Immune molecules, including complement proteins, C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement proteins localize to synapses and refine the developing retinogeniculate system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. RGCs grown in the presence of astrocytes significantly upregulated C1q compared to controls, implicating an astrocyte-derived factor in neuronal C1q expression. A major goal of my dissertation research was to identify the signals that regulate C1q expression and function in the developing visual system. In this study, I have identified transforming growth factor beta \((TGF-\beta)\), an astrocyte-secreted cytokine, as both necessary and sufficient for C1q expression in RGCs through an activity-dependent mechanism. Specific disruption of retinal \(TGF-\beta\) signaling resulted in a significant reduction in the deposition of C1q and downstream C3 at retinogeniculate synapses and significant synaptic refinement defects in the retinogeniculate system. Microglia engulfment of RGC inputs in the lateral geniculate nucleus (LGN) was also significantly reduced in retinal \(TGF\beta\)RII KOs, phenocopying the engulfment defects observed in C1q KOs, C3 KOs, and CR3 KOs. Interestingly, in C1q KOs and retinal \(TGF\beta\)RII KOs, microglia also failed to preferentially engulf less active inputs when retinal activity was manipulated, suggesting that retinal activity and \(TGF-\beta\) signaling cooperatively regulate complement mediated synaptic refinement. In support of this hypothesis, blocking spontaneous activity in RGC cultures significantly reduced C1q upregulation by \(TGF-\beta\). Moreover, manipulating spontaneous retinal activity in vivo modulated C1q expression levels in RGCs and C1q deposition in the LGN. Together these findings support a model in which retinal activity and \(TGF-\beta\) signaling control expression and local release of C1q in the LGN to regulate microglia-mediated, complement-dependent synaptic pruning. These results provide mechanistic insight into synaptic refinement and, potentially, pathological synapse loss which occurs in the early stages of neurodegenerative diseases concurrently with aberrant complement expression and reactive gliosis.

Breast cancer is the most common cancer in female population worldwide. In addition to mutations, the breast tumor microenvironment especially the tumor cell - stroma interactions through extracellular matrix components and multiple growth factors have been shown to promote tumor progression. Among those, increases in both TGF-β (transforming growth factor beta) activities and periostin expression were associated with tumor cell survival, proliferation and metastasis. TGF-β role in breast cancer progression including its ability to promote periostin expression has been extensively studied. In contrast, the role of periostin in cancer progression remains to be fully understood. Thus, the present study aimed to determine whether TGF-β and periostin have effect on each other’s expressions in breast tumor and stroma cells using in vitro cell models. Through Western blot analyses and ELISAs, the periostin and TGF-β expressions of both stroma and tumor cells were analyzed following TGF-β and periostin treatments, respectively. The results indicate that TGF-β treatments led to significant increase in periostin expression in fibroblasts (p<0.05). In addition, periostin was differentially expressed by human breast cancer cells following TGF-β1 treatment. The TGF-β activities involved activation of pSMAD2 in both L929 fibroblasts and MCF10A mammary cells. Taken together, all experimental data indicate that within the breast tumor TGF-β and periostin likely participate in a regulation loop. Whether this putative regulation loop is critical to metastasis remains to be determined. Should periostin play a critical role in breast cancer progression, it could become a specific target in the preventive and/or therapeutic development of breast cancer patients.

From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

Sleep fragmentation induces acute inflammation and increases glucocorticosteroids in vertebrates. Obesity and sleep fragmentation are often concurrent pro-inflammatory conditions in patients with obstructive sleep apnea. Despite the association between the two, their simultaneous effects on immune and endocrine profiles have not been explored. In the first experiment, we investigated changes in proinflammatory (IL-1β, TNF-α) and anti-inflammatory (TGF-β1) cytokine gene expression in the periphery (liver, spleen, fat, and heart) and brain (hypothalamus, prefrontal cortex, and hippocampus) in mice exposed to various intervals of sleep fragmentation. Serum corticosterone concentration was also assessed. Sleep was disrupted in male C57BL/6J mice using an automated sleep fragmentation chamber (Lafayette Industries), which involved movement of a sweeping bar at specified intervals. Mice were exposed to bar sweeps every 20 sec (high sleep fragmentation; HSF), 120 sec (low sleep fragmentation; LSF), or the bar remained stationary (control). Trunk blood and tissue samples were collected after 24 h of SF. It was found that HSF is a potent inducer of inflammation in the periphery (IL-1β: adipose, heart, and hypothalamus), but leads to upregulation of antiinflammatory cytokines in the brain (TGF-β1: hypothalamus and hippocampus), despite elevated serum corticosterone. Due to the association between obesity and SF, this experiment was replicated in male C57BL/6J mice (lean) and ob/ob KO mice (obese) using the previously described methods. We predicted the acute inflammatory response resulting from HSF would be different for the lean and the obese mice, with the greatest cytokine gene expression levels in the OB HSF group, due to a summative effect of the pro-inflammatory conditions. Obesity was the factor that most affected cytokine gene expression profiles. Additionally, the pro- vs anti-inflammatory gene expression profile varied with tissue type. While obesity resulted in neuroinflammation (hypothalamus, prefrontal cortex, hippocampus), it led to decreases in pro-inflammatory cytokine gene expression in the periphery (spleen, fat, heart). Serum corticosterone concentration was significantly elevated due to SF, but was not affected by obesity. As a result, the obese mice likely had neuroendocrine adaptations to combat the pre-existing pro-inflammatory condition of obesity, which impacted the acute inflammatory response to sleep loss.

