Andrew P. McMahon - US grants

DESCRIPTION: The applicant is interested understanding the molecular and cellular mechanisms that govern growth regulation, pattern formation and cell fate choice in the mammalian brain. His focus is on the roles played by intercellular signaling molecules, particularly members of the Wnt gene family. In his previous work, he demonstrated that Wnt-1 is essential for midbrain and anterior hindbrain development since both areas are missing in Wnt-1 mutants. Various studies indicate that Wnt-1 acts in the midbrain, and that the midbrain is required for development of the anterior hindbrain. The working model is that the brain develops as a series of segmental units, each with its own genetic program, but that interactions between adjacent segmental domains are also necessary to coordinate the development of each region. The applicant proposes the following: (1) to further explore the Wnt-1 signaling pathway by examining mutants in porcupine, an endoplasmic reticulum protein which is thought to facilitate Wnt-1 signaling, and to examine the possible later roles of Wnt-1 in the ventral midbrain; (2) to address the action of FGF8, an anterior hindbrain derived signal in reciprocal interactions with the midbrain; and (3) since the restricted expression of other members of the Wnt-family to specific areas of the embryonic brain supports a more extensive role for Wnt-signaling in brain development he proposes to examine strains of mice which lack these factors to unravel the function of these other Wnt-signals in the regulation of forebrain and hindbrain organization.

Patterning of the vertebrate brain depends on signals arising from the notochord as well as signaling operating within the induced neural plate. One such signal is encoded by the protooncogene Wnt-1. Wnt-1 expression demarcates the presumptive midbrain at presomite and early somite stages and its expression has been shown, by mutational analysis in the mouse, to be essential for midbrain development. Our goal is to understand how regional specification of the midbrain is established. To determine how the molecular regulation of midbrain specific gene expression is controlled, we have started to investigate the cis-acting regulatory sequences governing Wnt-1 expression. A 5.5 kb Wnt-1 enhancer has been identified which is capable of driving reporter gene expression within the normal Wnt-1 domain. We propose to use transgenic mice and P19 cell culture to identify the precise cis-acting regulatory elements within this enhancer. Founder transgenic mice injected with reporter constructs containing sub-elements of the enhancer will be assayed for reporter gene expression at 8.5 dpc. Reporter constructs will also be tested for activation in retinoic acid-treated P19 embryonal carcinoma cells which normally activate Wnt-1 on differentiation to neuronal cell types. We will also generate midbrain cell lines from transgenic embryos expressing a temperature sensitive T-antigen to induce conditional immortalization. These cell lines will be incorporated into functional and biochemical analysis of Wnt-1 expression. Cis-acting binding sites responsible for activating Wnt-1 in culture systems will be tested for their ability to activate Wnt-1 in vivo. On the basis of cell culture and transgenic experiments, we will use cDNA expression libraries from 8.5 dpc embryos, affinity chromatography with nuclear extracts from cell lines and a yeast transcriptional activation screen to identify putative regulatory factors. These experiments will further our understanding of how regional patterning in the brain is initiated and consequently how these structures are generated during normal development of the human CNS. They therefore should enhance our understanding of potential brain-related birth defects.

