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Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 48-51). Description: ix, 51 leaves : ill. ; 28 cm.

abstract: The development of the vertebrate musculoskeletal system is a highly dynamic process, requiring tight control of the specification and patterning of myogenic, chondrogenic and tenogenic cell types. Development of the diverse musculoskeletal lineages from a common embryonic origin in the paraxial mesoderm indicates the presence of a regulatory network of transcription factors that direct lineage decisions. The basic helix-loop-helix transcription factor, PARAXIS, is expressed in the paraxial mesoderm during vertebrate somitogenesis, where it has been shown to play a critical role in the mesenchymal-to-epithelial transition associated with somitogenesis, and the development of the hypaxial skeletal musculature and axial skeleton. In an effort to elucidate the underlying genetic mechanism by which PARAXIS regulates the musculoskeletal system, I performed a microarray-based, genome-wide analysis comparing transcription levels in the somites of Paraxis-/- and Paraxis+/+ embryos. This study revealed targets of PARAXIS involved in multiple aspects of mesenchymal-to-epithelial transition, including Fap and Dmrt2, which modulate cell-extracellular matrix adhesion. Additionally, in the epaxial dermomyotome, PARAXIS activates the expression of the integrin subunits a4 and a6, which bind fibronectin and laminin, respectively, and help organize the patterning of trunk skeletal muscle. Finally, PARAXIS activates the expression of genes required for the epithelial-to-mesenchymal transition and migration of hypaxial myoblasts into the limb, including Lbx1 and Met. Together, these data point to a role for PARAXIS in the morphogenetic control of musculoskeletal patterning. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2013

<p> Recent advances regarding commensals in the gastrointestinal tract point to an intimate &ldquo;accessory&rdquo; organ status. To study the cross-talk that an accessory organ must have, the Piletz laboratory began in 2014 developing a three-dimensional (3D) <i>in vitro</i> co-culture model system, whereby two differentiated cell lines are juxtaposed along with &ldquo;luminal&rdquo; contents. The model uses differentiated C2BBe1 cell line enterocytes grown to confluency on polycarbonate filters with 0.4 &micro;m pores over-layered atop SH-SY5Y cell line neurons to study cross-talk from either the lumen-side or the neuron-side. The focus is on an endogenous molecule, agmatine (1-amino-4-guanidobutane), made by gut bacteria at millimolar concentrations in the mucosa of the small intestine&mdash;yet in the brain known to be a neurotransmitter. Starting with each individual cell line in standard mono-cultures, agmatine was added at varying doses and varying times to replicate what is essentially dogma to the agmatine field, that of being anti-proliferative to all mammalian cells. Above 1 mM agmatine, the predicted anti-proliferative response was realized as a non-toxic, non-divisional state sustained for at least 4 days from single dosing. Moving to the 3D co-culture system, wherein the C2BBe1 cells were differentiated as per high transepithelial electrical resistance (TEER) over a 24-hour equilibration period, it was expected that agmatine would again be <i>anti-proliferative</i>. Yet, apical agmatine appeared to exert a <i>pro-proliferative</i> effect starting as low as 0.002 mM. A parallel decline in metabolism per SH-SY5Y cell was found using the color dye reaction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was therefore hypothesized that apical agmatine had caused the C2BBe1 cells to secrete a growth signal(s) impacting the underlying SH-SY5Y cells; and to test this, conditioned basal media collected from just C2BBe1 cells grown 4 days in the presence of apical 2 mM agmatine was taken to replace the media of na&iuml;ve SH-SY5Y cells growing in log phase mono-cultures. The expectation was that growth factors would be carried over, but to the contrary, an anti-proliferative response emerged from the conditioned media, mirroring the earlier studies with agmatine in mono-cultures. Cellular lysates were also prepared from treated cells exposed for 24 h to 2 mM agmatine, and these were probed on immune-blots to assess if any of 32 common receptor tyrosine kinases had phosphorylated /activated post-addition of apical mM agmatine. No evidence was obtained that agmatine (mM apical) had elicited such flags of cell activation. Next, the 3D co-culture condition was re-run for longer periods and with more controls, and from this came the realization that the model had hidden the existence of an anti-proliferative response from the C2BBe1 cells before agmatine was even added. In short, the starting hypothesis was disproven, but in doing so it was realized that micromolar apical agmatine is able to rejuvenate a cytostasis rendered by the C2BBe1 co-culturing. Two fundamentally different mechanisms must be invoked by agmatine, because the concentrations of agmatine at which these two processes occurred were 500-fold different (0.002 mM for the reversal of cytostasis vs. 1 mM for anti-proliferative, respectively). In summary, any microbial dysbiosis involving agmatine-producing bacteria is likely to act through two molecular signaling mechanisms from the &ldquo;accessory&rdquo; organ bacteria to enteric nervous system.</p><p>

