Syndrome, and miscarriages. Similarly, chromosome segregation errors during mitosiscan produce aneuploid somatic cells, which are often associated with cancer. Although theregulation of these two types of cell division is vital, the mechanisms that ensure theiraccuracy are still actively being studied. Among the proteins identified as key regulators ofboth mitotic and meiotic fidelity in various species, including Caenorhabditis elegans (C.elegans), is PCH-2. PCH-2 is a conserved AAA+ ATPase required for timely coordination ofthe meiotic prophase events pairing, synapsis and recombination, but the mechanism for howit accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shownto promote crossover formation and affect their distribution. Here I use C. elegans as a modelsystem to investigate the conserved meiotic role of PCH-2.In Chapter 1, I begin my investigation of PCH-2’s conserved role by observing it’s localizationto meiotic chromosomes in the mnT12 background, where two chromosomes, X and IV, arefused. In order to fully understand how defects in chromosomal rearrangements can affectPCH-2’s localization, I also studied its localization in heterozygous inversions, mIn1/+ andhIn1/+. I find that PCH-2 localization is extended in the chromosomal fusion backgroundmnT12, but not in mIn1 heterozygotes and hIn1 heterozygotes. I also find that the extensionof PCH-2 in mnT12 worms results in the promotion of crossover formation, providing anotherexample of how PCH-2 can affect crossover formation.In Chapter 2, I demonstrate that PCH-2 regulates the number and distribution of crossoversby opposing their formation. This antagonistic action has distinct effects at various stages ofmeiotic prophase. I show that in the early stages, PCH-2 restricts the conversion of doublestrand breaks (DSBs) into crossovers, thereby limiting crossover events at the initial sites ofDSB formation and homolog interactions. And then later in meiotic prophase, PCH-2 reducesthe number of crossover-eligible intermediates, aiding in the reinforcement and designation ofthese intermediates, and ultimately ensuring crossover assurance. Additionally, I show thatPCH-2 exerts this control through the meiotic HORMAD protein, HIM-3. The data I presentstrongly support a model where PCH-2's conserved function is to remodel meiotic HORMADsthroughout meiotic prophase, destabilizing crossover-eligible precursors, coordinating meioticrecombination with synapsis, and contributing to the progressive execution of meioticrecombination to ensure precise crossover regulation.Finally in Chapter 3, I test PCH-2’s role on modulating synapsis. Specifically by observinghow the loading and unloading of a central region component of the synaptonemal complex(SC) is affected in the absence of PCH-2. I show that loss of PCH-2 does not affect how thisprotein – SYP-3, is loading onto chromosomes during meiotic prophase.Added together, my work establishes a crucial framework for understanding the role of PCH-2in regulating the number and distribution of crossovers (Chapter 1 and Chapter 2), a functionI propose is conserved across species. While specific mechanisms may differ amongsystems (Chapter 3), I suggest that PCH-2 plays a key role in remodeling meiotic HORMADsthroughout meiotic prophase to destabilize crossover-eligible precursors. This processcoordinates meiotic recombination with synapsis, contributing to the stepwise progression ofrecombination and ensuring crossover assurance, interference, and homeostasis.

The neural crest is a multipotent transient cell population that migrates, proliferates, and differentiates in vertebrate embryogenesis and gives rises to a cornucopia of tissue derivatives such as craniofacial bone and cartilage, peripheral nerves, and glia. Congenital malformations including cleft palate and craniosynostosis arise from aberrant development of cranial neural crest cells and their derivatives. Neural crest derived tumors of Schwann cell lineages can arise from single genomic hits in the form of NF2 loss. Considering the low genomic burden of many neural crest derived tumors, we hypothesized that epigenetic regulators, which normally specify cell lineages and maintain cell fates in embryonic development, are mis-regulated in adult neural crest derived tissues, contributing to tumorigenesis and differential tumor radiation responses.Here we use mouse genetics and single cell sequencing to investigate how PRC2, a histone methyltransferase broadly involved in gene repression and maintenance of pluripotency in development, is involved in epigenetic control of post- otic neural crest derivates. We identified Eed, a PRC2 core subunit, as a potent regulator of craniofacial development. Eed deletion in post-migratory neural crest cells was perinatal lethal and knockout embryos presented with severe craniofacial abnormalities consistent with impaired differentiation of osteoblast derivatives. Using targeted primary cell culture gene expression analysis and unbiased scRNA-seq, we discovered changes in transcription factors involved in the proliferation and differentiation of neural crest derivatives including loss of Sox transcription factors and increase in Hox genes. Strikingly, we found an expansion of undifferentiated mesenchymal stem cells and decrease in differentiated osteoblast cells, indicating Eed controls proper differentiation of mesenchymal derivatives that comprise craniofacial structures. Taken together, we establish the embryonic, cellular, and molecular consequences of Eed loss in neural crest derived craniofacial tissues.Next, we investigated more broadly how epigenetic regulation contributes to cancer of neural crest derived cells including the tumorigenesis of schwannomas and schwannoma radiation responses. Using bulk and single-cell bioinformatics, functional genomic approaches, and mechanistic validation, we discovered schwannomas comprise 2 molecular subgroups marked by activation of neural crest signaling pathways or enrichment of immune cells in response to radiotherapy. CRISPRi radiation screening in human schwannoma cells identified the lysine demethylases KDM1A and KDM5C as drivers of radioresistance or radiosensitivity, respectively. Lastly, we integrated single-nuclei ATAC, RNA, and CRISPRi perturbation to identify chromatin accessibility motifs that drive schwannoma cell state evolution and radiation responses.

Transposable elements (TE) are mobile DNA elements that can replicate and re-insert themselves into different locations within the host genome. This retrotransposition has resulted in TEs accounting for more than 50% of the human genome. Much like other non-coding RNAs, TE insertions with multiple copies were once considered non-functional. But recently it has been shown to have much more regulatory role in different cellular pathways. The first part of this thesis delves in describing the different types of TE and other non-coding RNAs like non-coding RNA and their significance in inflammation and cancer. Noncoding and repeat-derived cell-free RNA transcriptome (TE) is a rich source of novel, abundant, and disease-specific RNA biomarkers. We show that repeat-derived cell-free RNAs, including simple repeat RNAs and TE RNAs transcribed from LINE, SINE, and LTR elements, are cancer-specific RNAs that are normally present at low or undetectable levels in healthy individuals.The second part explains about the role of RAS in regulating TE in the context of cancer. Here I describe about RAS effector transcripts give rise to a diverse array of non-coding RNAs that contribute significantly to cancer initiation and progression. Our study also reveals the transcriptomic and epigenomic impact of oncogenic KRAS signaling on TE RNAs and ISGs. Our study suggests that KZNF repression by mutant KRAS signaling leads to de-repression of TE RNAs, triggering an intrinsic ISG response. This model is supported by broad and significant downregulation of these same KNZFs in mutant KRAS-driven lung adenocarcinomas in vivo. We also identified mutant KRAS(G12C)-specific exRNA signatures secreted from LUAD cells by comprehensively analyzing the coding and noncoding RNAs secreted in extracellular vesicles (EV). The final section addresses the regulation of transposable elements (TEs) by KRAS during the pluripotency and differentiation of human induced pluripotent stem cells (hiPSCs). My research demonstrates that KRAS deficiency results in the differential expression of TEs, particularly LTR7, causing a defect in differentiation towards the neural lineage. Additionally, we have identified novel genes containing LTR7 within their gene bodies, which are differentially transcribed during the transition out of pluripotency.

Bone is one of the most complex tissues due to the numerous embryonic origins it is derived from to its ossification processes. The transcriptional network that regulates bone development has been extensively studied. However, the insight pertaining to microRNAs and osteogenesis is either limited or seen at later stages of development. MicroRNAs are small non-coding RNAs that are involved in RNA silencing and post-transcriptional regulation of gene expression. These negative epigenetic modulators bind to the 3’Untranslated Region of their target mRNAs to initiate mRNA degradation or translational inhibition. They have been linked to numerous biological processes ranging from maintenance of pluripotency in embryonic stem cells, to differentiation, and later stages of osteogenesis. We have identified a novel microRNA, miR361, that impacts early osteoprogenitors and promotes osteogenesis. MiR361 accomplishes this role by directly targeting negative modulators of non-canonical Wnt, Planar Cell Polarity (PCP) pathway, Prickle 1, and Prickle 2. PCP signaling, known for establishing polarity and directing cell locomotion, is a crucial aspect of proper bone development, as mutations have led to severe skeletal deformities and disorders such as Robinow Syndrome. Studies on both Prickles are typically centered around their responsibilities in neurogenesis. Recently, research has shown that Prickle 1 plays a necessary role in bone development and is a novel marker for Robinow Syndrome. Furthermore, Prickle 1 has been shown to act as a nuclear translocator for Re1-silencing transcription factor (Rest). Recently, Rest has been linked to osteoblast differentiation. Prickle 2’s osteogenic function has not been established. Therefore, the objective of this dissertation is to (i) determine the role of miR361 during early osteogenesis, (ii) delineate the individual functions of Prickle 1 and Prickle 2 in bone development. This will provide the first evidence for Prickle 2. (iii) Uncover the molecular mechanisms Rest plays in osteogenesis. Together these findings establish a novel miR361-Prickle-Rest osteogenic triad that could be used for further diagnostic and screening for bone development and disorders.

To orchestrate the complex events of craniofacial morphogenesis requires a concerted effort between gene expression and tissue specific events. In the embryonic midface, facial prominences must execute a coordinated fusion between maxillary, medial and lateral nasal prominences at the lambdoidal junction, requiring breakdown and removal of epithelium to permit coalescence of underlying mesenchyme. When this process is disrupted by genetic mutations, cleft lip, the most common craniofacial birth defect in humans, is created. Key to understanding cleft lip pathogenesis is the reliance on mouse models. Models of cleft lip and studies of the role of epithelium are rare in comparison to cleft palate and research on underlying mesenchyme. The epithelium possesses discrete roles that facilitate fusion at the lambdoidal junction, including apoptosis and epithelial to mesenchymal transitions. To better understand its heterogeneity, we sought to enrich for the midface epithelium and isolate it from mesenchyme to gain fuller resolution of the lambdoidal junction transcriptome. This dissertation contains the product of that endeavor to focus on the role of the epithelium alone both in normal conditions and in cleft lip pathogenesis. In doing so, an atlas of lambdoidal junction epithelium at timepoints before, during and after fusion of the midface was created and validated. Through these methods, a deep analysis of each cluster resulted in the localization of a cluster undergoing cell cycle arrest and located at the fusion site of each prominence. A series of experiments were conducted which demonstrated that cell cycle arrest is characteristic of this population that is altered in cleft lip models. Additional research yielded ties to normal midface morphogenesis as well as novel orofacial cleft risk candidates, including Zfhx3 a gene with ties to cell cycle arrest. This thesis also expands upon the phenotypic characterization of a novel mouse cross which conditionally removes Bmpr1a from the ectoderm of the embryo, creating a model of cleft lip and palate. This work collectively produces knowledge of the midface epithelium at a scale of resolution not seen before as well as describes in detail how cell cycle arrest is a characteristic of primary palate fusion.