DESCRIPTION (provided by applicant): Our goal is to understand the mechanisms by which information encoded in an extracellular signal is converted into complex patterns in the developing mammalian embryo. This proposal focuses on the role of Sonic hedgehog (Shh) signaling. The Shh signaling pathway is not only essential for the induction of clinically relevant neurons within the CNS, but inappropriate activation of Shh signaling has been linked to the development of several types of tumor, most notably basal cell carcinoma (BCC) (the most common form of skin cancer), medulloblastoma (the most common brain tumor of children) and rhabdomyosarcoma (the most prevalent soft tissue cancer in children). Further, loss of Hedgehog (HH) signaling underlies several birth defects that include holoprosencephaly. Consequently, understanding how a Shh signal is received, transduced and modulated is likely to lead to new biological insights with direct relevance to human health. Given the close human parallel and advantages of available genetic approaches, the mouse is our principle experimental system. Aim 1 proposes to explore the biological significance of cholesterol modification of Shh investigating the role of a Dispatched gene in Shh release and the requirement for cholesterol in Shh-mediated patterning of the neural tube. A GFP tagged Shh protein will be generated to study Shh movement in vivo. Aim 2 proposes to explore the genetic interactions amongst several Shh-binding proteins (Ptch1, Ptch2, Gas1 and Hip1) that appear to act in Shh-mediated feedback control as they relate to neural patterning. We will map the interaction domains between Shh and one of these, Hip1. Aim 3 will explore the roles of several novel targets of HH-signaling identified in transcriptional screens. Aim 4 proposes to characterize a new inducible model of HH-mediated tumorigenesis.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The kidney regulates water/salt balance, nitrogenous waste, blood pressure and blood composition. Kidney function is vital to homeostasis of the body fluids. Chronic kidney disease affects one in nine Americans, a similar number are at risk. The heath care, societal and individual toll of kidney disease is profound. A rigorous understanding of normal kidney development and repair should underpin logical efforts to design new therapeutic strategies for this vital organ system. The basic functional unit of the kidney is the nephron. The full complement of nephrons, approximately half a million in man, form over a lengthy period of fetal life. In this process, uncommitted mesenchymal progenitors are induced to form epithelial nephron precursors, the renal vesicle and other cellular components of the adult organ. The renal vesicle undergoes a complex morphogenesis and patterning process to establish the mature nephron with its distinct physiological domains positioned appropriately along the length of the epithelial tubule. Depending on the mammalian species, nephrogenesis is complete in fetal or early post-natal life. Thereafter, the only option is to maintain and repair these early-formed structures. The long-term goal of our research effort is directed at defining the molecular and cellular processes that establish a functional physiological system during the course of organ development. Given the clinical importance of the kidney, its well-defined physiological roles and the rich history of embryological study over several decades, this organ is an attractive model. We will combine genetic and genomic approaches to understand how the kidney is built from its constituent cell parts in the mouse. In Aim 1, we will define the cellular complexity of the developing kidney's progenitor pools, determine the progenitor-product relationships between uncommitted cells in the kidney and their mature descendants, and identify the cellular mechanisms that maintain progenitors thereby establishing the full complement of nephrons. In Aim 2, we will specifically address the transcriptional actions of opposing regulators, Six2 and _- catenin/canonical Wnt signaling, on the maintenance and commitment of purified nephron progenitors. The transcriptional programs of each will be examined by transcriptional profiling and direct DNA-binding studies and the regulatory networks mined from these data sets. In Aim 3, we will determine whether Notch, a proximalizing factor in the context of the epithelial nephron, can directly induce mesenchymal nephron progenitors to form clinically relevant proximal cell fates, notably podocytes. We will utilize similar strategies to those above to understand Notch's regulatory action in the critical early patterning processes that establish functional cellular asymmetry in the developing nephron. Project Narrative Chronic kidney disease affects one in nine Americans, a similar number are at risk. Determining the normal mechanisms of organ assembly and repair are high priorities. The proposed study utilizes mouse genetic models and genomic strategies to determine the molecular and cellular processes that establish a full complement of functional nephrons during mammalian development from pools of early renal progenitor/stem cells. [unreadable] [unreadable] [unreadable]

Understanding the molecular and cellular events which lead to normal bone formation is an essential prerequisite for understanding the aetiology of skeletal disorders in humans. Further, these insights will enable the design of rational, information-based therapeutic approaches for treating pathological or accidental damage to the skeleton. This proposal sets out to explore the mechanisms underlying key aspects of mammalian skeletogenesis using the mouse as a genetic model. Aim 1 will examine Ihh's regulatory role in the endochondral skeleton. We will address the significance of Gli3-repression in Ihh signaling and the direct or indirect role of Ihh in regulating PTHrP expression, and thereby chondrocyte maturation. Finally, we will use transcriptional profiling to identify putative targets of Ihh's multiple actions in endochondral skeletal development. Aim 2 will address the role of Ihh and Wnt signaling in the osteoblast lineage through a step-wise series of genetic manipulations. Aim 3 will fate map cranial neural crest skeletal precursors as a first step towards examining the relationship between a putative "Fox-code" and specific skeletal structures of the mammalian face.

Description: (Taken directly from the application) The aim of this Core is to develop transgenic mouse strains either by pronuclear injection of DNA or by blastocyst injection of genetically modified embryonic stem (ES) cells. These procedures not only require considerable skill they require dedicated equipment and facilities. The Core will facilitate genetic approaches which underpin each of the projects in this program. It will facilitate in training individuals from member laboratories who are unfamiliar with these procedures.

DESCRIPTION (provided by applicant): Kidney disease is one of the leading health-care costs. Despite the vital importance of the kidney to maintaining the normal physiological equilibrium of an individual, there is a relatively poor understanding of the developmental program that creates the functional organ. Our goal is to generate a detailed spatial map of the cellular expression of selected regulatory genes during mammalian (mouse) kidney development to generate a "molecular anatomy" of the developing kidney. We will also create a series of transgenic mouse strains that will allow the ready identification and genetic manipulation of key cell types. Aim 1: We will perform a genome-wide analysis of the expression of all genes encoding mouse transcriptional regulators, ligands and cognate surface receptors in the embryonic urogenital system. Aim 2: We will generate a high resolution section in situ hybridization (SISH) map of the expression of all genes in Aim 1 in the fetal and neonatal kidney. Aim 3: We will perform transcriptional array analysis of renal tubule deficient mouse kidneys and micro-dissected kidney tubules to identify, and subsequently map the expression of tubule specific genes. Aim 4: We will use BAC-mediated transgenics to express fluorescent reporter proteins and Cre recombinase in specific cell populations for cell marking, cell fate and genetic manipulation studies.

