2010 — 2014 |
Megason, Sean G |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
In Toto Imaging and Genomics to Decode Ear Hair Cell Formation and Regeneration
DESCRIPTION (provided by applicant): Loss of hearing and balance is a widespread and debilitating medical condition in humans, and is predominantly caused by a loss of hair cells, the primary sensory cell of the inner ear. Hair cells do not naturally regenerate in humans, in contrast to other animals such as zebrafish where hair cells readily regenerate. To understand hair cell formation during development and hair cell regeneration in response to damage we will undertake an integrative systems biology based approach. Our approach integrates in toto imaging which provides systematic high resolution analysis across the space and time of hair cell generation with omic approaches that allow systematic analysis of transcriptional activity across the genome. Specifically, we will use in toto imaging, a technology we developed, to generate a 4-dimensional, cell-based Digital Ear that comprehensively quantifies the cellular processes that form and regenerate hair cells in zebrafish. We will use cell-type-specific ChIP-seq of histone modifications to determine the enhancers, promoters, and insulators active across the entire genome at all the key steps of hair cell generation. Bioinformatic approaches will be used to map transcription factor binding sites within defined enhancers to the genes they control to construct a comprehensive cis-regulatory network within the virtual cells of our Digital Ear. This research will provide unprecedented insight into how the genome encodes the stepwise specification of hair cells, with potentially important implications for hair cell regeneration in humans. Relevance to Health Deficiencies in hearing and balance are widespread and debilitating. They are principally caused by loss of the sensory cells (hair cells) of the inner ear hair which cannot regenerate in humans but can in other animals. We seek to understand the genetic and cellular control of inner ear hair cell regeneration. PUBLIC HEALTH RELEVANCE: Deficiencies in hearing and balance are widespread and debilitating. They are principally caused by loss of the sensory cells (hair cells) of the inner ear hair which cannot regenerate in humans but can in other animals. We seek to understand the genetic and cellular control of inner ear hair cell regeneration.
|
0.934 |
2012 — 2013 |
Megason, Sean G |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Streamlined Cloning of Auditory and Vestibular Mutants by Whole Genome Sequencing
DESCRIPTION (provided by applicant): Over the last two decades zebrafish has emerged as one of the preeminent model organisms. This rise was in large part due to the promise of using forward genetic screens in a vertebrate animal to discover and elucidate the function of genes relevant to human development and health. Numerous genetic screens have now been performed both for general embryonic morpohological phenotypes as well as more specialized functional screens such as for balance and hearing. These screens have been very successful in identifying mutants and there are now over 6000 described chemically induced mutant lines in zebrafish. However, the gene mutated in these lines has only been identified in less than half the cases due to the current difficulty of positional cloning. The high cost and labor currently required for positional cloning prevents a molecular analysis of many current mutants as well as discouraging future genetic screens. Recent advances in next generation sequencing have reduced the cost of sequencing by several orders of magnitude such that it is now possible to routinely resequence entire genomes. These advances have already revolutionized many areas of genomics yet the way people do positional cloning in zebrafish and other model organisms has changed relatively little. Here we propose to apply next generation sequencing technologies to streamline positional cloning of mutants in model organisms focusing on zebrafish inner ear mutants. Our goal is to reduce the labor and cost of positional cloning by one to two orders of magnitude. We will compare two different approaches for cloning-by-sequencing based on linkage and homozygosity mapping by cloning 10 existing mutants in two mutant classes-semicircular canal morphogenesis and deafness.