In the developing vertebrate brain, Radial Glia Progenitor (RGP), the principal neural stem cells, undergo symmetric and asymmetric cell division (ACD), giving rise to both RGPs (self-renewal) and differentiated cell types (e.g., neurons, oligodendrocytes, and astrocytes) that will ultimately form the central nervous system. Perturbations of RGPs divisions will result in neurodevelopmental disorders and brain tumors. Understanding the basic biology of RGPs divisions will potentially have a significant impact on understanding brain development and may provide insights into treatments for developmental disorders or brain tumors. During ACD, the mother cell must establish the proper axis of polarity with respect to asymmetrically localized cell fate determinants. These processes are regulated by the evolutionarily conserved Partition defective protein (Par) complexes. The Par complexes have been extensively characterized in invertebrates, but much remains to be understood in vertebrates, especially in the context of RGPs ACD. Previous studies in invertebrates and vertebrates have demonstrated that Par-3 is essential for regulating ACD by localizing asymmetrically along the division axis. Par-3 has long been thought to function exclusively at the cell cortex. Recent findings have led us to investigate the role of Par-3 during ACD in RGPs. Therefore, one approach to gain insights into this question is the utilization of zebrafish (Danio rerio), a powerful model organism used in biological research. Proteins of interest can be easily targeted by both pharmacological agents and genetic alterations within zebrafish, allowing for examination of complex phenotypes resulting from desired perturbations. Unlike mammalian models, development is relatively fast, and breeding produces a large amount of progeny, allowing for quick generation progression and large sample sizes. Also, unlike mammals, development occurs externally, and the brain is transparent, allowing for accessible and clear visualization of neurodevelopment, neuronal activity, and fluorescent markers of interest throughout the whole brain. These attributes make zebrafish an excellent model for studying the in vivo the process of RGPs undergoing ACD in the developing forebrain. In this dissertation, we explore how polarity plays a role during ACD in RGPs.In Chapter 1, we delve into Par-3's role in regulating polarity along the cell cortex in RGPs, influencing Notch activity in daughter cell nuclei. We explore techniques, such as the antibody uptake assay and in vivo time-lapse imaging in zebrafish, revealing the dynamics of internalized Notch ligand DeltaD during RGP ACD. Our findings uncover the role of cytoplasmic Par-3 in localizing intracellular determinants.Next, in Chapter 2, we delve deeper into the role of Par-3 during active neurogenesis, particularly in ACD in RGPs. Using various techniques including in vivo time-lapse imaging, biochemical assays, and pharmacological studies, we investigate the interplay between cortical and cytoplasmic Par-3. Our focus is on understanding how Aurora Kinase A (AurkA) phosphorylation of Par-3 influences ACD in RGPs. Our findings highlight AurkA's role in regulating Par-3 dynamics between the cytoplasm and cortex during mitotic RGPs, ultimately impacting neural progenitor fate.In chapter 3, we pivot away from lab bench research and discuss a diversity, equity, and inclusion (DEI) project, DE-SILO (Diversity, Equity, and Sociology training in Laboratory Organizations), that I worked on with Dr. Melaine Jeske. We designed a course module that was tailored for laboratory meetings at academic institutions. Our aim was to intersect DEI topics and facilitate discussions in the context of lab meetings. Therefore, by designing core curriculum modules and community building modules, it would assist our “facilitators” (individuals tasked with leading DE-SILO modules at respective institutions) in leading discussions within in their lab. We piloted our project at four universities: UCSF, CalTech, University of Washington, and University of Washington, Saint Louis. In conclusion, this dissertation sheds light on the intricate processes of RGP during ACD in the developing vertebrate brain. By elucidating the role of Par-3 in regulating polarity and Notch activity during ACD, we contribute to a deeper understanding of regulation of cell polarity. Our findings underscore the significance of proper RGP divisions for normal brain development and highlight the potential implications for addressing neurodevelopmental disorders and brain tumors. Through our investigations using zebrafish as a model organism, we uncover novel insights into the dynamic interplay between cortical and cytoplasmic Par-3 localization, orchestrated by AurkA-mediated phosphorylation. This regulatory mechanism governs the fate of neural progenitors, offering new avenues for therapeutic exploration. Furthermore, our commitment to DEI is evident in our DE-SILO project, which aims to foster inclusive environments within academic laboratory settings. By integrating DEI principles into laboratory meetings, we promote meaningful discussions and cultivate a more equitable scientific community. In essence, this dissertation not only advances our understanding of fundamental neurobiological processes but also underscores the importance of inclusivity in scientific research and academia.

Proteotoxic stress drives the progression of numerous degenerative diseases including Type I diabetes, Huntingtin’s Disease, Parkinson’s Disease, and retinitis pigmentosa, a degenerative blinding disease. Cells respond to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated-protein-degradation (ERAD) and proteasomal degradation, but persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. From plants to humans, loss of the zinc transporter ZIP7 causes ER stress; however, the mechanism is unknown. We used Drosophila melanogaster to genetically manipulate ZIP7 and investigate its function in vivo. Here, we show that ZIP7-mediated Zn2+ transport enhances ERAD and that cytosolic Zn2+ is limiting for deubiquitination of client proteins by the Rpn11 Zn2+ metalloproteinase as they enter the proteasome. We then decided to test ZIP7’s effects in a fly model of retinitis pigmentosa. Interestingly, we found ZIP7 overexpression rescues defective vision and photoreceptor degeneration caused by misfolded rhodopsin in Drosophila. We have begun a collaboration looking into whether ZIP7 can prevent or suppress mutant Rhodopsin-mediated neurodegeneration in human retinal organoids. Additionally, these results prompted a screen for potential ZIP7 rescue of other aggregate-prone proteins associated with neurodegenerative diseases. So far, we found that ZIP7 rescues photoreceptor degeneration caused by Aβ42 and Vap33. Further study of ZIP7, a novel component of ERAD, holds significant promise for therapeutic advancements in protein-folding disorders.

Skeletal muscle is one of the most regenerative tissues in the human body. Skeletal muscle progenitor cells (SMPCs) contribute to developmental myogenesis, and skeletal muscle stem cells (satellite cells, SCs) contribute to postnatal muscle homeostasis and regeneration. Differentiating human pluripotent stem cells (hPSCs) into SCs is a valuable resource for developing cell replacement therapies for muscle wasting diseases. However, current hPSC directed myogenic differentiation protocols result in immature, embryonic-like SMPCs that do not engraft and promote muscle regeneration as efficiently as postnatal SCs. Because it is unclear how SMPCs and SCs molecularly differ, it is not known how to mature SMPCs into SCs. Chapter 2 provides an overview on the current understandings of muscle development and the state of the field in generating directed differentiation myogenic cultures from hPSCs. In chapter 3, we found that although SMPCs and SCs from across development have many functional similarities in their roles in muscle formation, profiling them from both in vivo and in vitro hPSC directed differentiations using single-cell RNA-sequencing has revealed that they are transcriptionally distinct. However, it was unclear what regulated their differences in cell states. The chromatin landscape has a direct role in dictating the state of a cell, particularly through modulations in accessibility at regulatory regions which mediate transcription factor (TF) binding and control gene expression. In chapter 4, we evaluated differences in chromatin accessibility using single-nucleus ATAC-sequencing in tissue-derived SMPCs and SCs and hPSC-derived SMPCs (hPSC-SMPCs). We identified several TF candidates as potential regulators of development from embryonic SMPCs to postnatal SCs. For example, nuclear factor I family members were found to have roles in establishing stage-specific enhancers in the maturation from embryonic to fetal SMPCs. We also found that reduction of HMGA1 expression during directed differentiation promotes maturation of hPSC-SMPCs, and thus we identified HMGA1 as a TF that maintains SMPC immaturity. Collectively, this work sheds light on the transcriptional differences between muscle progenitor and stem cell states and the gene regulatory mechanisms that mediate these differences. It provides a comprehensive, multiomic view on the progression of human myogenesis in single-cell and single-nucleus resolution. These findings enhance our understanding of how to promote the regenerative potential of hPSC-derived muscle cells and enable the development of improved cell therapies for muscle diseases.

The rhythmic contraction of the heart depends upon the conductive properties of a small population of highly specialized cardiomyocytes, known as pacemaker cells. In the early embryonic heart, pacemaker cells reside in a ring-like pattern at the venous pole of the atrial chamber, in a region known as the inflow tract (IFT). While the gene regulatory networks that contribute to pacemaker cell differentiation have been previously investigated, we do not fully understand the upstream signaling landscape that defines the dimensions of the IFT. Our analyses in zebrafish suggest an essential role for Bmp signaling in promoting the formation of an appropriate number of IFT cardiomyocytes. Diminished Bmp signaling activity in alk8 mutants results in a substantial reduction of the number of IFT cells. Oppositely, heightened levels of Bmp signaling in chordin mutants leads to an increased number of IFT cells. In both alk8 and chordin mutants, altered numbers of IFT cells are evident within the primitive heart tube, indicating that Bmp signaling impacts IFT development prior to heart tube formation. Indeed, treatment with DMH1, a pharmacological antagonist of Bmp signaling, during gastrulation results in a significant reduction of IFT cell number, but treatment with DMH1 during early somitogenesis or during heart tube formation does not alter the number of IFT cells. Intriguingly, pharmacological inhibition of Wnt signaling during IFT cell differentiation suppresses the enlargement of the IFT in chordin mutants, implying that Wnt signaling acts downstream of Bmp signaling during IFT development. Together, these data suggest a model in which Bmp signaling acts during IFT progenitor specification, upstream of Wnt-directed IFT differentiation, to produce an appropriate number of IFT cardiomyocytes.

Hematopoietic stem cell (HSC) transplantation can cure life-threatening blood disorders such as leukemias and inherited blood diseases. However, shortage of HLA-matched donors, which disproportionally affects minorities and patients of mixed ethnic backgrounds, limits the number of patients that can be treated. Ex vivo expansion or de novo generation of transplantable human HSCs has not been successful due to poor understanding of the basic biology underpinning key HSC traits - self-renewal, engraftment and multi-lineage differentiation ability (together referred to as “stemness”). Previous studies identified MLLT3 as a key regulator of stemness in human HSCs whose expansion declines in culture and differentiation, and demonstrated that maintaining MLLT3 expression in culture expands transplantable HSCs. The focus of this is the characterization of a truncated isoform of MLLT3 . Analysis of RNA-seq data and epigenetic marks associated with the MLLT3 gene in human HSCs revealed a second TSS linked to a novel MLLT3 isoform (MLLT3-S), which encodes a truncated protein that can interact with known MLLT3 protein partners such as the Superelongation Complex (SEC) and Dot1L, but is unable to bind chromatin. MLLT3-L and MLLT3-S expression has opposing effects on gene expression and expansion of human HSCs in culture, suggesting distinct but complementary roles for the two isoforms. However, both isoforms of MLLT3 are necessary for proper HSC function. The MLLT3-S enhancer is accessible prior to its induction in fetal liver (FL) HSCs during their maturation, and coincides with downregulation of IGFBP2 expression. IGFBP2 is typically downregulated during HSC maturation, but its renewed expression is required for HSC proliferation in culture and MLLT3-L driven HSC expansion. The interplay between long and short isoforms of MLLT3 in human HSCs may provide a mechanism by which mature HSCs balance between expansion and maintenance modes.