The gene expression Core will permit a high-throughput, high-resolution analysis of gene expression in the developing skeleton. This core will perform two genome-wide screens of broad interest and utility to map the expression of all transcriptional regulators, ligands and their cognate receptors in the context of the developing mouse skeleton. Further, we will perform a rapid analysis of large data sets emerging from transcriptional profiling approaches addressing underlying molecular mechanisms of skeletogenesis.

DESCRIPTION (provided by applicant): The proposed research plan is in response to RFA-DK-11-001, A Genitourinary Anatomy Project (GUDMAP). This continuation of the GUDMAP initiative (GUDMAP II) aims to define the molecular anatomy of the lower urinary tract. We propose to generate a series of novel mouse strains that will enable groups funded through this RFA to identify, isolate, and genetically manipulate target cell types to better understand the roles of these cells and their descendants in development of the lower urinary tract. We anticipate that the strains will enable antibody-based approaches for 2- and 3-dimensional imaging of organ anatomy, cell isolation for transcriptional analysis of gene expression, and cell fate analysis to explore cell relationships in developing and adult organs of the lower urinary tract. In Aim1, we will make use of the extensive repertoire of targeted embryo stem (ES) cells created by the NIH-funded KOMP initiative and its European counterpart, EUCOMM. These knock-out first conditional alleles will serve as a platform for secondary gene-targeting, utilizng a dual recombinase mediate cassette exchange approach. In this, novel cell markers (GPP) and genome modifying enzymes (CRE-ERT2) are placed under the control of specific genes selected for expression in cell populations of interest. In Aim 2, targeted ES cells will be used t generate chimeric mice and ultimately novel mouse strains harboring the new marker/modifier alleles. These strains will be distributed to GUDMAP II members to facilitate their analyses and to NIH-funded Mouse Mutant Resource Centers for distribution to the biomedical research community.

DESCRIPTION (provided by applicant): This Program Project consists of three closely related and integrated proposals focusing on signals that regulate skeletal morphogenesis during embryonic development. The projects are highly coordinated, founded in the last 10 years of a highly successful Program Project and with a long history of collaborative efforts before that. A unique feature of this team is the exchange of expertise between laboratories whose primary focus has been either developmental skeletal biology (Tabin, McMahon) or endocrine control of skeletogenesis (Kronenberg). The high-resolution histology core extends the technical capabilities of each individual lab. All three projects are focused on dissecting the roles of key regulatory pathways in skeletal development. Project 1 will examine the mechanisms used by parathyroid hormone-related protein (PTHrP) and associated G-proteins in regulating these processes and will determine the roles of novel regulators of Chondrocyte differentiation discovered in the last grant cycle, as well as characterizing the early cells of the osteoblast lineage and their relationship with the perichondrium. PTHrP works in a feedback loop with a second secreted protein; Indian hedgehog (Ihh). Project 3 will explore the role of extracellular matrix in modulating Ihh signaling and will also study the role of the perichondrium in signaling to and controlling growth and differentiation of the cartilage elements. Project 2 is devoted to defining the regulatory networks involved in chondrogenesis and osteogenesis within the skeletal elements themselves. These projects are knit together by common themes, complimentary approaches, shared reagents, and direct collaborations. Together these highly related projects will achieve a new level of understanding of the regulation of bone morphogenesis which could not be attained by independent efforts.

