Raphael Kopan - US grants

DESCRIPTION: The Principal Investigator proposes to address the important questions of the mechanisms of intracellular signaling by Notch and the role(s) of Notch in mammalian development.

Generation of tissue from progenitor cells (stem cells) is a complex process, often requiring elaborate communication events between cells located near each other or at some distance away. Studies of skin development, and in particular the development of the hair follicle, have served as a model for such interactions and yet no precise information exists regarding the molecular details underlying the mechanisms by which acquisition of diverse cell fates within the hair follicle occurs. In this proposal we offer to investigate the potential of individual progenitor cells to contribute to any of the six differentiated cell types within the follicle. In addition, we will investigate the role of the Notch pathway in follicular cell fate selection. The Notch receptor is involved in a short-range cell-cell communication pathway which is able to amplify small, sometimes stochastic differences between equivalent cells, into an all or nothing developmental outcome. This pathway have been shown to hold the ky for fate allocation during Drosophila and vertebrate neurogenesis, in vertebrate hematopoiesis and in large number of other systems. We will establish whether Notch, expressed in hair follicles, plays a similar role in determining the identity of hair follicle cells. Combined, we hope these studies will further our knowledge of how individual progenitor cells at the base of a follicle contributes differentiated progenies to the hair shaft and provide the basis for future studies, aimed at understanding how cells integrate signals from multiple singling pathways to differentiate properly.

Our goal is to complete a detailed history of Notch 1 activity in the mouse, extending beyond the early embryo into self-renewal and tissue maintenance in the adult. Notch signaling plays an important role in cell fate arbitration, in boundary formation and, less frequently, in fate induction during early development and tissue renewal. Due to early lethality, the role of Notch signaling during and after organogenesis remains to be resolved. Identifying signal-receiving cells, a subset of the Notch protein expressing cells, is key to understanding how Notch regulates cell fate. This goal has broader appeal due to the observation that Notch signals and Amyloid-plaque generating A beta42 peptides are both produced by the same enzymatic activity, presenilin dependent g-secretase. We will obtain an objective characterization of the role Notch core signaling plays in adult vertebrates to gain a better understanding of the possible consequences of the therapeutic use of g-secretase inhibitors. We have generated several reagents to enable a thorough investigation of where Notchl is activated. Cells engaged in Notch signaling can be detected with existing antibodies directed against g-secretase cleaved Notch 1. We developed protocols that permit detection of low levels of this antigen in tissue sections. In conjunction with the effort to map Notch activation using these antibodies, we derived ES cell lines in our laboratory, marked with ROSA-LacZ, that are either Notch 1 deficient or express a proteolysis-deficient Notch 1 allele (N1V->G/N1V->G). Comparing the contribution of these ES lines in different tissues of mouse chimera will map tissues and cell types whose formation in the embryo and renewal adult may not require g-secretase-dependent Notch signals. We propose to correlate Vail 744 antigen detection and ES contributions with two functional assays that will determine the requirements for Notch 1 activity. First, we propose to create a lineage tracer to mark cells experiencing Notch 1 activation via proteolysis. Second, we are generating conditional Not 1 loss- and gain of function alleles. Conditional removal or activation of Notch 1 will permit analysis of its role late in fetal development and in adult tissue renewal. Aiml: Survey of cells that experience core Notch signaling and express NICD. Aim2: Comparison of the developmental potential of N-/- EX cells. Aim3: Analyze conditional loss and gain of function Notchl alleles in the adult.

DESCRIPTION (provided by applicant): Developmental anomalies account for 40% of chronic renal failure in children under age 4. In adults, the kidney is a major collateral target organ of disease processes and drug toxicity. In addition, cardiovascular homeostasis depends on precise regulation of blood pressure and electrolyte balance, neither of which can be maintained without good renal function; therefore, identifying the molecular regulators of kidney development and regeneration are of critical importance to human health. Many signals (GDNF, LIF, FGF-2, TGFbeta, Wnt-4, and Notch) control nephron development and, not surprisingly, many are arranged in feedback loops, such that signal integration maintains the balance of signaling necessary for the differentiation and development of multiple renal cell types that compose the adult kidney. Fully elucidating the role of Notch signaling in kidney development and its integration with other signaling pathways is of great importance. The genetic evidence in support of a role for Notch signaling is that partial loss of the Notch ligand Jagged I affects kidney function in humans (Alagille-Syndrome) and hypomorphic mutations in Notch-2 impair glomemlar development in mice. Despite these excellent studies, the role of the Notch pathway during all stages of renal development is under-investigated due to early embryonic lethality and redundancy of Notch receptors. The dependence of all Notch receptor activation on proteases and the selectivity of DAPT for presenilin-dependent protease (7-secretase) permit us to pharmacologically manipulate Notch signaling in an organ culture system, circumventing lethality. Our experiments uncovered a requirement for Notch signaling operating at an earlier time during kidney development than was previously appreciated: we discovered that 7-secretase activity (and thus Notch signaling) is essential for specification of proximal epithelial cell fates (podocyte and proximal tubule); a role for Notch in this process suggests a possible role in kidney regeneration post injury in adults, where Notch and its ligands are up-regulated in a renal fibrosis model. This application Specific Aims to identify Notch-dependent processes and downstream Notch targets during development and during adult kidney regeneration in several renal injury models.