Proper establishment of cortical structures during early brain development is vital to normal brain function. A key component of normal lamination of the cerebral cortex is the migration of neurons after they are born from precursor cells to their specific destination in one of the six cortical layers. Neuronal migration involves dynamic changes to microtubules and other cytoskeletal components at the tip of an extending axon or dendrite with the aid of microtubule-associated proteins (MAPs). Doublecortin (DCX) is a MAP that is highly and specifically expressed in immature, migrating neurons. Dysfunction of X-linked DCX causes lissencephaly in males, a malformation characterized by a lack of gyri in the cortex, and subcortical band heterotropia (SBH) or a "double cortex" in females. The role DCX plays in neuronal migration is not well understood and studies in mouse models have only investigated the effects of a complete knockout (KO) of Dcx, which resulted in no cortical lamination phenotype in male or female mice, in contrast to the conspicuous phenotypes observed in humans. However, documented disease-causing human DCX mutations involve a missense mutation in one of DCX’s microtubule binding domains, which has been shown to not remove DCX function entirely.Despite characterization of human-specific mutations and mouse knockout models, there remains an unmet need for elucidating the cellular and molecular mechanisms of patient-specific mutations in their native genetic context. I hypothesize that the limited phenotypes in Dcx KO mice are due to compensation by other proteins in Dcx’s absence, and not due to intrinsic species differences. In particular, I predict that the mutant phenotype observed in the cortex is due to altered binding to microtubules during neuronal migration, producing a dominant negative effect. To begin to elucidate the role of Doublecortin in regulation of neuronal migration, I have introduced a human disease-causing DCX mutation (T203R) into the endogenous mouse Dcx locus for in vivo experiments. These studies involving a disease-causing, patient-derived mutation of the endogenous Doublecortin locus in mouse contexts have yielded important insights into the biology of Doublecortin and cortical development, addressing unresolved mechanisms of patient-specific DCX mutations at the molecular and cellular level.

Precise regulation of both the timing and dosage of gene expression is critical for thedevelopment of the early embryo and the extraembryonic tissues that support it. These signalsenable molecular communication within and between tissues, thereby allowing cells todifferentiate to the correct identity and migrate to the correct location for patterned organ systemsto emerge. Some of the genes active during early development orchestrate morphogenesis in adose-dependent manner, whereby reduced expression fulfills some of the gene’s functions, but isinsufficient to drive normal patterning. How gene dosage informs morphogenesis is not wellunderstood. This dissertation focuses on how varying expression levels of two transcriptionfactors with dose-dependent phenotypes, TBXT and CDX2, influence the patterning and kineticsof the embryonic and extraembryonic mesoderm populations during gastrulation. By utilizing invitro models of early human gastrulation and mesoderm development, I demonstrate that TBXTdosage directly influences the temporal progression of the epithelial-to-mesenchymal transition(EMT) in the nascent mesoderm and hypothesize that this influences the temporal and spatialmigration kinetics surrounding primitive streak morphogenesis. I additionally show that CDX2dosage influences the gene regulatory network (GRN) underlying extraembryonic mesodermdevelopment despite heterozygous expression being sufficient to maintain a wild-type-likechromatin accessibility profile. This work suggests that the regulation of downstream geneexpression is not solely dependent on chromatin remodeling and implies that proper regulation ofthis GRN is potentially critical for the development of extraembryonic structures such as theallantois. These findings clarify how varying dosages of specific transcription factors can influencethe gene regulatory networks underlying early gastrulation, thereby contributing to ourunderstanding of both dose-dependent gene regulation and early human development.

RhoU and RhoV are Rho GTPases a part of the Ras superfamily and are atypical due to fast-cycling of GTP/GDP, making them constitutively active. Their unique N and C terminals regulate their submembrane localization for cellular activity. This, along with their fast-cycling nature categorizes them into their own subfamily. Rho GTPases are well known for their regulation of cell migration, polarity, apoptosis, proliferation and many other processes through effector protein interaction and behave in a switch-like mechanism. Over the last 20 years, studies have been connecting RhoV and RhoU to regulating the cytoskeleton due to their involvement with adhesion protein localization and induction of migratory protrusions. Our lab has found that the Rho GTPase RhoV is dynamically expressed in the zebrafish endoderm; rhov expression is high during gastrulation and low during mesenchymal to epithelial transition (MET). The endoderm is an essential germ layer that will form the epithelial layer of organs such as the respiratory tract, gut, and intestines. How MET is triggered in endodermal cells is still unknown. RhoV is a Rho GTPase whose role has not been investigated within the endodermal cell migration to sheet formation. Here, we used CRISPR-generated RhoV mutants and rhov overexpression to investigate the role of RhoV in the mesenchymal to epithelial transition in zebrafish endoderm. This work, in addition to past studies investigating RhoU and RhoV in development provides compelling reasoning into how RhoU and RhoV may be regulating endodermal cell migration in zebrafish.

Organ boundaries serve several important roles in development, but the mechanisms downstream of boundary-defining factors are not well understood. In Chapter 1 of my dissertation, I show the Arabidopsis transcription factor LOB regulates leaf angle via a phototropism-dependent mechanism, reducing phototropic responses at the base of the leaf. LOB and other boundary-defining transcription factors also regulate numerous cell wall genes, implying a biophysical mechanism is an important aspect of boundary function. Given the inherent challenges of measuring cell wall mechanics in the Arabidopsis leaf-meristem boundary, I used a different system, the maize ligular region, which is highly amenable to AFM experiments. In Chapter 2, I characterized mechanical patterns during the development of the ligular region. My findings are consistent with the existence of a cell wall rigidification program in the boundary, which correlates with patterns of growth, cell division, and auxin dynamics. In Chapter 3, I measured epidermal cell geometry and cell wall stiffness over a wide area of the maize leaf. I found that the establishment of the boundary in the maize leaf was linked not just to local changes, but to broader growth and mechanical patterns outside the boundary. This suggests that coordination between the boundary and adjacent leaf zones is involved in the morphogenesis of the maize leaf. My findings further our understanding of how plant boundaries regulate growth.

Microtubule-based spindle formation is essential to faithful chromosome segregation during cell division. In many animal species, the oocyte meiotic spindle forms without centrosomes (the major microtubule organizing centers), unlike most mitotic cells. Even in mitotic cells, centrosomes are sometimes dispensable for bipolar spindle formation, indicating a redundant pathway initiating spindle assembly. In this study, we examined meiotic spindle assembly in C. elegans oocytes. We have demonstrated: First, metaphase I spindle formation is Ran-GEF and Ran-GAP independent in meiotic embryos. Second, free tubulin, also called soluble tubulin, concentrates in the nuclear volume during Germinal Vesicle Breakdown (GVBD) as well as in the spindle region during metaphase I and metaphase II. We then showed that the concentration of free tubulin at metaphase II spindle region is enclosed by dense ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules from the meiotic spindle. Similar observations are also shown in early mitotic cells. Together, this suggests free tubulin concentrating in the nuclear region might be a common mechanism promoting spindle formation through volume exclusion in both meiotic and early mitotic embryos in C. elegans. Moreover, the movement of molecules during GVBD depends on the size and the charge of the molecules.

Resting membrane potential (Vmem) and calcium signaling change as a function of developmental stage and location. Electrogenic genes, encoding ion channels and other proteins regulating bioelectric state, also change spatiotemporally. Although numerous genes regulate Vmem, it remains unclear how electrogenic gene dynamics are coordinated system-wide, particularly during early differentiation from embryonic stem cells (ESCs). We measured transcriptomes during differentiation of mouse ESCs towards forebrain-like cells in vitro and used bioinformatics to predict biological processes and upstream regulators of electrogenic genes. Electrogenic genes were dynamically expressed during differentiation, and predicted shared regulatory groups included genes related to cardiovascular development and cell adhesion (and neural). Identification of predicted regulators enables studies of the role of bioelectric signaling during differentiation and may offer a more fine-tuned understanding of Vmem mechanisms in development, including channelopathies. To complement analysis of electrogenic gene dynamics, we performed physiologic measurements for Vmem and calcium signal during early ESC differentiation. Changes in Vmem and intracellular calcium concentration [Ca2+]i regulate proliferation and differentiation of embryonic cells, with prior evidence suggesting calcium signaling is downstream of Vmem. However, it remains unclear whether changes in bioelectric state and calcium signaling in precursor cells are connected with their differentiation trajectory. To address this, we differentiated mouse ESCs in monolayer towards neuroectodermal (NE) or mesendodermal (MZ) like cells, measuring Vmem and [Ca2+]i every 24 hours for 4 days. Cultures featured morphologies characteristic of NE- and MZ-like differentiation, containing regions with both flattened and dense, three-dimensional morphologies. NE-directed cells were hyperpolarized, with lower [Ca2+]i relative to MZ-directed cells prior to germ layer marker acquisition (SOX1 or Brachyury). Colonies with clustered morphologies maintained relatively depolarized Vmem and higher calcium signals, closer to those of ESCs, despite their distinct cluster appearance. Our results demonstrate that divergences in Vmem and [Ca2+]i occur prior to germ layer marker protein expression and suggest that a cell’s bioelectric state is connected with differentiation trajectory from ES cells. The regional differences identified in both Vmem and [Ca2+]i suggest that even simplified stem cell model systems can provide a useful platform in which to study how stem cells regulate membrane potentials and calcium signals during early differentiation.

Metamorphosis is a radical morphological and environmental transition from a larval phase to an adult. This process is utilized by ~80% of all animal species and is a particularly common life history strategy among marine invertebrates. It has been well established that across species developmentally competent marine larvae utilize bacterial cues to indicate a suitable habitat conducive to adult survival and reproduction. Despite the high incidence of bacterially induced metamorphosis, the underlying mechanisms of how animals directly sense these signals remain under-studied, especially in Echinoderms (e.g., sea urchins). This study aims to a investigate component of the innate immune system, antimicrobial peptides (AMPs), as a potential candidate for connecting bacterial presence to metamorphosis. To explore AMPs as a player in bacterially induced metamorphosis, I utilized the rapid and synchronously developing sea urchin Lytechnius pictus. I first identified a homolog of the Echinoderm specific AMP Strongylocin 2 in L. pictus. I found that LpStrongylocin 2 (LpS2l) is only transcribed in early larvae in the event of bacterial infection. In nascent late-stage larvae, LpS2l transcription is upregulated and accumulates within cells in developing adult structures, likely a subtype of blastocoelar cells. The spatiotemporal regulation of LpS2l transcription in L. pictus larvae suggests a role for AMPs in the development of new structures and recognizing metamorphosis-inducing bacterial cues. Future experiments will be aimed at assessing functional role of LpS2l in metamorphosis by disrupting transcription using CRISPR/Cas9 sgRNA guides designed in this study.