DESCRIPTION (provided by applicant): The development of a functional mammalian nervous system requires the formation of a diverse array of neuronal cell types. These arise during embryonic life from specific pools of mitotically-active neural progenitors. How distinct neural progenitor populations are established at the outset of neural development is a central question for understanding the organizational principles underpinning a functional nervous system. Further, a detailed knowledge of critical regulatory mechanisms will facilitate rational regenerative strategies for treatment of neurodegenerative diseases, and enable the effective use of patient-specific iPS cells to model disease states and screen for drugs that may treat a specific disease. Sonic hedgehog (Shh) signaling specifies mammalian neural progenitors throughout most of the ventral central nervous system including dopaminergic and motor neuron progenitors, targets of Parkinson's and Lou Gehrig's disease. Shh is a morphogen; the concentration of signal and duration of signaling underlies the formation of spatially- and temporally-distinct classes of neural progenitors. In contrast, deregulated pathway activity results in cancers of the nervous system, and normal signaling supports cancers arising from several other organs including the pancreas, lung, and gut. Our goal is to understand how a dynamic signaling response is transduced into distinct transcriptional regulatory programs in the specification of neural progenitor types. The transcriptional response is directed by the Gli-family of transcriptional regulators. We have used an in vitro model system to comprehensively identify cis-regulatory modules (CRM) mediating Gli- dependent regulation of mammalian neural target genes within the developing central nervous system. Specific Aim 1 will examine how the affinity of Gli-factors for their target sites determines the Sonic hedgehog response. Specific Aim 2 will investigate how the neural context, transcriptional status, and chromatin organization interplay in the initiation and progression of Shh-directed neural patterning. Our approach combines the strengths of an in vitro, embryo stem cell-based model of Shh action with in vivo studies utilizing genetically-modified mouse strains and utilizes chromatin immunoprecipitation and RNA-sequencing to identify and interrogate the gene regulatory networks underpinning Shh action. The proposed studies will provide an unprecedented level of insight into morphogen-based mechanisms patterning the mammalian embryo and knowledge gained here from the stem-cell base should be readily translatable to similar human model systems. We anticipate that the findings will have broader significance with respect to cell-fate specification processes during development, repair, and regeneration of many other organ systems where Hedgehog signaling plays a critical role, and enhance our understanding of Hedgehog family signaling in human disease.

This Core functions to coordinate activities under the Program Project including organizing monthly meetings and annual visits by the Advisory Committee, facilitating Core utilization and preparing grant-related reports and applications. These activities support all the Projects equally.

PROJECT SUMMARY (See instructions): Secreted proteins direct the initiation, growth and patterning of the developing skeletal elements. Indian hedgehog (Ihh) is an important example of such a factor. Like many inductive signals, Ihh physically interacts with extracellular matrix components in addition to receptors on it's target cells. Such extracellular interactions can sequester the signals, influence morphogen movement across tissues and/or mediate ligand-receptor interactions. Hedgehog proteins have previously been shown to specifically interact with Heparin Sulfate Proteoglycans (HSPGs). While this has not been directly explored for Ihh in particular, mutations in two genes involved in HSPG chain elongation, Ext1 and Ext2, cause skeletal dysplasias known as exotoses and Ihh signaling is compromised in Ext1 mutants. To directly address the importance of HSPG interacting for Ihh function, we will construct an allelle of Ihh that lacks the puttaive HSPG-binding Cardin- Weintraub (C-W) domain. Ihh acts on skeletal development through direct effects on prolioferating chondrocytes, and also through indirect effects via the perchondrium a membrous sheath of flattened cells encapsulating the skeletal aniagen. Indeed, the perchondrium is known to play important roles in a number of aspects of regulating the differentiation of subjacent skeletal elements. We recently identified a series of markers indicating a surprising level of complexity in the organization of this tissue. We will construct transgenic mice in which cre-recombinase is placed under the transcriptional control of promoters driving expression in different perichondrial domains. These will be used to fate-map the cells in different layers of the perichondrium and to test the roles of skeletal regulators in this tissue, including Ihh and Hox genes.

PROJECT SUMMARY (See instructions): Early in bone development, chondrocytes and cells of the osteoblast lineage proliferate and differentiate in a regulated fashion. Parathyroid hormone-related protein (PTHrP), a key modulator of these processes, signals through a G protein-coupled receptor, the PTH/PTHrP receptor (PPR) to delay hypertrophic differentiation of chondrocytes in fetal life. In preliminary studies we have found that in postnatal life, the removal of the PPR leads quickly to the disappearance of the growth plate (growth plate fusion). In Aim 1 we will confirm these findings, use genetic tools to determine the roles of specific G proteins in mediating the postnatal actions of the PPR, and determine the mechanism of growth plate fusion in this model. In Aim 2, we will continue to determine the ways that chondrocyte hypertrophy is regulated. In the first sub-aim we will use genetic tools both in vivo and in bone explants to test the hypothesis that PTHrP regulates hypertrophy by stopping the interaction of HDAC4 with MEF2c. In the second sub-aim, we will study the role of a novel transcriptional regulator, limb bud and heart (LBH) in regulating chondrocyte hypertrophy. Our studies in fetal chick limbs have demonstrated that LBH regulates both chondrocyte and osteoblast development by suppressing expression of the transcription factor, Runx2. Here we will use the cre-lox approach to knock LBH out of chondrocytes (collagen 11 promoter) and osteoblasts (osterix promoter) to determine to roles of LBH in bone development. We will verify in the mouse knockout model that LBH regulates hypertrophy and explore possible connections between the actions of LBH and the PTHrP-HDAC4-MEF2c axis. In the previous grant cycle, we used tamoxifen-regulated osterix-cre and collagen l-cre constructs to trace the osteoblast lineage in early bone development. In Aim 3, we will focus on earlier steps of the osteoblast lineage by generating further tamixofen-regulated cre lines driven by the Msx2 and Runx2 promoters. These mice will be used to assess the roles and regulation of very early cells of the osteoblast llneage.assess the roles and regulation of very early cells of the osteoblast lineage..