Notch signaling is an evolutionary conserved mechanism used by metazoans to direct cell fate decisions, proliferation and apoptosis at all stages of development, including self-renewing adult tissues. Aberrant Notch signaling is implicated in cancer, especially in the emerging field of cancer stem cells. Mammalian cells contain four Notch receptors and five Delta and Jagged cognate ligands;the overall goal of this proposal is to elucidate the significance of context on individual Notch receptor subtype-mediated signals using non-invasive molecular imaging strategies. We propose to develop enabling technologies that will facilitate high throughput screening for agents and gene products that are capable of modulating the Notch signaling pathway in cancer, inherited diseases and facilitate tissue engineering. A comprehensive mechanistic understanding of how to manipulate individual receptors in a context-dependent manner still eludes investigators. We propose to develop a real time imaging system that will go beyond the currently available reporters in providing real time, quantitative accounting of the activation status of individual Notch receptor subtypes. The reporter system we are developing is based on the luciferase complementation imaging technology developed in the ICMIC Molecular Reporter Core at Washington University School of Medicine and enables visualization of the interactions between a specific Notch intracellular domain (NICD) and the common nuclear cofactor RBPjk. The system is versatile;it can be adaptedfor studying the pathway in different cell types and can be easily modified to monitor interaction with other cancer-relevant partners such as components of the NF-kB pathway. While the imaging technology being developed in this application is not directly applicable to imaging in patients as it requires protein engineering, these methodologies will help develop and evaluate novel therapies for Notch-related diseases.

DESCRIPTION (provided by applicant): Research Area: (06) Enabling Technologies Challenge topic: New technologies for neuroscience research (06-MH-103) Project Title: Development of Split-DamID as an alternative methodology to chromatin immunoprecipitation Project Abstract A wide variety of genetic or drug-induced mental health disorders have been linked to altered functions of transcription factors, be it drug-induced or caused by genetic defects. Although it is known that the expression patterns of many genes are altered in the brain after exposure to a psychotropic drug, ultimately we must determine which genes were directly activated by the relevant transcription factors to uncover the expression hierarchy and prioritize druggable targets. Progress towards identification of these "direct targets" has been slow since the traditional methods used to identify the immediate targets of a transcription factor (chromatin IP or ChIP) are expensive, labor-intensive and technically demanding. Most dauntingly, they require very good antibodies and a large amount of starting material, largely limiting their use to cells in culture. In order to provide an approach that avoids these issues, we have optimized the sensitivity and specificity of an alternative technology, DamID, for broad use in neuroscience. Our solution (Split-DamID) relies on our ability to successfully split the bacterial DNA adenine methyltransferase (Dam) into N-terminal (D) and C-terminal (AM) halves. Each half is inactive;however, when fused to interacting transcription factors, the halves will reconstitute enzymatic activity only at the binding sites where these transcription factors interact. The reconstituted Dam marks adenine (A) by addition of a methyl group (Am) in the sequence GAmTC found in proximity to most cognate binding sites of any transcription factor. It is important to note that this epigenetic modification is never found in eukaryotic DNA and appears to have no consequence to cellular or organism survival. This GAmTC mark allows isolation of the transcription factor bound DNA from total DNA through digestion with Dpn1, an enzyme that digests only DNA methylated at GAmTC sites, and adaptor mediated PCR. We have successfully reconstituted Dam activity when fused to Notch or Mef2c. Importantly, we have observed enrichment of known Notch targets over other DNA fragments in cells reconstituting split-Dam fused to Notch. Split-DamID has several key advantages: much improved signal to noise ratio compared with ChIP or DamID;adenine methylation indelibly marks DNA bound by the fusion protein, allowing identification of transient interactions;the recovery of marked DNA is independent of antibody availability;and the amplification of the marked DNA by adaptor mediated PCR makes it amenable to working with the endogenous expression level of the fusion protein and low amounts of starting material. Moreover, Spilt-DamID permits precise spatial and temporal control of Dam reconstitution. We propose to generate multiple Split-Dam pairs with mental health related factors (Notch, DeltaFosB, CREB, NF[unreadable]B, and Mef2) and common transcription co-activators (p300, others) to reconstitute Dam activity only at sites where transcription takes place. These targeting vectors, expression vectors, cells and animals will create a resource for investigators interested in identifying sites directly bound by transcription factors implicated in mental health in animal models exposed to psychotropic drugs or carrying a mutation known to affect behavior in humans. We will develop resources allowing investigators interested in brain function to mark and identify key target genes bound by transcription factors of interest (e.g., those involved in mental health, cancer, and other transcription-driven processes in neuroscience). They will have control over when the marking occur, enabling precise correlation of transcriptional activity and behavior. In contrast to the current technology, our novel method will work for any transcription factor and requires only a few animals since the method allows amplification of the recovered target sites. It should allow the rapid characterization of the vast array of transcription factors involved in mental health and facilitate identification of therapeutically relevant targets altered by disease or drug abuse. Additionally, all of the reagents used will be obtained from companies within the USA, and the resource will be made available to the community through American depositories.