Cardiovascular disease is now the leading cause of death worldwide. Serum amyloid A (SAA), a positive acute phase reactant, along with C-reactive protein is used clinically as a marker of cardiovascular disease risk. However, recent data has shed light on a possible causal role of SAA in the development of atherosclerosis, the most pervasive form of cardiovascular disease. Several inflammatory diseases such as diabetes and obesity are known to confer increased risk of developing cardiovascular disease. Individuals with these diseases all have modest but persistent elevation of SAA. To determine if SAA caused the development of atherosclerosis, apoe-/-chow fed mice were injected with either an adenoviral vector expressing human SAA1 (ad-hSAA1), a null adenoviral vector (ad-Null) or saline. Human SAA levels rapidly increased, albeit briefly then returned to baseline within 14 days in mice that received ad-hSAA1. After 16 weeks, mice that received ad-hSAA1 had significantly increased atherosclerosis compared to controls on the aortic intimal surface (p<0.0001), aortic sinus (p<0.05) and the brachiocephalic artery (p<0.05). According to the “response to retention” hypothesis; lipoprotein retention by vascular wall proteoglycans is a key initiating event in the development of atherosclerosis. We previously reported that SAA-stimulated vascular smooth muscle cells expressed biglycan with increased glycosaminoglycan chain length and increased binding affinity for low density lipoprotein. To further test the role of biglycan on the development of atherosclerosis we generated biglycan transgenic mice. These mice were crossed to the ldlr-/- mouse on a C57BL/6 background and fed a pro-atherogenic western diet for 12 weeks. There was a significant increase in atherosclerotic lesion area on the aortic intimal surface (p<0.05) and the aortic sinus (p<0.006), as well as a significant correlation between vascular biglycan content and aortic sinus atherosclerotic lesion area (p<0.0001). These data demonstrate that transiently increased SAA resulted in increased atherosclerosis compared to control mice, possibly via increased vascular biglycan content. In support of this we found that biglycan transgenic mice had significantly increased atherosclerosis compared to wildtype controls, likely through increased lipid retention in the vascular wall.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Gastrulation is the process by which the three germ layers are generated during vertebrate development. Nodal ligands, which form a subgroup of the Transforming Growth Factor β (TGFβ) superfamily, regulate the expression several transcription factors implicated in gastrulation. Among these are the paired-like homeodomain transcription factors Goosecoid (Gsc) and Mixl1. At the molecular level, Gsc has been described to function as a transcriptional repressor by directly binding to paired homedomain binding sites on target promoters. Here, I describe a novel mechanism of transcriptional repression by Gsc. Using a molecular and embryological approach, I demonstrate that the forkhead transcription factor Foxh1, a major transducer of Nodal signaling, associates with Gsc which in turn recruits histone deacetylases to negatively regulate Mixl1 expression during early mouse development. Post-translational modification of transcription factors by SUMO proteins represents an important mechanism through which their activity is controlled. Here, I also demonstrate that Gsc is sumoylated in mammalian cells by members of the PIAS family of proteins and this modification potentiates the repressive activity of Gsc on direct targets such as the Xbra and Gsc promoters, but not on indirect targets such as Mixl1. Taken together, work presented in this thesis describes two novel mechanisms of transcriptional repression by Gsc.