bone; Development; Morphogenesis; Regulation;

PROJECT SUIVIMARY (See instructions): A mineralized endoskeleton supports the mammalian body. Bone also serves as a vital mineral repository and nurtures non-skeletal cell types, notably stem cells of the hematopoietic system. Skeletal anomalies are amongst the most common birth defects, problems in fracture repair are observed in non-union fractures and bone disorders are increasingly prevalent in an ageing population. Two key biosynthetic cell types generate the specific extracellular matrix of the mammalian skeleton: cartilage-secreting chondrocytes and bone secreting osteoblasts. These cells have diverse origins (neural crest, somitic and lateral plate mesoderm), and generate bone by distinct processes: directly from a mesenchymal stem cell (membranous bone) or via a cartilage template (endochondral bone). Mouse and human genetics have identified a number of critical signaling pathways, and three key transcription factors that are master regulators of chondrocyte (Sox9) and osteoblast (Runx2 and Osx) programs. Haploin sufficiency for Sox9 and Runx2 associates with human skeletal deficiencies. While the importance of these factors is well documented, their regulatory actions are not. To this end, we will determine the gene regulatory networks that underpin the regulatory actions of each of these critical skeletal determinants in chondrocyte and osteoblast development. Specific Aim 1: We will use gene-targeting in mouse embryo stem cells to create mice whose endogenous Sox9, Runx2 and Osx proteins are modified by appending a small peptide at their C-terminus. This epitope will enable subsequent analysis of the expression and activity of each factor through common approaches. Specific Aim 2: ChlP-seq analysis will be performed with each tagged protein to directly identify their DNA targets in primary chondrocytes or osteoblasts. Specific Aim 3: Data from microarray-based transcriptional profiling of normal chondrocytes and osteoblasts, or these same populations following knock-down of Sox9, Runx2 and Osx, will be intersected with ChlP-seq data to predict the targets of each regulatory factors actions. Selected regulatory models will be examined in transgenic mouse studies. These data will provide the first genome scale insight into the gene regulatory networks driving mammalian skeletogenesis.

? DESCRIPTION (provided by applicant): The functional unit of the mammalian kidney is the nephron - a complex, composite structure comprising an epithelial renal tubule with a closely associated specialized vascular network. Nephrons arise from a nephrogenic stem/progenitor population that only exists during fetal life. Consequently, the entire complement of nephrons is formed prior to birth. Thereafter, restoration of existing nephron structures is the organ's intrinic strategy for kidney repair following acute, and likely chronic, injury episodes. From studies in non-human model systems, we have a general framework for the molecular and cellular processes at play during kidney development and an understanding is emerging of intrinsic repair processes that maintain normal kidney function. Unfortunately, normal repair processes are inadequate: 1,000,000 patients are currently diagnosed with end state renal disease (ESRD), approximately 400,000 of these are on dialysis with a mean 5-year survival rates of 38%. The only option beyond dialysis for the ESRD patient is kidney transplant, but the number of transplants - approximately 20,000/year - does not match patient need. The long-term goal of the (Re)Building the Kidney Consortium is to harness our knowledge of nephron development and repair to forge innovative new approaches to treat injury and disease of the kidney. The consortium is being developed with the underlying thesis that a coordinated, focused, adaptive and interactive program of research by several groups has the potential to yield results in a timeframe that would not be possible through a static collection of independent single investigator sponsored research projects. Coordination across the research consortium demands the ability to rapidly integrate new research projects, communicate results, and share knowledge and data across the consortium. It is not enough to publish results at the end of a study, but rather, it must be possible to incrementally share at all stages of the research process. To address these issues, we propose to create a ReBuilding the Kidney Coordinating Center (RBKC) to ensure the consortium operates effectively as a coordinated research network, maximizing the value of individual projects, and communicates consortium activities to the broad research community. Specifically, the RBKC aims to: 1) create organizational structure and processes that will enable seamless and frequent communication and collaboration between RBKC researchers, the NIDDK and the broader research community, 2) create a data portal that will empower changing collections of researchers to rapidly create, analyze, share and discover diverse types of data, 3) broaden the research community and create a framework for targeted shorter term research results by establishing and managing an opportunity pool program, 4) disseminate consortium results to the broader research community. In total, the impact of these elements of the RBKC will accelerate the rate of discovery within the consortium and by the broader community.