The ability to move without pain is essential to many occupations and sports. When chronic tendon pain becomes limiting, it is diagnosed as tendinopathy. At a minimum, this tendon pain limits quality of life. For athletes engaged in professional sport, tendinopathy can be devastating and potentially career ending. The exact etiology of tendinopathy is unknown. However, it is especially common in activities that involve repetitive movements and high jerk (typing, jumping, etc.). Since this describes many sports and professions, it should not be surprising that tendon sprains, strains, and pulls are the leading cause of time away from work. The prevalence of tendon injury is highest in professional basketball with ~75% of players having patellar tendon lesions (Benítez-Martínez et al., 2019). Despite the prevalence and high personal and economic costs, there has been no progress in treatments for chronic tendon injuries over the last 25 years. This is in part due to a lack of understanding about basic tendon biology and adaptations that occur with growth, injury, and repetitive loading. This dissertation investigated post-natal development of the Achilles and patellar tendons, developed a rat model of patellar tendinopathy, determined the molecular response of different types (isometric and dynamic) of loads on a tendinopathic tendon, and characterized spatial gene expression in a healthy adult rat patellar tendon. The molecular and mechanical changes that occur during rat post-natal Achilles and patellar tendon development were characterized at P7 (before walking), P14 (shortly after the onset of walking), and P28 (motor maturity). The results are presented in Chapter 2. From P7 to P28, The Achilles increased 3-fold in length, whereas the patellar tendon increased largely in cross-sectional area. Despite the different modes of growth there was a ~10-fold increase in ultimate tensile strength of both the Achilles and patellar tendons. The increase in Achilles length resulted in a greater increase in modulus, whereas the greater cross-sectional area of the patellar meant a larger increase in maximal tensile load. The tendons shared transcriptional similarity at P7 and P14 but diverged at P28. However, there were still many shared processes indicative of tendon post-natal development. These changes included increased extracellular matrix (ECM) gene expression and decreased cell cycle and mitochondrial gene expression. Ribosomal gene expression also significantly decreased in the Achilles tendon and tended to decrease in the patellar tendon. From the genes that were highly expressed at P28 in the patellar tendon, STAT signaling was identified as a downregulated pathway. We hypothesized that these genes contributed to tendon radial growth. STAT inhibition in engineered human ligaments caused an increase collagen content and maximal tensile load (MTL). These results suggest that our developmental transcriptomic data may provide insight into targets to improve tendon function both after injury and during development/training.There are many models of rodent tendon injury; however, no molecular comparison had been made to the human condition. We modeled tendinopathy in the rat using a biopsy punch to remove the central third of the patellar tendon, followed by two weeks of cage activity to allow scar formation. The scar tissue was sequenced with 3’ Tag RNA-seq. Differential gene expression analysis of the healthy versus scar tissues identified an increase in ECM gene expression, decrease in mitochondrial gene expression, and no change in inflammatory pathways. The increase in ECM and decrease in mitochondrial genes meant that the scar was not recapitulating the developmental pattern of gene expression observed in Chapter 2. There was also a visible increase in vascularity and cellularity in the scar tissue. These changes within the tendon are nearly identical to human tendinopathy (Jelinsky et al., 2011). Next, we studied the effect of two distinct types of mechanical load (dynamic and isometric) on gene expression in tendinopathy. The isometric load (4 x 30 s) caused an increased in scleraxis and collagen Ia1 expression relative to the contralateral control leg. In contrast, the time under tension-matched dynamic load (360 x 0.33 s) caused in increase in type II collagen expression relative to the isometric loaded group. These data suggest that isometric loads may be used to treat a tendon injury and that the type of load, rather than the time under tension, is an important consideration when exercising a tendinopathic tendon. The last chapter of the dissertation presents the first complete report of spatial gene expression in tendon. I used the 10X Visium Platform to profile cell type and gene expression location in two adult rat patellar tendon samples. The cell type classification was performed with an anchoring analysis to a single cell RNA sequencing data set from mouse Achilles tendon (de Micheli et al., 2020). There were four cell populations identified: tendon fibroblasts (two main subpopulations), red blood cells, immune cells, and pericytes. The tendon midsubstance contained one tendon fibroblast subpopulation, while the other tendon cell subpopulation was located to the periphery of the main population and within the strip of connective tissue to the side of the tendon. This loose connective tissue also contained red blood cells, immune cells, and pericytes. The top expressed spatially variable genes in both samples were genes with known function in tendon (Col1a1, Dcn, Fmod, Sparc, and Comp). Interestingly, the two most common collagens 1a1 and 3a1 were expressed in separate populations of fibroblasts. I also identified one gene (AABR07000398.1) without a known function that was highly expressed in a pattern opposite to Col1a1. These data are important because they describe the spatial separation of two distinct fibroblast populations and provide the basis for better understanding spatial gene expression following injury.This dissertation provides novel insight into tendon post-natal development and spatial gene expression. It also validates a rodent model of tendinopathy and identifies molecular changes that occur after two different types of exercise on a tendinopathic tendon. Together, these data support a model where force is passed through the center of a healthy tendon and this force is transmitted through a matrix that is maintained by a population of tendon fibroblasts. Following injury, the scarred portion of the tendon is shielded from stress/strain and this may allow the infiltration of the tendon by a second population of fibroblasts. In order to return normal function of the tendon after injury, long holds are necessary to overcome stress shielding. Further, my data suggest that JAK/STAT inhibitors may improve tendon regeneration after injury. These data fill important knowledge gaps in the field of tendon biology, provide evidence for the first drug to treat tendinopathy, and establish the basis for future studies to reverse tendinopathy.

Asymmetric cell division is the process in which one cell divides to give rise to two daughter cells with different cell fates. This process is important throughout development and during stem cell maintenance. Defects in this process can lead to developmental defects and cancer. There are three important steps for a cell to divide asymmetrically. First the cell must generate a polarity axis, such as an anterior-posterior axis. Next the cell must distribute cell fate determinants, such as transcription factors, along this polarity axis. Finally, the cell must orient its spindle along this polarity axis so that when the cell divides each daughter cell receives the correct cell fate determinants. My dissertation work aimed to better understand this process. Specifically, I examined how a cell generates a polarity axis and how the spindle orients along this axis using the early C. elegans embryo as a model.In the one-cell C. elegans embryo, polarity is established by the highly conserved PAR proteins which form two mutually exclusive domains on the membrane, in the anterior and posterior. These domains are maintained by the kinase activity of PKC-3 in the anterior and PAR-1 in the posterior. Polarity establishment and maintenance at the one-cell stage have been well studied, but the mechanisms of polarity establishment in the P1 cell had not been examined. In my work, I found that there are two redundant pathways for polarity establishment. First I identified a novel early pathway in which PAR-1, and its downstream cytoplasmic factors MEX-5 and PLK-1 are required. Then through double mutant analysis, I identified a redundant pathway, similar to the P0 cell pathway, in which AIR-1 and actomyosin flow are required. The PAR polarity proteins also control cytoplasmic polarity and the orientation of the spindle to generate an asymmetric cell division. One of the downstream targets of the PAR proteins is LET-99, which localizes into a posterior-lateral band and acts as the link between cortical polarity and spindle positioning. LET-99 locally inhibits the force generating complex in its band causing asymmetric pulling forces which orient the spindle along the anterior-posterior axis. My work aimed to identify the mechanism by which LET-99 is localized to the membrane and how it is restricted from the anterior. Through a structure function analysis of LET-99, I found that the C-terminus of LET-99 is required for its cortical localization. I also found that PAR-3, PKC-3, and CDC-42 are all required for LET-99 localization. Our analysis of different LET-99 deletions is consistent with a role for PKC-3 in phosphorylating LET-99 to inhibit anterior localization. Overall, my studies contribute to our understanding of how cells generate a polarity axis and regulate spindle positioning.

Confined cell migration is a critical part of many biological processes. It is essential in developmental events such as neurogenesis, it’s important during the immune response as immune cells must migrate through endothelial tissue, and it is also a hallmark of cancer metastasis. The rate of confined cell migration is usually limited by the rate of nuclear migration as the nucleus is usually the largest and most rigid organelle of the cell. There are several factors that contribute to nuclear deformability such as lamin composition, heterochromatin levels, and the cytoskeletal network. Much of what we know about confined nuclear migration and the factors that regulate this process comes from in vitro studies. Most of these in vitro experiments utilize fabricated 3D matrices with constrictions that cultured cells can migrate through. While these experiments have been vital in our understanding of how this process is regulated, it is still unclear how this process is modulated in vivo.Our lab has developed an in vivo model to study nuclear migration through constricted spaces by observing hypodermal precursor cells called P cells in Caenorhabditis elegans. During the early L1 larval stage, P-cell nuclei have to move from a lateral position to a ventral position by migrating through a narrow constriction that is about 5% the diameter of the nucleus. If this process is successful, the P cells are able to divide and develop into vulval cells and GABA neurons. The canonical pathway for P-cell nuclear migration is through the Linker of the Nucleoskeleton and Cytoskeleton (LINC), which is made up of the SUN protein (UNC-84) found at the inner nuclear membrane and the KASH protein (UNC-83) found at the outer nuclear membrane. UNC-84 binds to UNC-83 which is able to interact with microtubule motor proteins. I show that conditional knockdown of DHC-1 leads to a significant nuclear migration defect and therefore dynein is the main microtubule motor protein involved.Our lab has shown that disruption of the LINC complex leads to a temperature sensitive phenotype, suggesting there is an alternative pathway that functions parallel to the LINC complex pathway. From a forward-genetics screen, we were able to identify several putative actin regulators that are involved in this alternative pathway, highlighting the major role that actin plays in P-cell nuclear migration. One of the genes identified is cgef-1, which codes for a GEF that is able to activate CDC-42. I hypothesized that CGEF-1 activates CDC-42, which indirectly activates the Arp2/3 complex to generate branched actin. I show that CDC-42, the Arp2/3 complex, and non-muscle myosin II (NMY-2) are required for P-cell nuclear migration in the absence of the LINC complex at the restrictive temperature. I also show constitutively active CDC-42 is able to partially rescue the nuclear migration defect of the cgef-1(-); unc-84(-) double mutant.I propose future work to determine if other GEFs are involved in regulating CDC-42 and if CDC-42 regulates both the Arp2/3 complex and NMY-2. It would also be informative to observe localization of CGEF-1, CDC-42, the Arp2/3 complex, and NMY-2 to further understand the mechanism by which these proteins function. An area of interest for further studies would be to determine how much nuclear actin plays a role in confined nuclear migration.