Doctor of Philosophy (PhD) / Background: The development of the blood supply in the primate retina has been extensively studied; however the relationship of the differentiating retina to the choroidal blood supply is less well known. The interaction of astrocytes and vascular endothelial cells promotes the development of the retinal vasculature from 14 weeks’ gestation (WG). Initially, astrocytes lead the developing capillaries from the optic nerve towards the macular area. However, neither astrocytes nor endothelial cells enter a prescribed avascular area, within which the fovea later forms. This may be attributed to expression of a factor that inhibits astrocyte and endothelial cell proliferation in the fovea. A factor found in the CNS that is already known to have these effects is transforming growth factor-β (TGF-β). Aims: This thesis investigated the relationship between retinal maturation and choroidal blood vessel supply and the possible role for TGF-β as an antiangiogenic factor in maintaining an avascular fovea during primate retinal development. Methods: Human eyes between 11 WG and 40 years were obtained with ethical approval from Prince of Wales Hospital and the NSW Lions Eye Bank and fixed and sectioned for histological procedures or prepared for polymerase chain reaction (PCR). Macaque eyes from foetal day (fd) 64 to postnatal 11years (p11y) were obtained from Bogor Agriculture University, Indonesia with the approval of the Ethics Committee of the University of Washington, Seattle, USA. Macaque eyes were also fixed and sectioned for immunohistochemistry and in situ hybridisation. RNA was extracted from human foetal retinas and used for RTPCR (Reverse Transcriptase PCR), QPCR (Quantitative PCR) and preparation of riboprobes. PCR products were analysed using both restriction digest and sequencing. RTPCR was used to identify TGF-β1, TGF-β2 and TGF-β3 in the developing human and in the developing and adult macaque retinas whilst QPCR was used to quantify the TGF-β isoforms in central compared to peripheral retina and in foetal compared to adult retina. In situ hybridisation was performed according to a standard protocol and visualised using Roche HNPP Fast Red detection set with designed riboprobes for TGF-β1, TGF-β2 and TGF-β3 (DIG RNA labelling kit). Some sections were counterstained with vimentin antibody. Immunohistochemistry was performed on human retina and choroid sections using antibodies to CD34 and Ki67 and on human and macaque retina using antibodies to synaptophysin, vimentin, GFAP, calbindin, S-opsin, RG-opsin, rhodopsin, TGF-β1, TGF-β2, TGF-β3 and their receptors TβRI and TβRII. Sections of the retina were imaged and analysed using either a Leica Confocal microscope and TCSNT software or Zeiss Confocal microscope and LSM 5 Pascal software. Data from the human retina and choroid sections corresponding to different regions (foveal, parafoveal nasal, parafoveal temporal, nasal and temporal) was collected to measure the number of Ki-67 immunolabelled mitotic endothelial cells and the area of CD34 immunolabelled choriocapillaris using Adobe Photoshop version 5.0.2, NIH software version 1.62 (measurement macros) and Excel. In the human and macaque sections the intensity of TGF-β protein and mRNA expression was captured from different regions of the retina (foveal, parafoveal nasal, parafoveal temporal, nasal, temporal, nasal to disc) to compile montages. Montages were then re-imported into LSM 5 Pascal software to measure the optical density across each montage along the ganglion cell layer, outer neuroblastic zone and photoreceptor layer collecting data in Excel for graphical representation. In addition to the montages, individual sections were assessed for co-localisation of TGF-β and TβR to various retinal cell types. Results: Analyses of choriocapillaris area and endothelial cell (EC) proliferation were able to demonstrate that the area of choriocapillaris endothelium is greater in the foveal region at all ages (14-18.5WG), that the rate of choriocapillaris EC proliferation declines dramatically over this same period and that the lowest rates of EC proliferation are at the incipient fovea. Most importantly these findings indicate that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons which are more developed with higher oxygen and nutrient demands, which is the mechanism widely thought to regulate development of the retinal vasculature. PCR showed all TGF-β isoforms to be present in the human developing and adult retina. QPCR revealed that TGF-β2 was the most predominant isoform, followed by TGF-β3 with very small amounts of TGF-β1 seen. The isoforms were more abundant in developing rather than adult retina and in central rather than peripheral retina. Studies of the distribution of TGF-β protein and mRNA using immunohistochemistry and in situ hybridisation confirmed the low levels of TGF-β1 protein and mRNA observed in QPCR and demonstrated distinct centroperipheral gradients in the photoreceptor layer for TGF-β2 and TGF-β3. Relative high amounts of TGF-β in the fovea could affect vascular patterning due to TβRI seen in astrocytes which lead the blood vessels at the foveal rim at the level of the ganglion cell plexus. TGF-β2 and TGF-β3 expression is detected before formation of the foveal avascular zone (FAZ) at fd64 (~15WG) - fd73 (~17WG) with levels peaking in the foveal region at fd105 (~25WG) by the time the FAZ forms. Conclusions: This thesis has shown that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons as reduced rates of EC proliferation in the ‘foveal’ chorioretinal location were observed at all ages studied between 14 and 18.5WG. The findings suggest that mechanisms regulating proliferation and growth of the choroidal vasculature are independent of differentiation in the neural retina and are therefore different to those governing the formation of the retinal vasculature. All TGF-β isoforms are expressed in developing and adult human and macaque retina with TGF-β2 being the predominant isoform. TGF-β isoforms are more abundant in central compared to peripheral retina and in developing compared to adult retina. Centro-peripheral gradients of TGF-β2 and TGF-β3 across the photoreceptor layer and TβRI on astrocytes support the presence of TGF-β in the fovea as an antiproliferative and antiangiogenic factor by helping to define the FAZ early in development, well before 23-25 WG in humans and before fd100 in macaques.