A major goal of the this AADCRC program is to define the role of the epithelial cell barrier in the pathogenesis of asthma and allergic disease and develop new preventative strategies. In that context, this project aims to investigate means by which to block progression from atopic dermatitis to asthma (often referred to as the atopic march). Patients with a history of severe atopic dermatitis (AD) exhibit a 8-to-10-fold greater incidence of developing asthma. Our recent observations demonstrated that in mice, epidermal-derived thymic stromal lymphopoietin (TSLP) was secreted by AD skin. Moreover, circulating levels of TSLP were sufficient to sensitize the lung airways to inhaled allergens in animals lacking any AD-like pathology, preexisting inflammation, or previous exposure to the allergen. In a pilot study in infants we uncovered a correlation between serum TSLP and aeroallergens. Based on these observations in mice and humans, we hypothesize the following mechanism for the atopic march. (1) Epidermal defect/injury during early childhood is sensed by an unknown mechanism that initiates production of TSLP in keratinocytes; (2) keratinocytes secrete TSLP into the serum. (3) Subsequently, circulating TSLP facilitates Th2 immune responses by dendritic cells and T-cells towards innocuous allergens (inhaled or introduced epicutaneously); and (4) this exaggerated adaptive Th2 response results in hypersensitivity to aeroallergens and consequent allergic asthma. We further hypothesize that interrupting some of these events in a model organism will lead to strategies for blocking the development of allergic disease and asthma in humans. To achieve this goal, we propose the to (I) examine how epidermal differentiation/barrier formation defects (intrinsic factors) as well as allergen or pathogens (extrinsic factor(s)) drive TSLP overexpression; (II) with the help of Cores B and C ask how TSLP secretion is regulated by human skin and lung cells in vitro and in patients. Next, (III) we will ask if we can blunt the effects of TSLP in the serum with small molecule adjuvants capable of manipulating the immune responses and (IV) analyze the contribution of the skin microbiome to the maintenance of skin barrier, TSLP expression, and airway hyper sensitivity. Finally, (V) we will confirm TSLP as a risk factor for asthma in a birth cohort (URECA) and, in collaboration with project1, compare its role in an RSV bronchiolitis in early life cohort (RBEL). Achievement of these aims will open up novel therapeutic approach to prevent asthma development in AD patients.

Mammals express four Notch receptor paralogs (Notchi -4) that have been implicated in a broad range of functions during embryogenesis, post-natal development, and vartous pathologies, including cancer. As a result, there is growing interest in therapies aimed at blocking or activating Notch signaling in specific cellular or pathological contexts. During the previous funding period, we explored the spectrum of effects triggered by loss of Notchi activity with a novel genetic approach combining a knock-in Notchi allele (Nl ::CreLo), in which Cre replaced the NICD, and a conditional Notchi target allele (Niflox). The trans-heterozygous animals (Nl :CreLo/flox) were bred to reporter mice developed in the Molecular Reporter Core which contained floxed-stop click beetle red luciferase and dual reporter (lac-Z or eYFP) genes on respective alleles downstream ofthe ROSA26 locus. A systematic, life-long in vivo screen with bioluminescence imaging of animals wherein a random, progressive and age-dependent Notchi loss occurred identified a role for Notchi in suppressing neoplasia ofthe vascular system. Our findings caution against the Genetech approach of therapies targeting Notchi and provide a model in which to assess strategies to delay, prevent or control vascular tumor formation and thereby widen the therapeutic window of anti-Notch therapies. We will examine age, diet and organ dependence in vascular tumor formation using new Nl ::CreERT2 alleles permitting temporal control over loss of Notchi. We will use another new Notch trans-heterozygous combination, N2::CreLo/flox, to monitor risks associated specifically with loss of Notch2. An additional benefit from the proposed experiments will be a detailed map of all stem cell compartments where Notchi and Notch2 maintain stemness or promote differentiation. These studies will ultimately lead to safer Notch- targeting therapies.