Type one diabetes is an autoimmune condition characterized by the loss of insulin producing beta cells of the pancreas. A loss of beta cells leads to a dysregulation in glucose homeostasis and results in prolonged hyperglycemia. If left untreated, numerous secondary complications can result such as neuropathy, nephropathy, retinopathy, limb amputation, and eventually death. Current treatments for type one diabetes rely on the administration of exogenous insulin or cell transplantation methods which have a variety of problems such as donor scarcity, a high cost, and requires immunosuppressants to be taken. Our lab is interested in inducing the body to produce its own beta cells endogenously via neogenesis. Since the mammalian pancreas is limited in its capacity to regenerate beta cells, zebrafish function as an excellent model to study neogenesis because they have a beta-cell progenitor called a centroacinar cell. Zebrafish centroacinar cells are like mammalian Ngn3+ progenitor cells of the developing pancreas. However, zebrafish centroacinar cells retain their progenitor capacity in adult fish. From mammalian models, centroacinar cells have also been suggested as a potential origin of pancreatic cancer. This is because centroacinar cells express similar pathways utilized by pancreatic cancer. These pathways are also conserved in zebrafish centroacinar cells. In this thesis, I am interested in further understanding the biology of centroacinar cells in order to elucidate (1) how zebrafish centroacinar cells can function as an in vivo model to better study the pathways involved in pancreatic cancer and (2) how the pathways utilized by zebrafish centroacinar cells allow them to behave as a beta-cell progenitor compared to mammalian centroacinar cells. In Chapter 1 I focused on the importance of the SOX9 pathway in pancreatic cancer and related this pathway back to zebrafish centroacinar cells. Previous work in our lab used RNA-seq and ChIP-seq in PANC-1 cells to identify the targets of SOX9. One of these targets was EPCAM. Using zebrafish, I demonstrated that epcam is expressed in centroacinar cells and is regulated by levels of sox9b. In Chapter 2 I focused on improving our lab’s nitroreductase/metronidazole transgenic zebrafish line to facilitate beta-cell regeneration. Using this line, I have shown that a lower dose of prodrug is required to ablate beta cells. As a result of this lower dose, it reduces the toxic side effects experienced by both larvae and adult fish. Unlike our old nitroreductase transgenic line, adult fish expressing this improved nitroreductase can achieve complete beta-cell loss by simply immersing them in prodrug. Additionally, my improved nitroreductase model can also be used to study chronic hyperglycemia which could not be done using our old nitroreductase model. In Chapter 3, I took advantage of using single-cell RNA sequencing to uncover centroacinar cell heterogeneity. Uncovering heterogeneity is important because it has allowed us to further uncover the role of centroacinar cells as a beta-cell progenitor but also as a potential origin for pancreatic cancer. From my genomics study, I have identified various subpopulations of centroacinar cells including a potential transitioning subpopulation of acinar cells toward a centroacinar cell fate, an immune-like cell population of centroacinar cells, and a potential endocrine precursor cell population. Additionally, I used both an in silico and in vivo approach to uncover new marker genes of centroacinar cells that may have an important role in their progenitor and cancerous behavior.

Collective cell migration is crucial for development and the preferred mode of migration by metastatic tumors, but much about it is unknown. The border cell cluster in the Drosophila ovary is an ideal model for collective cell migration, as border cells migrate on and between nurse cells. The cytoskeleton is a critical regulator of cell migration. Septins, now considered the fourth cytoskeletal element, remain unexplored in collective systems. The objective of this study was to understand the functions and necessity of septins in border cell migration. Using RNAi, mutants, and over-expression, we investigated the impact of septins on border cell migration and cluster morphology through fixed and live imaging. We found that knocking down septins significantly impacted border cell migration and cluster morphology through detachment failure and failure to form stable forward-directed protrusions. Overexpressing septins also dramatically impacted migration, with an inverse effect on morphology. We used high-resolution Airyscan imaging paired with Tissue Cartography to generate 3D models of the surface of the cluster and measure spectral decomposition with changes in septin expression. We also created 2D binary masks of border cells and quantified morphological changes due to septin expression. To uncover the mechanistic role of septins in border cell migration, we tested candidates that may interact with septins. Myosin colocalized dynamically with septins, but surprisingly they do not directly influence each other. Instead, septins and myosin are independently regulated by Rho. Septins and myosin regulate border cell membrane texture and contractility to allow for proper cortical tension, protrusion formation, and migration.

Prostate inflammation is associated with prostate cancer (PCa) development. Basal-to-luminal cell differentiation, a process partly regulated by canonical Wnt signaling, plays a crucial role in PCa initiation. Here, using a basal cell lineage tracing mouse model, I investigated how loss of Wnt/β-catenin signaling in basal cells affects basal-to-luminal differentiation during E. coli-induced prostatitis. Results revealed that β-catenin-null basal cells still gave rise to luminal cells, albeit with reduced capacity compared to wild-type cells. Further exploration using single-cell RNA sequencing (scRNA-seq) could unveil distinct subpopulations arising from these basal cells during prostatitis. Future studies should also aim to minimize inflammation variation in bacterial prostatitis models.Additionally, I examined how knockout of androgen receptor (AR) in stromal cells affected stromal-to-epithelial signaling. Bulk RNA sequencing of WT and AR-KO stromal cells and differential gene expression analysis identified potential gene candidates for future investigations into the impact of stromal AR deletion on PCa progression. This study provides insights into the intricate cellular and molecular processes underlying PCa initiation and development, shedding light on potential therapeutic targets.

The acquisition of cell fate is dependent on gene regulatory networks that are regulated spatiotemporally by cell type specific transcription factors (TFs). In binding to a class of cis-regulatory elements known as enhancers, TFs stimulate transcription at target genes. Studies on enhancers have revealed that enhancers exist in multiple different states and interconvert between them. The establishment of an active, transcriptionally promoting enhancer state from an inactive state is a stepwise process initiated by TFs and then facilitated by chromatin regulators. Here, we investigate the molecular processes required to establish an active, transcriptionally promoting enhancer state. To study enhancer activation, we utilize an in vitro embryonic stem cell differentiation system known as the naive to formative transition that recapitulates gene regulatory events that occur in vivo during mouse early embryogenesis. We first investigate the generalizability of current molecular models of enhancer activation in Chapter 2. Using the naive to formative transition and genetic deletions we find that the homologous enzymes MLL3/4 (KMT2C/D) are required for activation of some but not all enhancers as previously thought. Moreover, surprisingly, there is an underwhelming impact on gene expression changes during the transition despite loss of MLL3/4 and the loss of “active” molecular signatures at many enhancers. This work demonstrates the existence of multiple modes of enhancer activation and suggests a more cell-context specific role for the key chromatin regulators MLL3/4 than known before. In ongoing work in Chapter 3, we are investigating the current prevailing molecular model of enhancer activation more deeply. We built a new synthetic system using the TF Grhl2 for rapid and conditional induction of enhancer activation. We are currently employing this system to identify the mechanistic relationships between Grhl2, MLL3/4, other key chromatin regulators, and transcription. Our discoveries on the molecular fundamentals of enhancer state dynamics serve as a foundation on which to better understand drivers of human development and disease.

Projection neurons underly cortical computation and are necessary for proper cortical function. Understanding how these cell types arise is a central question in neuroscience and has wide-ranging implications for treating brain diseases. Genes encoding for transcription factors or chromatin remodeling proteins have been identified that are necessary for generating projection neuron cell types in the cerebral cortex. Exactly how they function is unknown. Using genetically engineered mouse models combined with immunohistochemistry, bulk and single-cell RNA sequencing, chromatin profiling, circuit tracing, and electrophysiology, this work uncovers the molecular mechanisms by which the transcription factors Fezf2, Satb2, and the transcriptional corepressor Tle4, specify cell fate in the developing cerebral cortex. Fezf2 specifies cell fate by functioning as a selective repressor. It regulates subtype-specific identities of corticothalamic and subcerebral neurons by selectively repressing the expression of genes inappropriate for each neuronal subtype. Tle4 works collaboratively with Fezf2 in layer 6 to inhibit layer 5 subcerebral neuronal genes. On the other hand, Satb2 is involved in gene activation and repression; these functions are likely mediated by interactions with ATP-dependent chromatin remodeling complexes. This work also demonstrates that Satb2 dosage affects cortical development, shedding light on the etiology of patients with a mutant SATB2 allele.Fezf2, Tle4, and Satb2 all function to specify subtype identity in postmitotic neurons. These results indicate that a cortical glutamatergic identity is specified by progenitor cells, but subtype-specific identity is achieved through postmitotic fate refinement.

Despite current understanding of transcriptional and epigenetic programs regulating transitions of human embryonic stem cells between distinct stages of pluripotency, our knowledge of the cell biology of changes in pluripotency states remains limited. We report a dynamic remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency that includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. The actin ring requires activity of the Arp2/3 complex and traction force microscopy suggests a role in limiting cell-substrate tensional forces. Arp2/3 complex activity is also necessary for effective transition to the naïve pluripotency state, including translocation of the Hippo pathway effectors YAP and TAZ from the cytosol to the nucleus. In hESCs with inhibited Arp2/3 complex activity, expressing a nuclear-localized YAP-S127A restores assembly of the actin ring and the naïve pluripotency state, including established markers and colony formation. Together these new findings on the cell biology of hESC reveal a signaling network of Arp2/3 complex activity for dynamic remodeling of contractile actin filaments and YAP/TAZ activity for transition to the naïve pluripotency state.

Astrocytes play essential roles in the developing nervous system, including supporting synapse maturation. These astrocyte support functions emerge coincident with brain maturation and may be tailored in a region-specific manner. For example, gray matter astrocytes have elaborate synapse-associated processes and are morphologically and molecularly distinct from white-matter astrocytes, raising the question of whether there are unique environmental cues that promote their identity and synaptogenic function. We previously identified adrenergic receptors as preferentially enriched in developing gray versus white matter astrocytes, leading us to hypothesize that noradrenergic signaling might promote the functional maturation of gray matter astrocytes. We first characterized noradrenergic projections during postnatal brain development in mouse and human, finding that they are preferentially enriched in gray matter and increase in density in concordance with astrocyte maturation. RNA-sequencing revealed that astrocytes in both species expressed ɑ and β adrenergic receptors. We found that stimulation of β adrenergic receptors increased the primary branching of rodent astrocytes in vitro, while conditional knockout of the β1 adrenergic receptor in astrocytes in vivo reduced the size of gray, but not white matter, astrocytes, and led to dysregulated sensorimotor integration in female animals. These studies suggest that adrenergic signaling during development directly impacts astrocyte morphology and can impact behavior. More broadly, they demonstrate a mechanism through which environmental cues impact astrocyte development. Given the key roles of norepinephrine in brain states such as arousal, stress, and learning, these findings could prompt further inquiry into how developmental stressors impact astrocyte development and adult brain function.

Asymmetric cell division (ACD) is an important mechanism that generates cellular diversity during development. Not only do asymmetric cell divisions produce daughter cells of different fates, many can produce daughters of different sizes, which we refer to as Daughter Cell Size Asymmetry (DCSA). In C. elegans, apoptotic cells are frequently produced by asymmetric divisions that exhibit DCSA, where the smaller daughter dies. We focus here on the divisions of the Q.a and Q.p neuroblasts, which produce apoptotic cells and divide with opposite polarity using both distinct and overlapping mechanisms. The PIG-1/MELK and TOE-2 proteins both regulate DCSA and specify the apoptotic cell fate in both the Q.a and Q.p divisions. In many asymmetric cell divisions, the non-muscle myosin NMY-2 is involved in properly positioning the cleavage furrow to produce daughters of unequal size. It was previously reported that NMY-2 is asymmetrically distributed and required for the DCSA of Q.a but not Q.p. In this study, we examined endogenously tagged reporters of NMY-2, TOE-2, and PIG-1 and found that all were asymmetric at the cortex during both the Q.a and Q.p divisions. TOE-2 and NMY-2 were biased toward the side of the dividing cell that would produce the smaller daughter, whereas PIG-1 was biased toward the side that would produce the larger daughter. We used temperature-sensitive nmy-2 mutants to determine the role of nmy-2 in these divisions and found that these mutants only displayed DCSA defects in the Q.p division. We generated double mutant combinations between the nmy-2 mutations and mutations in toe-2 and pig-1. The nmy-2 mutations did not significantly alter the DCSA of the toe-2 and pig-1 mutants but did alter the fate of the Q.a and Q.p daughters. This finding suggests that NMY-2 functions together with TOE-2 and PIG-1 to regulate DCSA but plays an independent role in specifying the fate of the Q.a and Q.p descendants.