|
0.934 |
2015 — 2021 |
Megason, Sean G |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dynamic Regulatory Mechanisms of Robust Pattern Formation in the Neural Tube
Abstract The long-term goal of our research is to understand the principles that permit developmental systems to robustly construct embryos of the correct pattern, shape, and size. Developmental systems face a gamut of variations from different sources including environmental, genetic, and stochastic, which manifest at multiple levels from molecules to cells to organs. In the face of these challenges, organisms have been designed through evolution to buffer the phenotype against these variations in order to robustly achieve a developmental norm, a process Waddington termed canalization. As our knowledge of the molecular and cellular details of patterning systems has expanded, there is now the opportunity to understand the systems level mechanisms that give rise to robust pattern formation. Here we focus on pattern robustness through the lens of scaling and size control. Scaling is a remarkable process in which the size of a pattern can be adjusted to the available size of the tissue. Scaling has fascinated and baffled embryologists since the time of Hans Driesch who in 1885 found that when the blastomeres of a two-cell stage sea urchin embryo are separated, the result is not two partial embryos but rather two complete embryos in which all their pattern is scaled by half. Similar results have since been found in a variety of organisms, but the surgical manipulations required to generate size- reduced animals are generally difficult and result in a lot of variability, thus limiting quantitative investigation. Recently, we have developed a new method for generating zebrafish eggs of different size that is robust and reproducible. Such embryos have qualitatively normal but scaled patterning and can give rise to viable adults. At a molecular level we find that most gene expression patterns (e.g. morphogens and their targets) scale with the tissues they pattern; however, a small subset of genes, the ones that sense tissue size to regulate scaling (e.g. by interacting with morphogens), do not. Thus, these size altered embryos represent a powerful and unique method to identify and determine the mechanisms of pattern scaling. Ultimately, tissue size is determined by balancing the rates of proliferation and differentiation over the course of development. We have found that the balance of proliferation and differentiation in the neural tube is under negative feedback control by mechanical pressure/tissue packing. Here we will use a combination of quantitative imaging, molecular and mechanical perturbations, and computer modeling to determine the systems-level mechanisms that allow: 1) morphogen patterning to scale to fit the available space, and 2) proliferation and differentiation rates to be balanced to cause a tissue to grow to fit the available space. These questions will be addressed in the zebrafish neural tube, but we expect the resulting mechanisms to be widely applicable. Such an integrated understanding is important for diagnosing and treating birth defects such as neural tube defects and in the rational design of engineered tissues.
|
0.934 |
2017 — 2021 |
Megason, Sean G |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Mechanism of Inner Ear Pressure Homeostasis by the Endolymphatic Sac
Abstract Hearing and balance loss is prevalent in every population and poses significant challenges to those affected. For many types of hearing and balance loss including Meniere's Disease, Enlarged Vestibular Aqueduct Syndrome, and Pendred Syndrome, the mechanism underlying the disease is currently unknown but is suggested to result from the loss of endolypmh volume and pressure control in the inner ear. Our long-term goal is to understand how the exquisite morphology of the inner ear is created during development and maintained in adults. Here we focus on the role of inner ear fluid pressure regulation by the endolymphatic sac in this process. The endolymphatic sac is a deeply conserved yet mysterious and poorly studied part of the inner ear. It has previously been suggested that the endolymphatic sac absorbs excess endolymph but through an unknown mechanism. Our preliminary data using state of the art timelapse imaging on larval zebrafish reveals that the endolymphatic sac pulses: the lumenal volume slowly increases over 1-3 hours and then rapidly decreases over several minutes. Endolymph pressure is necessary and sufficient for the expansion of the endolymphatic sac, and breaches in the epithelial barrier are necessary and sufficient for its collapse. These breaches occur at a novel cell-cell junction we term ?basal lamellar junctions? that seem to act as pressure relief valves. These preliminary data support our central hypothesis that regulated breaches in the epithelial barrier of the endolymphatic sac at specialized pressure relief valves are essential for proper fluid homeostasis in the inner ear; failure of these pressure relief valves causes endolypmh pressure to build up leading to inner ear swelling, death of sensory cells, and unregulated tearing of the otic epithelium called endolymphatic hydrops. We plan to test our central hypothesis using three specific aims: 1) identify the molecular and cellular mechanisms of valve formation; 2) determine the role of the valve in homeostasis of endolymph pressure and composition; and 3) determine the structure and function of the valve across species and developmental stages. Our experimental approach will use functional studies on zebrafish and quail and descriptive studies on mouse and human. Our studies will employ state of the art 3D, timelapse, confocal microscopy and serial section electron microscopy along with genetic, pharmacological, and physical perturbations. At the completion of this project, we will have a deeper understanding of the normal physiology of the endolymphatic sac and how disruptions to this physiology may lead to disease. Better knowledge of pressure homeostasis as well as the small molecule reagents we develop will provide a foundation for development of potential therapeutic interventions for these diseases. We are optimistic that this work will establish a new causal mechanism for inner ear pressure diseases such as Meniere's.
