<p> Celsr1 is an atypical cadherin central to the asymmetric cell-cell complexes that define planar cell polarity (PCP). Previous work has shown that Celsr1 undergoes bulk endocytosis during cell division in the basal layer of the mouse skin. Here, we report the unexpected finding that Celsr1-mediated intercellular complexes remain intact during mitotic internalization, resulting in uptake of Celsr1 and associated PCP components into dividing cells from their neighbors in a process known as trans-endocytosis. Our observations suggest that the bulk of this internalized pool of Celsr1 is targeted for degradation. Furthermore, Celsr1 internalized from neighboring cells carries with it additional core PCP proteins, including the posteriorly-enriched Fzd6 and anteriorly-enriched Vangl2. However surprisingly, Vangl2 originating from the dividing cell is excluded from mitotic endosomes and remains associated with the membrane. Overexpression of Vangl2 in vitro is sufficient to interfere with Celsr1 internalization, and mitotic internalization of Celsr1 within the skin is delayed at anterior cell surfaces. We propose that Vangl2 stabilizes Celsr1 at the membrane and its dissociation from Celsr1 is a prerequisite for Celsr1 turnover. Together our results indicate that mitotic turnover of Celsr1 depends on the displacement of Vangl2 from PCP complexes and results in the non-autonomous turnover of PCP proteins from neighboring cells.</p><p>

<p> Early embryonic cells in many species polarize radially by distinguishing their contacted and contact-free surfaces. Radial polarization is a critical patterning event driven by cell-cell contact and is required for developmental processes, such as the first differentiation event in the early mammalian embryo. The homophilic adhesion protein E-cadherin is required for contact-induced polarity in many cells. However, it is not clear whether E-cadherin functions instructively as a spatial cue, or permissively by ensuring adequate adhesion so that cells can sense other contact signals. In <i>C. elegans,</i> radial polarity begins at the four-cell stage, when cell contacts restrict the PAR polarity proteins to contact-free surfaces. We previously identified the RhoGAP PAC-1 as an upstream regulator that is required to exclude PAR proteins from contacted surfaces of early embryonic cells. PAC-1 is recruited specifically to sites of cell contact and directs PAR protein asymmetries by inhibiting the Rho GTPase CDC-42. How PAC-1 is able to sense where contacts are located and localize to these sites is unknown. We show that HMR-1/E-cadherin, which is dispensable for adhesion, functions together with HMP-1/&alpha;-catenin, JAC-1/p120 catenin, and the previously uncharacterized linker PICC-1/CCDC85/DIPA to bind PAC-1 and recruit it to contacts. Furthermore, we show that ectopically localizing the intracellular domain of HMR-1/E-cadherin to contact-free surfaces of cells recruits PAC-1 and depolarizes cells, demonstrating that HMR-1/E-cadherin plays an instructive role in polarization. Furthermore, we show that radial polarity is defective in embryos lacking HMR-1/E-cadherin. Our findings identify an E-cadherin-mediated pathway that translates cell contacts into cortical polarity by directly recruiting a symmetry-breaking factor to the adjacent cortex.</p>

<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>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>

<p>Over the course of development, cells and tissues of the embryo must take on the correct fates and morphologies to produce a functioning organism. The patterning events and morphogenetic processes that accomplish this task have been the subject of decades of research, the consequence of which has been a detailed comprehension of the molecular mechanisms that regulate each. Equally important is an understanding of the mechanisms that coordinate patterning with morphogenesis, such that they occur with the correct relative spatiotemporal dynamics. My thesis work sought to characterize such co-regulation in the context of two developmental events in a vertebrate model, the African clawed frog Xenopus laevis: induction of bottle cell formation at the onset of gastrulation after germ layer induction, and regulation of the morphogenetic movements of neurulation in relation to neural plate patterning. Chapter 1 of this dissertation provides a general introduction to the patterning and morphogenetic events of early development relevant to my thesis. Chapter 2 presents a discussion of my work to characterize the function of two signaling pathways, namely Nodal signaling and Wnt/Planar Cell Polarity, in the induction of bottle cells. My experiments confirm the requirement for Nodal signaling in bottle cell induction, but do not support a role for the individual transcriptional targets of Nodal signaling tested here or for Wnt/PCP. Chapter 3 summarizes my work to address the function of two transcription factor-encoding genes, sall1 and sall4, in neural development, including their roles in anteroposterior neural patterning, neural tube morphogenesis, and neural differentiation. My work shows that both sall1 and sall4 are required for all three processes, and supports the hypothesis that their key role in this context is to transcriptionally repress stem cell factors of the pou5f3 family, allowing progression through neural development. As a whole, this work summarizes my research to characterize molecules that co-regulate early patterning and morphogenetic events in the X. laevis embryo.