<p> Mitochondrial metabolism of reactive oxygen species (ROS) is tightly regulated during brain development. Imbalance has been correlated to neuropsychiatric disorders. Nevertheless, the contribution of ROS accumulation to aberrant cortical circuit organization and function remains unknown. Individuals with 22q11 deletion syndrome (22q11DS) are highly susceptible to psychiatric disorders; therefore, 22q11DS has been suggested as a model for studying the neurodevelopmental origins of these disorders. Six genes &ndash;<i>Mrpl40, Tango2, Prodh, Zdhhc8, Txnrd2</i> and <i>Scl25a1</i>&ndash; located in the 22q11DS commonly deleted region encode proteins that localize to mitochondria. This project aimed to characterize the effects of altered mitochondrial function, due to diminished dosage of these genes, on cortical projection neuron development, using the <i>LgDel</i> mouse model of 22q11DS. I found growth deficits in <i>LgDel</i> neurons that are due to increased mitochondrial ROS and are <i>Txnrd2</i>-dependent. Antioxidant treatment, by n-acetyl cysteine (NAC), rescues neuronal morphogenesis in <i>LgDel</i> and <i> Txnrd2</i>-depleted neurons <i>in vitro</i> and <i>in vivo.</i> Electroporation of <i>Txnrd2</i> restores ROS levels and normal dendritic and axonal growth. <i>Txnrd2</i>-dependent redox regulation underlies a key aspect of cortical circuit differentiation in a mouse model of 22q11DS. These studies define the effects of mitochondrial accumulation of ROS on neuronal integrity, and establish the role of altered pyramidal neuron differentiation in the formation of circuits in 22q11DS. These data provide novel insight into the role of redox imbalance in aberrant development of cortical circuits.</p><p>

<p>Fibroblast growth factors (FGFs) and their receptors (FGFRs) play a vital part in the proper development and maintenance of the brain. FGFR1, which is one of four FGFRs total and one of three found in the brain (FGFR1-3), has been shown to be important in cellular proliferation, cellular migration, synaptogenesis, cellular morphology, and has also been implicated in multiple neuropsychiatric disorders. Understating the role FGFR1plays in these and other cellular processes is vital to our understanding of the human body and in the prevention and treatment of some neuropsychiatric and developmental disorders. Although previous studies have produced groundbreaking findings in the field, they have fallen short in the accurate identification of which cell type express Fgfr1. Therefore, to validate the use of a transgenic mouse line in the accurate and efficient study of Fgfr1 expression during experimental manipulations and cellular localization, we utilized the tgFGFR1- EGFPGP338Gsat BAC mouse line (tgFgfr1-EGFP+) obtained from the GENSAT project. By utilizing the tgFgfr1-EGFP+ mouse line, we were able to accurately identify which cell types in the embryonic and perinatal mouse brain express Fgfr1. Furthermore, we were able to measure relative changes in Fgfr1 expression via GFP fluorescence as a proxy after both exposure to chronic stress and the chemical demyelinator, cuprizone. The combination of our results lead us to conclude that the tgFgfr1-EGFP+ mouse line is a very useful tool in the study of FGFR1 and may aid in the identification of potential targets for therapeutic treatment of the many disorders associated with FGFR1 signaling.