|
0.934 |
2018 — 2021 |
Klein, Allon Moshe Megason, Sean G |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mapping the Signaling Landscape of Vertebrate Development At Single Cell Resolution
Abstract (Project Summary) A major goal of developmental biology is to understand the detailed molecular progression of all embryonic cell lineages, from pluripotency to adulthood, and how signalling pathways control lineage choices at every step of differentiation. Such an understanding addresses several fundamental questions in developmental biology, while having practical implications for re-programming cells in disease, and for in vitro differentiation for cell therapy. Recently, we developed droplet microfluidic single cell RNA-Sequencing (scRNA-Seq) technology, which allows profiling the transcriptome of tens of thousands of single cells at low cost, and we additionally developed a method to combine droplet scRNA-Seq with lineage tracing, and the computational methods required to reconstruct time series of differentiation from scRNA-Seq and lineage data. In preliminary work, we applied these tools to generate a comprehensive map of cell state trajectories in zebrafish development through the first 24 hours post fertilization. In this proposal, we will extend our map of zebrafish development, combining scRNA-Seq with clonal analysis to track every cell state in the developing zebrafish embryo up to 7 days post-fertilization. We will then use staged, acute perturbations of seven major signaling pathways, followed by scRNA-Seq, to define which signaling pathways control the flow of cells down different trajectories throughout development, as well as their context dependent and invariant gene targets. Focusing deeply on neural tube patterning, we will then dissect the transcription factor networks that integrate signaling pathways, by CRISPR/Cas9 perturbation coupled to scRNA-Seq. This proposal builds on a multi-year collaboration between two labs with strong and synergistic expertise -- the Megason lab in the use of zebrafish for developmental systems biology and the Klein lab in single cell genomics and analysis.
|
0.934 |
2020 |
Megason, Sean G |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Microscope Purchase For R01 Dynamic Regulatory Mechanisms of Robust Pattern Formation in the Neural Tube
Project Summary The long-term goal of our research is to understand the principles that permit developmental systems to robustly construct embryos of the correct pattern, shape, and size. Developmental systems face a gamut of variations from different sources including environmental, genetic, and stochastic, which manifest at multiple levels from molecules to cells to organs. In the face of these challenges, organisms have been designed through evolution to buffer the phenotype against these variations in order to robustly achieve a developmental norm, a process Waddington termed canalization. As our knowledge of the molecular and cellular details of patterning systems has expanded, there is now the opportunity to understand the systems level mechanisms that give rise to robust pattern formation. Here we focus on pattern robustness through the lens of scaling and size control. Scaling is a remarkable process in which the size of a pattern can be adjusted to the available size of the tissue. Scaling has fascinated and baffled embryologists since the time of Hans Driesch who in 1885 found that when the blastomeres of a two-cell stage sea urchin embryo are separated, the result is not two partial embryos but rather two complete embryos in which all their pattern is scaled by half. Similar results have since been found in a variety of organisms, but the surgical manipulations required to generate size-reduced animals are generally difficult and result in a lot of variability, thus limiting quantitative investigation. Recently, we have developed a new method for generating zebrafish eggs of different size that is robust and reproducible. Such embryos have qualitatively normal but scaled patterning and can give rise to viable adults. At a molecular level we find that most gene expression patterns (e.g. morphogens and their targets) scale with the tissues they pattern; however, a small subset of genes, the ones that sense tissue size to regulate scaling (e.g. by interacting with morphogens), do not. Thus, these size altered embryos represent a powerful and unique method to identify and determine the mechanisms of pattern scaling. Ultimately, tissue size is determined by balancing the rates of proliferation and differentiation over the course of development. We have found that the balance of proliferation and differentiation in the neural tube is under negative feedback control by mechanical pressure/tissue packing. Here we will use a combination of quantitative imaging, molecular and mechanical perturbations, and computer modeling to determine the systems-level mechanisms that allow: 1) morphogen patterning to scale to fit the available space, and 2) proliferation and differentiation rates to be balanced to cause a tissue to grow to fit the available space. These questions will be addressed in the zebrafish neural tube, but we expect the resulting mechanisms to be widely applicable. Such an integrated understanding is important for diagnosing and treating birth defects such as neural tube defects and in the rational design of engineered tissues.
|
0.934 |