Cell growth and differentiation are highly regulated to allow organisms to develop properly. During differentiation, cells often switch entire gene transcription and translation programs in order to accommodate changing metabolic and functional needs. One way differentiation is regulated is through changes to the cell’s ribosome concentration. Decreases in ribosome concentration tend to move cells away from proliferation and towards differentiation. This regulation of cell physiology at the level of ribosome concentration has important implications for human diseases, including cancer and neurodegenerative disorders.This work describes a mechanism influencing cell differentiation in the developing Drosophila nervous system. While it is generally common that progeny cells have lower concentrations of mature ribosomes than their progenitors, the mechanisms behind this change are poorly understood in the nervous system. I demonstrate that decreased ribosome concentration in neurons compared to neuroblasts is driven by a significant decrease in ribosomal RNA transcription in these post-mitotic cells. My data also show that high levels of translation in newborn neurons is supported exclusively by ribosomal RNA that is inherited from their progenitors.This work adds to a growing body of evidence linking changes in ribosome concentration to changes in the cell cycle and to the proteome. Additionally, it lays the groundwork for future studies of this kind to proceed with confidence using the developing brain of Drosophila as a model.

In multicellular organisms, asymmetric cell division generates cell type diversity during both development and tissue homeostasis. For successful asymmetric division to proceed, a cellular axis of polarity, or asymmetry, must be established via any of several molecular mechanisms. This step is followed by two sequential processes requiring precise coordination of the various components of the cytoskeleton. First, the mitotic spindle must be positioned in alignment with the axis of polarity; second, cytokinesis must occur at the right position and time, as instructed by the spindle, to segregate chromosomes and bisect the axis of polarity. In this dissertation I present my contributions to two research projects related to cytoskeletal regulation during asymmetric cell division. Chapter I reviews the state of the field prior to my studies and introduces the two research projects in context. Chapter II is a manuscript in revision for Journal of Cell Science that relates our findings from the first project, which aimed to understand the role of the DEPDC1 homolog LET-99 during cytokinesis. LET-99 was previously characterized as a negative regulator of the dynein-containing force generating complex during spindle positioning, but had also been shown to play a separable role in cytokinesis. With others, I demonstrated that during the first mitotic division LET-99 and the Rac1 homolog CED-10 have antagonistic roles in regulating the balance of branched vs linear actin during cytokinesis to achieve robust and timely furrowing. These roles appear to be global rather than spatially restricted to the contractile ring. However, the findings suggest that CED-10 does not interact with LET-99 to promote spindle positioning in the first mitotic division, further confirming that LET-99’s role as a regulator of the actomyosin cytoskeleton and cytokinesis is separable from its role in spindle positioning. Chapter III is a manuscript in preparation that documents our investigations into the role of CED-10 in spindle orientation of the asymmetrically dividing EMS cell. Two signaling pathways that regulate this spindle orientation were previously identified: a Wnt/Frizzled pathway and a MES-1/SRC-1 pathway. I determined that CED-10 works upstream or at the level of SRC-1 in the MES-1/SRC-1 signaling pathway and furthermore that CED-10 contributes to cortical localization of the same dynein-containing force generating complex involved in spindle positioning at the one-cell stage. I also found that CED-10’s molecular function in this context may be to promote branched actin formation at the EMS/P2 contact, but this evidence is partially contradicted by our findings that loss of the branched-actin regulator GEX-3 does not enhance the rate of EMS spindle rotation defects in Wnt/MOM-2-depleted embryos. An additional curious finding is that CED-10 has a previously unreported role in P1 spindle rotation, as do the branched actin nucleator Arp2/ARX-2 (Arp2) and the Frizzled ortholog MOM-5.Although this research was performed using nematodes as a model organism, the results have broad implications for all animals because most of the molecular components involved are evolutionarily conserved. By understanding in detail the mechanisms of different types of asymmetric divisions, we can understand both normal development and diseases such as cancer, which involves the dysregulation of asymmetric division.

Pancreatic islets contain several types of hormone-expressing cells to help regulate glucose homeostasis. Chief among these is beta cells which secrete insulin to lower blood glucose levels. Diabetes, which currently impacts over 37 million Americans, ensues when beta cells are destroyed by the body’s own immune system or when they become dysfunctional caused by a myriad of genetic and environmental factors. Consequently, the major question becomes how best to replace the lost and impaired beta cells. To this end, we discovered a novel population of immature virgin beta cells which may potentially serve as a new source to generate functionally mature beta cells as a therapeutic treatment for diabetes.In this dissertation, we further characterized virgin beta cells to learn more about their unique properties that make them distinct from other known beta cell subpopulations. Using various transgenic mice, complex lineage-tracing techniques, and immunohistochemical analyses, we determined several novel insights about virgin beta cells. Chapter 1 reviews the discovery of virgin beta cells and the many unanswered questions remaining about them. Chapter 2 examined the proliferation capacity and maturation potential of virgin beta cells. Chapter 3 elucidated the role of pancreatic alpha and delta cells in the formation and maintenance of virgin beta cells. Chapter 4 interrogated the contribution of artemether, an anti-malarial drug, in the generation of new beta cells via beta cell transdifferentiation from alpha cells. Lastly, Chapter 5 summarizes all the original, including unpublished, work performed in this dissertation, concluding remarks, and various exciting direction for future studies.

Entorhinal cortex (EC) cooperates with the hippocampal formation (HPF) in episodic memory encoding and spatial navigation, and it is one of the first human brain regions to degenerate in Alzheimer’s disease (AD). EC layer II (L2) contains unique projection neurons, including Reln+ stellate neurons (SNs) and clusters of Calb1+ pyramidal neurons (PNs), that are selectively vulnerable to AD and remain poorly understood. Therefore, we aim to better characterize these neuron types with a focus on neurogenic trajectories and gene expression patterns.Chapter 1 (Introduction) provides key background on the following: cortex organization; EC structure, function, and neuron types; classic “inside out” neocortical development versus current understanding of EC development; EC in disease; and transcriptional profiling of EC cells.Chapter 2 is an original study into developmental timelines and trajectories of EC principal neurons, in which we address the hypothesis that some EC neurons undergo specialized migration and differentiation. Through thymidine analog birthdating, we demonstrate that murine EC diverges from classic “inside out” neocortical neurogenesis with a population of early-born L2 SNs (but not PNs) that develops concurrently with deeper layers. We also dismiss the possibility precocious SNs may derive directly from early, potentially multipotent neuroepithelial cells (NECs) or radial glial cells (RGCs) by lineage tracing both PNs and SNs from neuron-fated intermediate progenitor cells (IPCs) despite their differing birthdates. Chapter 3 details preliminary work in establishing a comparative atlas of EC neurons with the aim of better understanding (1) intrinsic properties that may influence differential disease susceptibility across neuron types and (2) functional significance of an early-born SN population. We perform single nucleus RNA sequencing (snRNAseq) on neuronally-enriched nuclei from murine and human EC, annotating clusters based on marker gene expression and laminar positioning. In a new collaboration with the Scheuermann Lab at J. Craig Venter Institute, we determine minimal marker genes that define each cell cluster, elucidating several new PN and SN markers. We also begin to probe differentially-enriched biological pathways between neuron types.Finally, Chapter 4 provides a Summary and Future Work, including proposed experiments for understanding early SN development and plans for ongoing snRNAseq analysis.

The separation of embryonic and extraembryonic lineages is a process that has evolved in amniotes to adapt for fetus survival on land. One of the major roles of the extraembryonic tissues is to provide nutritive support sourced from the maternal environment, since development occurs in utero. The initial differentiation of the extraembryonic lineages occurs first prior to embryonic cell differentiation to prioritize cell survival. Thus past studies have extensively focused on understanding these lineage segregation mechanisms which take place in three major waves of lineage differentiation events. The key commonality of these events begins with a bipotent progenitor that co-expresses lineage-specific transcription factors (TFs), which is resolved by mutual inhibition of TFs and key signaling pathways. During the first lineage breaking event, differential Hippo signaling activation and cross-antagonism of POUF51(OCT4) (ICM TF) and CDX2 (TE TF) forms the trophectoderm (TE) and the inner cell mass (ICM). Likewise, in the second lineage breaking event, differential Fgf signaling activation and cross-antagonism of NANOG (Epi TF) and GATA6 (PrE TF) regulates the ICM bifurcation to the epiblast (Epi) and primitive endoderm (PrE). Next, the extraembryonic trophectoderm differentiate to facilitate the implantation process. Concurrently, during this rapid and dynamic event, the PrE differentiates into the parietal endoderm (PE) and the visceral endoderm (VE). However, less is known about the regulation of these extraembryonic endoderm (ExEn) lineages partly due to inaccessibility of these cells during implantation. Therefore, the molecular basis of PrE cell fate divergence to PE or VE and the concerted actions of TFs that moderate PE- versus VE-cell gene regulatory programs remains to be elucidated. To investigate the regulative behavior of ExEn cells, we analyzed scRNA-seq datasets of E4.5 mouse embryos. We delineated PrE, PE and VE cell states and resolved TFs associated with PE or VE. Comparative motif analysis of the enhancer repertories of VE cells and PE cells suggests that GATA6, SOX17, and FOXA2 are core TFs in the ExEn gene regulatory network. To test this model, we performed transcriptome analyses on cXEN cells using degron tagged GATA6 and SOX17 combining with FoxA2 knockout revealed that PE development requires positive GATA6 and SOX17 inputs, whereas VE development requires FOXA2 to activate VE gene program suppressed by GATA6 or SOX17. Next, we found that BMP signaling cues instruct the PE-VE lineage decision. These data reveal a core gene regulatory module that underpins PE and VE cell fate choice. Lastly, we compare GATA4 and GATA6 functional activities in PE cells, which suggest they have overlapping functions in ExEn development. Overall, this dissertation will present a characterization of the ExEn gene regulatory network (GRN), while also proposing new models and regulators that govern ExEn development.

Duchenne muscular dystrophy (DMD) is caused by an out-of-frame mutation in the DMD gene that results in the absence of a functional dystrophin protein, leading to a devastating progressive lethal muscle-wasting disease. Muscle stem cell-based therapy is a promising avenue for improving muscle regeneration. However, despite the efforts to deliver the optimal cell population to dystrophic muscles, little is understood on the role endothelial cells play during systemic delivery. Recent single cell RNA sequencing (scRNA-seq) advances has permitted the unraveling of cellular composition and phenotypes in multiple mouse tissues, including skeletal muscle. Here we describe the development of an optimized protocol for systemic delivery of skeletal muscle progenitor cells (SMPCs) which showed limited ability to escape the endothelial barrier in dystrophic and severely dystrophic muscle. To further understand the role of the microenvironment as a barrier to systemic cell delivery to skeletal muscle we explored skeletal muscle-resident cell populations in healthy, dystrophic and severely dystrophic mouse models utilizing scRNA-seq. We found an increased frequency of activated fibroblasts, activated fibro-adipogenic progenitor cells and proinflammatory macrophages in dystrophic and severely dystrophic gastrocnemius muscles. Moreover, employing a computational intercellular interaction method, we show an upregulation of extracellular matrix and platelet aggregation genes on endothelial cells in dystrophic and severely dystrophic muscles. We further show an increased risk of clotting especially in the severely dystrophic environment. This work extends our understanding of the severe nature of DMD, which should be taken into account when considering stem cell-based systemic delivery platforms.