Project Summary/Abstract The Genito-Urinary Developmental Molecular Anatomy Project (GUDMAP) has generated information and research tools to enhance our understanding of the development of urogenital system, both informing and stimulating research into urogenital development, and associated disorders and disease. Our proposed studies focus on the developing kidney complementing existing data available through the GUDMAP website. The functional unit of the kidney is the nephron ? hence, our focus on obtaining a thorough understanding of mammalian nephrogenesis. The current GUDMAP view of this process is largely two-dimensional and mouse focused. We will harness recent advances in both static and live dynamic cell imaging, the access to human kidney specimens and the development of pluripotent stem cell (PSC) culture-seeded nephrogenesis to extend our understanding of both mouse and human nephrogenesis. In Aim 1, we will develop high-resolution 3D views of mouse and human nephrogenesis through selective imaging approaches comparing mouse and human nephrogenesis to develop comparative 3D molecular atlases of nephron patterning and nephron morphogenesis. In Aim 2, we will take advantage of improvements in imaging and organ culture, and a bank of genetically modified mouse strains, to perform time lapse imaging of the nephron progenitor niche, and the formation, morphogenesis and patterning of the mouse nephron. Dynamic imaging studies will be applied to pluripotent stem cell (PSC)-derived models of human nephrogenesis. Together, these studies will significantly enrich the GUDMAP resource generating comparative 4D views of nephron development in the mouse and human kidney. Given the recent advances in the directed differentiation of PSCs into nephron-like structures, the studies will provide a benchmark for normal nephrogenesis that will guide the emerging field of regenerative kidney biology.

Abstract Chronic kidney disease affects over 26 million Americans. For the one million patients with end stage renal disease, dialysis and kidney transplant are the only therapeutic options. However, dialysis is palliative and kidney donors are in short supply. Thus, there is a critical need for new therapeutic strategies. The basic unit of the kidney is the nephron, a highly vascularized filtration and recovery unit. In the nephron, the plasma filtrate generated in the glomerulus passes into the proximal tubule (PT). The PT is lined by cuboidal proximal tubule epithelial cells (PTECs), the major resorptive cell type of the nephron characterized by the polarized distribution of channels and transporters that recover essential molecules and ions from the plasma filtrate. In acute kidney injury, PTECs are highly susceptible to damage. Surviving PTECs can repair the injured nephron, but endogenous repair mechanisms are not well-understood. This slows the development of new therapeutic strategies to accelerate PT repair in both acute and chronic kidney disease. Recently, the McMahon lab identified that the transcription factor SOX9 is up-regulated in PTECs after acute kidney injury in mice. This SOX9+ population of PTECs repopulates the nephron and restores function. However, whether a similar mechanism underlies repair of the human PT is unclear. One of the only practical approaches to identify mechanisms of human PT regeneration is to study human PTECs cultured in vitro. However, conventional culture substrates are highly artificial and lack physical cues present in the native PT that impact PTEC phenotype and survival, such as fluid shear stress. As a result, PTECs in conventional 2-D culture lose polarity and functionality. Recently, ?Organ on Chip? approaches have been developed to expose PTECs in vitro to physical cues similar to those in vivo, such as fluid shear stress. PTECs cultured within these platforms form differentiated structures and have improved functionality. However, existing platforms require specialized equipment that is not accessible to most research groups, neglect to include supporting cell populations (such as endothelial cells), and have not been used as tools for identifying mechanisms of PT regeneration. Thus, in Aim 1, we will use off-the-shelf equipment to engineer a scalable platform for engineering and maintaining a human PT, leveraging the McCain lab?s experience in engineering ?Organ on Chip? models of striated muscle. Our key design parameters are to apply fluid shear stress to primary human PTECs cultured as a tubule within a protein-derived extracellular matrix (ECM) hydrogel with relatively low elastic modulus. After validating that our engineered PT recapitulates key structural and functional phenotypes, we will add supporting cell populations (endothelial cells, fibroblasts) into the ECM hydrogel and establish any further improvements in PTEC viability, structure, and/or function. In Aim 2, we will induce global and local injury to our engineered PT and examine the expression of SOX9 throughout the PT during repair. We will then determine whether manipulating SOX9 activity can augment PT repair. This project is especially well-suited for the EBRG funding mechanism because we have established a multidisciplinary team (Prof. Megan McCain: junior investigator in biomedical engineering; Prof. McCain Andy McMahon: established investigator in kidney development) to develop a new engineered PT tissue platform to enable our hypothesis-driven research into SOX9-mediated mechanisms of human PT regeneration.