Gastrulation is a fundamental process that reorganizes the primary germ layers to shape the internal and external features of an early embryo. Morphogenetic movements underlying this process can be classified into a variety of different types of cellular movements. I will focus on investigating in this thesis two types of cell movements in the dorsal mesoderm; mediolateral cell intercalation and convergence and extension. During gastrulation, mesoderm cells send protrusions to gain traction on neighboring cells and the surrounding extracellular matrix; a process called mediolateral cell intercalation. Mesoderm cells use this type of cell movement to converge and extend the dorsal mesoderm tissue during gastrulation; a process called convergence and extension. These morphogenetic movements are essential to form the early embryo and are important for later development. There are a number of different proteins involved in regulating the morphogenetic movements during gastrulation. The Planar Cell Polarity Signaling Pathway helps establish individual cell polarity and is activated in dorsal mesoderm cells undergoing convergence and extension. In addition, dorsal mesoderm cells migrate by using integrin receptors and the surrounding extracellular matrix to correctly position the mesoderm in the embryo. I will focus my efforts on analyzing the function of ADAM proteins during Xenopus laevis gastrulation. The ADAM family of metalloproteases is important for a variety of biological processes. ADAM proteins function as ectodomain sheddases by cleaving membrane bound proteins involved in signal transduction, cell-cell adhesion, and cell-extracellular matrix adhesion. I will focus on investigating the roles of two ADAM family members; ADAM13 and ADAM19 during gastrulation. Both ADAM13 and ADAM19 are expressed in the dorsal mesoderm during gastrulation. Throughout early embryonic development, ADAM13 is expressed in the somitic mesoderm and cranial neural crest cells. ADAM19 is expressed in dorsal, neural and mesodermal derived structures such as the neural tube, notochord, the somitic mesoderm, and cranial neural crest cells. Since ADAM13 and ADAM19 are expressed in similar tissues, I investigated if both proteins functionally interacted. I show that a loss of ADAM13 protein in the embryo reduces the level of ADAM19 protein by 50%. In the opposite experiment, a loss of ADAM19 protein in the embryo reduces the level of ADAM13 protein by 50%. This suggests that both ADAM13 and ADAM19 are required to maintain proper protein levels in the embryo. This might be explained through their physical interaction in a cell. The ADAM19 Proform binds to the ADAM13 Proform in cultured cells. Through domain analysis, I show that ADAM19 binds specifically to the cysteine-rich domain of ADAM13. When co-overexpressed in a cell, the level of Mature ADAM13 (compared to the Proform) is reduced suggesting a complex form of regulation. I propose a few models that discuss how ADAM19 may function as a chaperone to stabilize and regulate the further processing of ADAM13 protein. Some of the unpublished work discussed in this thesis focuses on the roles of ADAM13 and ADAM19 in the dorsal mesoderm during gastrulation. Specific emphasis is made on investigating the axial mesoderm during notochord formation. I show that ADAM19 affects gene expression important for the A-P polarity of the notochord while ADAM13 does not. The changes in gene expression can be partially rescued by the EGF ligand Neuregulin1β, a known substrate for ADAM19 in the mouse. ADAM13 and ADAM19 are important for convergence and extension movements of the axial mesoderm during gastrulation. Specifically, a loss of ADAM13 or ADAM19 causes a delay in mediolateral cell intercalation resulting in a significantly wider notochord compared to control embryos. These defects occur without affecting dishevelled intracellular localization or the activation of the PCP signaling pathway. However, a loss of ADAM13 or ADAM19 reduces dorsal mesoderm cell spreading on a fibronectin substrate through α5β1 integrin. To conclude, the work presented in this thesis focuses on the similarities and differences of ADAM13 and ADAM19 in the early embryo. Although ADAM13 and ADAM19 are required for normal morphogenetic movements during gastrulation, my data suggests they have different functions. ADAM13 appears to function in regulating cell movements while ADAM19 appears to function in regulating cell signaling. I propose a few models that discuss how each ADAM metalloprotease may function in the dorsal mesoderm and contribute to convergence and extension movements during gastrulation.