Organelle positioning is important in many cellular and developmental processes, especially for the function of polarized and spatially extended cells. For example, in mammalian myofibers, most nuclei are evenly spaced out at the periphery of the fiber, except for a few that group into small clusters anchored under the neuromuscular junction. Disruption of nuclear positioning is found in many muscular diseases. Nuclear positioning is mediated by the conserved bridge termed the LINC (Linker of nucleoskeleton and cytoskeleton) complex, which is formed by the interaction between inner nuclear membrane SUN domain proteins and the outer nuclear membrane KASH domain proteins. The SUN proteins pair with different KASH proteins which contains various cytoskeleton interacting domains, to perform distinct functions. For example, in Caenorhabditis elegans, SUN protein UNC-84 either interacts with the KASH protein UNC-83 to move nuclei during development, or the KASH protein ANC-1 to anchor nuclei in the hyp7 syncytium. Interestingly, UNC-83 KASH is shorter and lacks the conserved cysteine predicted to be important to stabilize SUN/KASH interaction by structural and molecular dynamic simulations. In collaboration with other lab mates, we showed that the conserved intermolecular disulfide bond plays important roles for UNC-84/ANC-1 mediated nuclear anchorage but is dispensable for UNC-84/UNC-83 mediated nuclear migration. In addition, the longer KASH containing the DDEY residues predicted to be associated with the membrane also is necessary for ANC-1 KASH to function.ANC-1 has been proposed to physically tether nuclei to the actin cytoskeleton through its C-terminal KASH domain and the N-terminal calponin homology (CH) actin-binding domains. However, I found that deletion of the KASH domain causes less severe nuclear anchorage defects than null mutations. Moreover, the short isoform anc-1b lacking CH domains is sufficient for hyp7 nuclear anchorage. It was completely unknown how ANC-1 functions without CH and KASH domains. Interestingly, other than nuclear anchorage defects, the ER, mitochondria, and lipid-droplets were also severely unanchored and sometimes moved throughout the cytoplasm in anc-1 but not unc-84 null mutants. Unlike most KASH proteins which are enriched on the nuclear envelope, I found that GFP-tagged ANC-1 was mostly colocalized with the ER. To understand the mechanism by which ANC-1 regulates organelle positioning, I used CRISPR/Cas9 gene editing to delete different domains of ANC-1 and found that the spectrin-like repeat structures and the transmembrane domain play important roles in organelle anchorage. Deletion of the spectrin-like repeat region disrupted the protein’s ER-localization. These results led to a cytoplasmic integrity model where ANC-1 localizes to the ER and functions in positioning nuclei, the ER, mitochondria, and likely other organelles in place.The cytoplasm is a crowded environment filled with macromolecules. To test if ANC-1 anchors organelles through regulating the intracellular crowding, I expressed and analyzed the diffusion of 40nm GEMs (genetically encoded multimeric nanoparticles) in C. elegans hypodermal and intestine tissues. The diffusion of the 40nm GEMs is significantly increased in the cytoplasm of anc-1 null mutants in both tissues, indicating that ANC-1 may regulate the crowding of the cytoplasm. Ribosome concentration has been reported to play an important role in cytoplasmic crowding. RNAi of small ribosome protein rps-15 or rps-18 caused nuclear anchorage defects. In addition, in anc-1 mutants, the ribosome distribution is altered. These results indicate that ANC-1 may regulate the biophysical properties of the cytoplasm through the ER and ribosomes.

The proper organization and coordination of cells is essential to the integrity and function of tissues, organs, and multicellular organisms. While diverse organs and organisms have been used to study these foundational principles, the shear structural and compositional complexity of many in vivo experimental models raises numerous challenges to dissecting the cellular mechanisms underlying their formation and function. Conversely, in vitro models, despite limiting structural complexity, frequently fail to recapitulate the complete physiology of the organs from which they are derived. To ameliorate these tradeoffs, we exploited the anatomical simplicity and extraordinary regenerative capacity of the basal metazoan Hydra vulgaris to probe the mechanisms of tissue patterning and coordinated cellular physiology. In Chapter 2, we apply live microscopy and physical cellular manipulation to investigate a decades-old observation in which a disordered aggregate of Hydra cells sorts into inner and outer epithelial compartments, a prerequisite to regenerating a functional body plan. We show that differences in the rates of epithelialization between two primary cell types give rise to the bilayered structure of the intact animal. In Chapter 3, we investigate the function of these assembled tissues. Specifically, through the identification and dissection of a fluid engulfment phenomenon known as macropinocytosis, we reveal a role for tissue mechanics in regulating fluid uptake and membrane recycling in homeostatic Hydra epithelia. Together, these studies illustrate how the emergent properties of cell collectives, such as cell polarity and tissue mechanics, can contribute to the structure and function of living animals.

The tympanic membrane (TM) is the central component of the conductive apparatus of the ear and is the first major organ sound waves hit in the process of being transmitted to the brain. The TM receives sound from the external auditory ear canal (EAC) and appropriately vibrates to transmit this sound to the middle ear. Notably, the TM has the remarkable ability to rapidly repair itself, with perforations typically closing in days to weeks in all mammalian species studied. However, in a subset of cases, these perforations do not close, and patients present with conductive hearing loss. Though this function of the TM has been known for many decades, lack of understanding of the basic biology of the TM has hindered our ability to explain the pathophysiology of chronic perforations because the cellular and molecular mechanisms underlying this repair ability remain largely unknown. This study looked to thoroughly characterize the repair process of the injured TM as epimorphic regeneration and uncover mechanisms of epithelial regeneration that could serve as future targets for treatments for disorders of the tympanic membrane and be applied broadly to epithelial wound repair.

Embryos develop in dynamic environments and are subject to myriad stressors – both biotic and abiotic. Among those stressors are encounters with pathogens and toxic molecules. The systems for sensing and responding to the environment include a robust suite of molecular machinery that protects the cell, and a heterogeneous population of immune cells that mount coordinated responses. Establishing a whole-animal echinoderm embryo model to address embryo-environment interactions and how stressors influence such systems is the focus of this dissertation work. Accomplishing this task began with identifying a need for a sea urchin model species that grew more rapidly that other commonly used echinoderms, and establishing a comparable developmental staging scheme and culturing practices for rearing animals in the lab. The painted white urchin, Lytechinus pictus, was a clear choice for the optimal model. Generating molecular tools was also essential, and yielded a fully sequenced genome with chromosomal resolution and transcriptomic tools pooled across developmental stages. To assess the quality of these molecular resources I annotated the suite of ATP-binding cassette transporters (ABCs). ABCs are expressed in, and play important roles in handling of toxic molecules as well as in the differentiation and migration of, immune cells. In addition to the ABCs, I combed the genome for other components involved in immunity – including cytokines, transcription factors, enzymes, effectors, and signal mediators. This suite of annotations establishes a foundation of potential targets of exposure in the immune system. From there, I challenged sea urchin larvae with bacterial infection and characterized the first phenotypic responses of an echinoderm model of immunosuppression. The outcome of this research is a series of tools and resources that open doors for future investigation. With a reliable, dose-dependent model of immunosuppression and complimentary molecular tools in a rapidly developing echinoderm species, it is now possible to address the influence of toxic molecules on whole-animal immune responses. It is also possible to investigate how early life exposures alter later life health or the health of subsequent generations. Finally, we can begin to tease apart the cross-talk of developmental and protective systems in the embryo contributing to environmental sensing and signaling.

The centrosome functions as the main microtubule-organizing center in metazoan cells and orchestrates diverse cellular processes critical for development, including cell division, polarization, and migration. Centrosome dysfunction is associated with cancer and microcephaly in human, warranting a mechanistic understanding of its assembly in vertebrates. The centrosome is a membraneless organelle that consists of a pair of centrioles embedded in a proteinaceous network named pericentriolar material (PCM). The PCM is a complex ensemble of proteins enriched with coiled-coil domains (CCs) and low-complexity regions (LCRs). At the onset of mitosis, centrosomes expand the PCM to maximize their microtubule nucleation capacity. This process, termed centrosome maturation, ensures proper mitotic spindle assembly and chromosome segregation in mitosis. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. This dissertation work primarily focuses on understanding the functional contribution of pericentrin (PCNT), a conserved PCM component, to centrosome assembly in vertebrates. I found that endogenously expressed PCNT condenses into dynamic aliphatic alcohol-sensitive granules around centrosomes during late G2/early mitosis in human cells. These data suggest that full-length PCNT may undergo liquid-liquid phase separation (LLPS) in physiologically relevant conditions during centrosome maturation. Furthermore, the N-terminal and middle segments of PCNT, enriched with conserved CCs and LCRs, undergo concentration-dependent condensation. Formation of PCNT “condensates” exhibits characteristics of LLPS, including a sharp phase transition at a concentration threshold, as well as coalescence, deformability, and rapid fluorescence recovery after photobleaching. Moreover, these PCNT condensates selectively recruit endogenous PCM components and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate LLPS, a process that bestows upon the centrosome distinct properties critical for its assembly and functions. Another part of the dissertation centers on developing computational methods for image analysis to study centrosome assembly in a quantitative manner. To accurately detect and quantify centrosome-localized mRNAs and proteins at single cell resolution, I developed a streamlined workflow for performing, acquiring, and analyzing sequential immunofluorescence and single-molecule RNA fluorescence in situ hybridization data via 3D reconstruction in Imaris, followed by quantifications using MATLAB and R. Moreover, to automatically track the dynamics and spatiotemporal distribution of condensates relative to centrosomes, I developed TRACES (Tracking of Active Cellular Structures), a customizable and open-source pipeline capable of detecting, tracking, and quantifying fluorescently labeled cellular structures in up to three spectral channels simultaneously at single-cell resolution. Overall, my studies provide a molecular and biophysical framework, as well as quantitative image analysis methods, for dissecting mechanisms underlying centrosome assembly in vertebrates.

Hematopoiesis, the process of generating all blood and immune cells from hematopoietic stem cells (HSCs), is a tightly regulated process orchestrated by cell-intrinsic and cell-extrinsic cues. This process occurs in “waves” during fetal development, with windows of distinct and transient hematopoietic stem and progenitor cells (HSPCs) giving rise to “non-traditional” mature cell types. These cells are referred to as non-traditional because they arise during fetal development from these unique HSPCs, and then self-maintain for the lifespan without contribution from the adult (or life-long definitive) HSC pool. In contrast, adult hematopoiesis occurs in a very well-characterized hierarchy, where HSCs differentiate in a more lineage-restricted progression into more lineage restricted progenitor cells before terminally differentiating into “traditional” mature cell types. These cell types are referred to as traditional mature blood and immune cells because they are constantly replenished from the life-long definitive HSCs for the remainder of life. This body of work identifies cell-intrinsic and cell-extrinsic regulators of this process both during steady state health development and maintenance, as well as how dysregulation of this process alters hematopoietic output and function for life.