PROJECT SUMMARY Our interdisciplinary team proposes to develop and apply a novel method to systematically identify proteins involved in inter-organ communication. While a number of hormone peptides have been identified in mammals, many physiologically important factors involved in energy homeostasis, water-salt balance, pH, and blood pressure remain to be identified. As existing methods of identifying secreted factors from the blood using mass spectrometry (MS) have serious limitations, we will use an engineered promiscuous biotin protein ligase to biotinylate secreted proteins in a specific organ, and then identify biotinylated proteins in distant target organs through affinity enrichment followed by quantitative MS. These experiments will be performed in two organisms, Drosophila and the mouse. Drosophila facilitates the rapid development and optimization of the basic research strategy, generating evolutionary insight into conserved factors in organ communication. The mouse provides a more direct discovery model for biomedically-relevant, inter-organ regulatory communication in humans. In addition to identifying factors involved in organ communication, both in normal and disease conditions, our findings will provide fundamental insight into novel proteins that could be used in the future as therapeutics. Given our goal to broadly interconnect organ systems, the project is relevant to most NIH Institutes.

Project Summary/Abstract: Deafness and balance disorders resulting from the loss of sensory hair cells of the inner ear are a major cause of disability and morbidity in the US. In mammals, the cells of the various sensory epithelia of the inner ear arise embryonically and subsequently do not regenerate if damaged (Rubel et al., 2013). Hearing loss resulting from the death of hair cells in the organ of Corti is thus permanent, and treatments aimed at reversing hearing loss through stimulated regeneration of hair cells are badly needed. However, experimentation on the cells of the inner ear is difficult due to their small number and extreme inaccessibility in the adult. As a result, modern techniques of cell and molecular analysis and drug discovery have been difficult to apply, and aside from prosthetics, treatment options for sensorineural deafness remain few. To overcome these problems, and in pursuit of new treatments for hair cell loss, we have used powerful new ?direct lineage reprogramming? technologies, originally developed for neuron-specific reprogramming (Son et al., 2011; Vierbuchen et al., 2010), to generate hair cell-like cells in vitro, directly from mouse and human somatic cells (fibroblasts and inner ear supporting cells). This advance allows a new range of experimentation into the mechanisms of hair cell differentiation, the development of preclinical models of genetic hearing loss (disease modeling), high-throughput screening for drug discovery related to regeneration and ototoxicity, and the application of gene therapy approaches to the problem of hair cell regeneration. The Aims of the proposal include: 1) Development of improved reprogramming strategies to induce mouse and human hair cell-like cells. 2) Development of a drug screen for ototoxicity, and an in vitro disease model of genetic hearing loss. 3) A test of reprogramming in a preclinical model of hair cell regeneration/replacement in long-deafened mice.

Summary Acute kidney injury (AKI) has a wide spectrum of outcomes from recovery to a long-term transition to chronic kidney disease (CKD). Between 2000 and 2014, AKI hospitalizations have increased from 3.5 to 11.7 per 1000 persons. Medicare patients aged 66 years and older hospitalized for AKI have a 35% cumulative probability of a recurrent AKI hospitalization within one year and 28% will be diagnosed with CKD in the same time frame. Men have a higher risk of AKI, and of developing progressive CKD, although the mechanisms are poorly understood. In the mouse, males also show a heightened vulnerability to AKI. Recent single cell RNA-seq studies from the McMahon and Kim groups have highlighted marked differences in gene expression between the sexes in proximal tubule segments, the region of the nephron most susceptible to AKI. Preliminary studies in the Humphreys and McMahon laboratories using single nuclear sequencing identified a cell type resulting from failed repair of proximal tubule cells (FR-PTC) following mild to severe AKI with a pro-inflammatory, pro- fibrotic signature. FR-PTCs are hypothesized to drive progressive kidney disease following AKI. This proposal centers on the postulates that an understanding of sex differences in response to AKI, and the application of genetic approaches to target proinflammatory properties of FR-PTCs and to eliminate FR-PTCs following renal repair, will be effective routes to ultimately benefit patient outcomes post AKI. To this end, we have assembled a complementary team, with prior collaborative experience: Humphreys (Washington University), Kim (University of Pennsylvannia) and McMahon (University of Southern California). All team members have participated in the ReBuilding a Kidney Consortium. In Specific Aim 1: we will characterize successful versus failed proximal tubule repair with single nucleus transcriptomics (snRNA-seq) and single nuclear chromatin accessibility studies (scATAC-seq) in male and female mouse models examining key findings in human kidney biopsies. In Specific Aim 2: we will harmonize multimodal datasets generated in Specific Aim1 to facilitate viewing and interrogation of these data by the broad research community. Mining of these data by the group will focus on defining the regulatory logic of repair strategies and outcomes in the male and female kidney. In Specific Aim 3: we will examine the hypothesis that adverse outcomes in the male kidney following AKI are driven by NF-kB pathway components Nfkb1 and TNIK in FR-PTCs, genetically eliminating the action of these genes. We will generate and validate a new transgenic mouse resource for the community, enabling genetic modification and elimination of FR-PTCs. We will determine whether FR-PTC removal has a favorable outcome, as we predict, on progressive kidney disease following AKI.

