Project Title: Epigenetic regulation of liver regeneration
Adult mammalian liver exhibits the remarkable ability to regenerate following traumatic injury. We hypothesize that this regenerative response will be deficient in the absence of the epigenetic regulator CXXC Finger Protein 1 (Cfp1) due to abnormalities in the modulation of chromatin structure. Transgenic mice were developed that are homozygous for a conditional (ÒfloxedÒ) Cfp1 allele and carry the Cre recombinase transgene under control of the liver-specific albumin promoter. These animals are viable and exhibit a specific ablation of Cfp1 expression in liver.
Partial hepatectomy will be performed on adult animals, which will be euthanized at 0, 1, 2, 3, and 7 days following surgery. This will be done using Cfp1f/f mice carrying the albumin-Cre transgene (experimental) and Cfp1f/f mice lacking the Cre transgene (control). Regenerating liver mass will be determined, and liver sections will be analyzed for cell proliferation and apoptosis. Plasma will be collected and analyzed for levels of liver enzymes. Regenerating liver tissue will also be collected for recovery of protein, DNA, and RNA for molecular analyses such as quantitation of epigenetic marks and gene expression patterns.
We expect that completion of these studies will firmly document a regenerative defect in Cfp1-depleted liver that is associated with epigenetic perturbations, and may also document baseline changes in liver function in the absence of Cfp1.
Project Title: Kinocilia mediated hedgehog signaling is necessary for stereocilia actin organization
Stereocilia are extremely stable actin-based protrusions on sensory hair cells of the inner ear. Proper hearing requires that stereocilia be grouped into highly organized rows of bundles on the apical surface of the hair cell. The development and establishment of these stereocilia bundles depends on another cellular appendage, the primary cilium, which in hair cells is called the kinocilium. Like most primary cilia, kinocilia are microtubule based structures thought to serve as subcellular signaling centers. In particular they regulate the mammalian hedgehog pathway, which communicates extracellular cues to the nucleus via the Gli family of transcription factors. This type of cilia mediated signaling is critical to several developmental processes.
The objective of this collaboration between the Perrin and Berbari labs is to test the importance of kinocilia-mediated hedgehog signaling in stereocilia morphogenesis. To address this question, we are utilizing transgenic and knockout mouse models of hedgehog pathway members to modulate signaling in the developing hair-cell. The results of this project will give valuable insights into the physiological role of kinocilia-mediated hedgehog signaling in regulating stereocilia formation. We will also determine if altering the hedgehog pathway in adults independent of the kinocilium can subsequently impact what appears to be the most stable actin network in the mammalian body. A better understanding on how to direct changes to this actin network will serve future studies looking at regeneration of stereocilia in age related hearing loss.
Project Title: Angiomotins and the Hippo Signaling Pathway in Murine Retinal MŸller Glial Injury-Induced Responses
Gliosis is a global response from the astrocytes of the central nervous system (CNS) characterized by an initial regenerative response that is replaced by a fibrotic scarring response in chronic disease states. During gliosis, astrocytes and MŸller glial cells of the retina hypertrophy, lose polarity, undergo de-differentiation, and, in some cases, proliferate. This response accompanies virtually every form of disease that occurs in the CNS. An increase in the regenerative proliferation response coupled with less of a fibrotic response would be ideal for optimizing the regenerative capacity of the CNS and would be beneficial for retinal diseases such as glaucoma, diabetic retinopathy, and age-related macular degeneration.
This proposal targets a novel group of proteins in the gliosis process, known as the angiomotins (AMOTs). AMOTs are scaffolding proteins that stabilize apical-basal polarity by connecting to tight junctions in cells. These proteins regulate the balance between cell-contact inhibition and proliferation by stabilizing the tight junctions between cells and sequestering the proto-oncogene yes-associated protein (YAP) to keep it from entering the nucleus to direct proliferation. The AMOTs have not been studied in the gliosis process to date; however the preliminary data generated by the Wells and Belecky-Adams laboratories indicate AMOTs are found primarily in the retinal MŸller glia and astrocytes and are responsive to the inflammatory signals that can trigger gliosis. We hypothesize that a loss of AMOT activity is the primary driver of the de-differentiation and proliferation evident in gliosis.
This proposes focuses on establishing a link between a loss of AMOT stabilization and the formation of gliosis by 1) determining changes in AMOT, AMOT-like proteins, and dominant negative forms of AMOT during gliosis, 2) showing that an AMOT gain-of-function following introduction of inflammatory molecules will lead to a decrease gliosis, and 3) showing that an AMOT loss-of-function in wild type retina will lead to an increase in gliosis.
Project Title: Adult Liver Stem Cells Associated with Pregnancy
The maternal liver exhibits robust growth response to pregnancy to accommodate physiological needs of both the mother and the fetus. The cellular mechanism behind the maternal hepatic adaptation to pregnancy is unclear. We found a small cell population expressing a basic helix-loop-helix transcription factor achaete-scute complex homolog-like 1 (Ascl1), a master regulator of neurogenesis, residing around the central veins in the liver pre-pregnancy. As pregnancy advances, these Ascl1-expressing cells migrate, expand, and eventually repopulate the maternal liver, exhibiting stem cell-like behaviors. These findings allow us to hypothesize that hepatic Ascl1-expressing cells are adult liver stem cells repopulating the maternal liver during gestation. The goal of this CDRB grant is to isolate and identify this cell population. We will isolate maternal hepatic Ascl1-expressing cells from pregnant mice by fluorescence-activated cell sorting (FACS) and analyze their transcriptome by RNA sequencing (RNA-Seq). By performing these studies, we will be able to (1) obtain living Ascl1-expressing cells for establishing an in vitro culture system to study the activities of Ascl1-expressing cells; (2) reveal the unique transcriptome of these cells to define their identity; (3) uncover their signature genes in addition to Ascl1; and (4) find the clues to investigate their regulation and biological properties. The results will enable us to lay a solid foundation for advancing our studies on gestation-dependent functions of Ascl1 and Ascl1-expressing cells in maternal liver.