<p> Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42. They regulate many cellular functions, including cell polarity, cytoskeletal dynamics, and transcription. Pak1 and Pak2 are redundantly essential for embryonic skeletal myoblast fusion in <i> Drosophila</i>, with Pak2 playing the more important role. Both are expressed in mammalian skeletal muscle, but little is known as to their function in myogenesis. We find that Pak1 and Pak2 are expressed in mammalian myoblasts and are activated specifically during differentiation. Individual genetic deletions of <i>Pak1</i> and <i>Pak2</i> in mice show no overt defects in muscle development or regeneration. However, young adult mice with muscle-specific deletion of <i>Pak1</i> and <i>Pak2 </i> together (dKO mice) present with reduced muscle mass and a higher proportion of myofibers with smaller cross-sectional area compared to controls. This phenotype is exacerbated after repair to acute injury. Primary myoblasts from dKO animals show delayed differentiation, with lower expression of myogenic markers and inefficient myotube formation. Additionally, with age, dKO mice develop a chronic myopathy. Histological analyses of resting muscle show the presence of central nuclei in the majority of fibers, as well as significant fibrosis, inflammation, necrosis, and hypertrophy with fiber splitting. Ultrastructural analysis revealed grossly elongated and branched intermyofibrillar mitochondria, known as megaconial mitochondria, along with occasional accumulation of subsarcolemmal mitochondria. Moreover, dKO mice show impaired mitochondrial function, with significantly reduced Complex I and II activity. These characteristics are absent in control animals. We conclude that the role of Pak1 and Pak2 in embryonic myoblast fusion, first identified in the fly, is not conserved in mammals. Rather, our data demonstrate that Pak1 and Pak2 function redundantly in regulating myoblast differentiation, thereby impacting overall postnatal muscle size. Furthermore, their major function appears to be in muscle homeostasis. Few protein kinases have been implicated in muscle disease. Group I Paks have wide roles in cell regulation, and the generation of dKO mice provides a genetic system to gain new mechanistic insights into muscle maintenance, as well as to discover the substrates of Paks that regulate this process.</p><p>

Transition zones are regions in the animal body where two types of epithelial tissue meet. Many transition zones are known high-risk sites for tumorigenesis. However, little is known on why transition zones are more susceptible to tumor formation, mainly due to the lack of a suitable study model. In this dissertation, I report that the Drosophila salivary gland imaginal ring can be used as a model to study tumorigenesis in transition zones. Drosophila melanogaster imaginal rings are larval tissues composed of progenitor cells that are essential for the formation of three adult tubular structures, including the salivary gland, foregut, and hindgut. In the first part of this dissertation (Chapter 2), I show that during the larval stage, Notch signaling is activated in all three imaginal rings and canonical Notch signaling positively controls cell proliferation in these imaginal tissues. In addition, Serrate (Ser) is the ligand provided from neighboring imaginal ring cells that trans-activates Notch signaling, whereas both Ser and Delta could cis-inhibit Notch activity when the ligand and the receptor are in the same cell. In the second part of this dissertation (Chapter 3 and 4), I demonstrate that constitutive activation of Notch signaling in the imaginal ring during the third larval instar stage is sufficient to induce neoplastic tumorigenesis in the tumor hotspot at the posterior end of salivary gland imaginal rings, which is also a transition zone between diploid salivary gland imaginal ring cells and polyploid salivary gland cells. In this region, local endogenous JAK-STAT and JNK activation creates a tissue microenvironment that is susceptible to oncogenic Notch induced tumorigenesis. JNK activates a matrix metalloprotease, MMP1, to determine where the neoplasms form. Moreover, ectopic MMP1 can transform the anterior area of the salivary gland imaginal ring, which is normally refractory to oncogenic Notch-induced tumorigenesis, into a tumor "hotspot". In the third part of this dissertation, I further report that the cells in tumor hotspot of salivary gland imaginal ring adopt an endoreplicative cell fate after the second instar larval stage. These endoreplicating cells are normally lost during the pupal stage. However, overexpression of Notch induces re-mitosis in these polyploid tumor hotspot cells, which results in aneuploidy contributing to advanced tumor development. Loss of endoreplication or re-mitosis activity is sufficient to rescue the malignancy of Notch-induced tumors. Taken together, these findings reveal how endogenous signaling creates tumor-favoring microenvironments, and post-endoreplication mitosis promotes neoplastic tumorigenesis at the Drosophila transitional zone model, and ultimately establish the salivary gland imaginal ring as an in vivo model for studies of site-specific tumorigenesis. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2018. / November 05, 2018. / Imaginal ring, Notch, Transition zone, Tumor hotspot, Tumorigenesis, Tumor microenvironment / Includes bibliographical references. / Wu-Min Deng, Professor Directing Dissertation; Timothy Megraw, University Representative; Michelle Arbeitman, Committee Member; Hongchang Cui, Committee Member; Karen McGinnis, Committee Member.

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