The goal of my thesis was to discover how basal cell multipotency is is regulated in the adult, homeostatic prostate. During development, basal stem cells actively differentiate to produce mature basal, luminal and neuroendocrine cells. Some basal cells retain this ability in adulthood, however the stem cell activity is heavily downregulated. Most of this thesis focuses on understanding cellular mechanisms that are responsible for modulation of basal stem cell activity. Chapter 2 examines a potential inhibitory signal emanating from luminal cell contact. Chapter 3 follows by investigating how the canonical Wnt and androgen receptor (AR) signaling pathways interact to induce basal stem cell activity. The 4th chapter takes a brief look into the internal programing of adult basal stem cells. A better understanding of the regulatory processes that govern stem cell activity is necessary in order to understand the pathogenesis of malignancies that arise from the alterations of these processes. To this end, the final chapter of this thesis uses mouse models of prostate adenocarcinoma to investigate the effect of stromal AR activity on cancer progression.

The goal of my thesis work was to resolve the molecular mechanisms governing mammary gland alveologenesis during pregnancy. Alveologenesis is the essential process by which alveolar progenitor cells undergo rapid expansion during early pregnancy and subsequent differentiation into mature milk-producing alveolar cells during late pregnancy, generating a functional mammary gland capable of providing nourishment for offspring. How the mammary gland maintains a balance between massive proliferation and functional differentiation, however, remains unclear. Here, we show that the mammary gland couples proliferation with differentiation by means of endoreplication, the cellular process of replicating DNA in the absence of cell division to generate a polyploid cell. Through pharmacological inhibition and transgenic loss-of-function studies, we show that the DNA damage response to replication stress is activated during the rapid proliferation of early pregnancy. This, in turn, results in early mitotic arrest through WEE1-mediated CDK1 inhibition and subsequent endoreplication during early lactation, generating a population of terminally differentiated polyploid alveolar cells that are essential for efficient milk production. Furthermore, Notch signaling has long been known to be essential for maintenance of the alveolar progenitor population, and it must be attenuated to achieve alveologenesis. Despite the importance of Notch signaling, however, the mechanisms that regulate Notch activity to ensure proper alveolar proliferation and differentiation during pregnancy are not well understood. Through transgenic loss-of-function and 3D organoid studies, our preliminary data suggest ROBO2 functions from the luminal epithelial compartment to promote expression of the Notch ligand JAG1 in the basal epithelial compartment, potentially through the direct inhibition of ROBO1 in basal cells. This, in turn, induces the Notch activity of alveolar progenitor cells in a juxtacrine manner, maintaining them in the progenitor state to inhibit their differentiation. Moreover, our preliminary data also suggest NOTCH1 activation inhibits the DNA damage response to replication stress and alveolar endoreplication through the interaction of its intracellular domain with the DNA damage response kinase ATR. Taken together, the results of my thesis work elucidate multiple molecular mechanisms by which the mammary gland achieves a balance between the rapid proliferation of alveolar progenitor cells and their functional differentiation into mature milk-producing alveolar cells during late pregnancy and lactation.

Despite nearly 5000 years of study and treatment, breast cancer remains the most common form of cancer in women. Breast cancer is the leading cause of malignant mortality in women, and its treatments are a significant cause of morbidity. Women with mutations in the gene Breast Cancer 1 (BRCA1) have a 70% chance of developing breast cancer in their lifetimes. While the molecular subtyping of breast cancer has formed the basis of targeted therapies, the majority of breast cancers that develop in women with a BRCA1 mutation will be ‘triple-negative’, with no targeted therapies, poor prognosis, and decreased overall survival. Changes in the BRCA1 breast epithelium have been observed before tumors are detected, however, BRCA1 mutations are carried in all cells of mutation carriers, not just the epithelium. The mesenchymal stromal cells pericytes, fibroblasts, and adipocytes have been observed to be an important, albeit enigmatic contributor to breast tumorigenesis and metastasis. These less well studied connective tissue stromal cells and the preneoplastic changes that occur in them before tumorigenesis are the focus of this dissertation. The overarching hypothesis of this dissertation is that the breast microenvironment is distorted in women with BRCA1 mutations, and that stromal cells contribute to cancer initiation. A persistent challenge in elucidating the roles of pericytes, fibroblasts, and adipocytes in the breast cancer microenvironment has been the lack of distinguishing molecular phenotypic markers of these cell types. To overcome this challenge, our lab has performed single cell RNA sequencing (scRNAseq) of more than 200,000 stromal and epithelial cells from a 22-patient cohort of high-risk BRCA1 mutation carrier and non-mutation carrier patient surgical breast samples. I show that scRNAseq has the resolution to distinguish fibroblasts and pericytes from one another, and develop a novel flow cytometry strategy to isolate these cells for the first time from the human breast and find that they have distinctive differentiation capacities. Using our scRNAseq data of preneoplastic breasts I identify pro-proliferative changes in stromal cell communications with epithelial cells in BRCA+/mut tissues. We test these signals from pericytes and fibroblasts and find that they have the potential to alter the cell proliferation and cell fate of breast epithelium. Finally, to determine the transcriptomic profile of adipocytes, which are not amenable to standard methods of droplet based single-cell sequencing, I develop a method to isolate the nuclei from adipocytes and submit them to scRNAseq. Using this method, I distinguish vast differences between the adipocytes of human breast tissues and those of murine mammary glands. As a body of work, this dissertation emphasizes the importance of mesenchymal stromal cells in the breast microenvironment and highlights the individually important roles they play in breast cancer tumorigenesis.

Infection in the adult organism drives cytokine-mediated inflammation that directly influences hematopoietic stem cell (HSC) function and differentiation within the bone marrow, but much less is known about the fetal hematopoietic response to maternal inflammation and infection during pregnancy. Here, we demonstrate that fetal hematopoietic stem and progenitor cells (HSPCs) respond to prenatal inflammation in utero and that the fetal response drives long-term changes to HSC function after transplantation. First, we modeled maternal immune activation (MIA) with the IFN-a stimulating viral mimetic, poly(I:C), and showed that fetal HSPCs exhibit changes in quiescence, expansion, and lineage-biased output in response to prenatal inflammation. Single-cell transcriptomic analysis of fetal HSPCs in response to MIA revealed specific upregulation of inflammatory gene profiles in discrete, transient HSC populations that propagated expansion of lymphoid-biased progenitors in the fetal liver.Next, we investigated the fetal hematopoietic response to maternal Toxoplasma gondii infection. T. gondii is an intracellular parasite that elicits Type II, IFNγ-mediated maternal immunity to prevent vertical transmission and promote parasite clearance. The production of excessive IFNγ during congenital toxoplasmosis has dire consequences for the developing fetus, such as lowered birth weights and premature abortion, but the effects to the developing immune system and the signals that mediate these interactions have not been investigated previously. Our examination revealed that the fetal inflammatory repertoire is distinct from the maternal response and is directly influenced by parasite virulence. We show that maternal IFNy crossed the fetal-maternal interface and was perceived directly by fetal HSCs, and that the response of fetal HSCs was dependent on the fetal IFNy receptor. Functionally, the heterogenous fetal HSC pool responded to aberrant inflammation with virulence-dependent changes in proliferation, long-term multi-lineage reconstitution, and self-renewal potential. By directly comparing the effect of maternal IFNy injection with congenital T. gondii infection of varying virulence, our observations delineate both a direct effect of IFNy on fetal HSCs and illuminate the independent role of additional inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors. Finally, in direct contrast to the adult hematopoietic response to infection and the fetal response to Type I interferon-mediated MIA, exposure to Type II interferon-mediated inflammation in utero did not impair fetal HSC function, even in response to severe infection. Our findings provide insight into the cues that direct fetal hematopoiesis in response to inflammation and begin to tease apart inflammatory cues that promote a beneficial hematopoietic response versus those that may ultimately be detrimental.

All eukaryotic cells depend on faithful cell division to give rise to daughter cells that contain the correct number of chromosomes. Chromosome segregation errors during meiosis in germ cells can lead to aneuploid gametes, which can cause infertility, genetic disorders such as Down Syndrome and miscarriages. Additionally, chromosome segregation errors during mitosis can lead to aneuploid somatic cells, which are a hallmark of cancer. Despite the importance of tightly regulating these two types of cell division, the mechanisms that ensure fidelity during these processes remain active areas of investigation. Proteins that have been shown to be important regulators of fidelity during meiosis in Caenorhabditis elegans (C. elegans) include ZHP-3 and PCH-2. ZHP-3 is a putative SUMO or ubiquitin ligase required for crossover recombination, but whether or not ZHP-3 is regulated to promote recombination remains poorly understood. PCH-2 is a conserved AAA+ ATPase required for timely coordination of the meiotic prophase events pairing, synapsis and recombination, but the mechanism for how it accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shown to be required for both silencing and activation of the spindle assembly checkpoint during mitosis, but how it regulates the checkpoint is still an active area of investigation. Here, I use C. elegans as a model system to investigate the molecular mechanisms of these two proteins during meiotic and mitotic chromosome segregation. In Chapter 1, I investigate whether ZHP-3 is phosphorylated by a conserved DNA damage kinase, CHK-1, to determine if this phosphorylation event is required for ZHP-3’s localization or function during meiosis. I find that while ZHP-3 is phosphorylated by CHK-1 kinase in vitro, mutation of these phosphorylation sites in vivo does not affect ZHP-3’s localization to meiotic chromosomes, or its function in promoting crossover recombination. In Chapter 2, I investigate a different regulator of meiosis, PCH-2, to determine how it coordinates meiotic prophase events and whether it acts on meiotic HORMADS to accomplish this. I find that mutations in PCH-2 genetically interact with mutations in the meiotic HORMADS HTP-3 and HIM-3, suggesting that these proteins work together to coordinate meiotic prophase events. Additionally, I find that PCH-2 acts on HTP-3 to coordinate pairing and synapsis, but acts on HIM-3 during recombination, indicating that the meiotic HORMADS are cleanly delegated to regulate specific meiotic prophase events. Finally, in Chapter 3, I explore PCH-2’s role during somatic cell division and investigate how PCH-2 regulates the strength of the spindle assembly checkpoint. I find that PCH-2 is more enriched in P1 cells compared to AB cells in the early C. elegans embryo, potentially explaining why cells that give rise to the germline have stronger checkpoints than somatic cells. Additionally, we find that this enrichment depends on cell fate factors and PCH-2’s adaptor protein CMT-1. Taken together, these results contribute mechanistic insight for how these proteins promote fidelity during meiosis and mitosis by determining post-translational modifications of meiotic recombination factors (Chapter 1), identifying potential substrates of meiotic prophase regulators (Chapter 2), and demonstrating how spindle checkpoint regulators are differentially inherited in specific cell types to give rise to differences in spindle checkpoint strength (Chapter 3).