Project Summary/Abstract: Sensory hair cell regeneration does not occur spontaneously in the mature mammalian organ of Corti, making hearing loss permanent. The goal of this proposal is to identify the causes and mechanisms behind the failure of hair cell regeneration, as well as ways to stimulate regeneration in surviving populations of inner ear supporting cells in deafened individuals. Our primary hypothesis is that regeneration requires the re- engagement of developmental gene networks to guide supporting cells to a hair cell fate, and that during postnatal inner ear maturation, epigenetic barriers arise that block the re-activation of these gene networks. The goal is to develop methods to overcome these epigenetic barriers, and to establish new cell fates with regenerative potential within the organ of Corti. Experimentally, perinatal mice retain a latent capacity for direct supporting cell transdifferentiation to a hair cell-like fate, but this capacity is rapidly lost in the first weeks after birth. This age-dependent change in regenerative potential provides a window through which to investigate the transition from a permissive to a non-permissive state for this form of regeneration in the normally maturing organ of Corti. The work of this proposal is to elucidate the mechanism(s) of epigenetic control of transdifferentiation in perinatal supporting cells (Aim1) and to identify the machinery of maturation-related changes in epigenetic/chromatin structure in supporting cells of the inner ear. (Aim 2). We hypothesize that these changes are responsible for the failure of regeneration. Finally, to investigate the epigenetic structure of adult supporting cell chromatin in normal and deafened mice (Aim 3). We hypothesize that manipulation of the epigenetic state is an important approach for future regenerative medicine approaches to restoring lost hair cells.

Project Summary/Abstract There is a growing consensus that men and women differ in their response to kidney injury, and their susceptibility and progression to chronic kidney disease. Similar findings have come from the analysis of different sexes in rodent models. Historically, females have been under-represented in animal modeling and clinical studies. Redressing this imbalance and understanding how sex-related differences in gene expression are generated, and how these influence normal and pathological actions within mammalian organ systems, is a priority. Recent single cell RNA-seq studies in the McMahon group have highlighted extensive sexual dimorphism within proximal tubule segments of the adult mouse kidney. Proximal tubule cells share a major role in chemical modification of circulating metabolites with hepatocytes of the kidney. Proximal tubule cells also have kidney specific actions in resorption, transport and removal of beneficial or harmful molecules. Comparative analysis shows both similar and distinct sexually dimorphic gene sets between the liver and kidney, with potential differences in hormonal interplay (androgens, estrogens, growth hormone) underlying how each organ establishes dimorphic cell states. Pregnancy and nursing present additional demands on the female, specifically. How these demands may impact dimorphic cell states in the female kidney is not clear, even in the mouse model. Due to the absence of comparable, high quality, comparative data for the human kidney, there is no clear idea of the extent of sexual dimorphism in the human kidney, and consequently, which regulatory actions may be shared with mouse models, or are human specific. In this proposal, we will use single nuclear (sn)RNA-seq, snATAC-seq and genetic approaches to determine the regulatory processes establishing sexually dimorphic cell types in the mouse kidney, and those modifying gene activity within proximal tubule cell in the reproductive process. Comparable datasets emerging from worldwide efforts applying single cell technologies to human systems will be co-analyzed for shared and distinct regulatory processes. Specific Aim 1 will determine regulatory mechanisms, including the action of direct hormone signaling (androgens, estrogen and growth hormone), in generating distinct proximal tubule cell types in the male and female mouse kidney. Kidney datasets will be contrasted with similar data for overlapping gene cohorts within sexually dimorphic hepatocytes. Specific Aim 2 will determine the regulatory interplay of pregnancy, nursing and prolactin signaling in modifying sexually dimorphic cell states in the mouse kidney. Specific Aim 3 will compare sexual dimorphism in the mouse with human kidney biopsies, integrating data generated in the proposal into the framework of KidneyCellExplorer (https://cello.shinyapps.io/kidneycellexplorer/) for viewing and analysis of the data.