1985 — 1991 |
Hatten, Mary Elizabeth |
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. |
Influence of Neurons On Astroglial Differentiation
The objective of this project is to study the influence of neurons on astroglial differentiation. To analyze the contribution of neuronal interactions to glial morphology and proliferation, we will purify granule neurons and astroglia harvested from early postnatal mouse cerebellum into highly enriched cell fractions and recombine them in varying ratios. We will then correlate the number of neurons and occurrence of neuron-glial contacts with astroglial morphology by quantitative morphometric analysis of immunostained cells. The proliferation rate of the astroglia will be measured by a cytofluorograf modular flow cytometer system. Time-lapse video microscopy will be used to analyze whether individual astroglial cells, cultured alone or in the presence of varying numbers of neurons, change their shape or have stable, identifiable morphological types over time in culture. To begin to analyze the mechanism of neuronal regulation of astroglial differentiation, we will test for trophic factors in the medium, use a fluorescence assay for gap junctions and analyze whether the intracellular cAMP or Ca++ levels of the astroglia change when neurons are present. To distinguish the contribution of the astroglial genome versus that of neuronal interactions, we will recombine the enriched granule neuron fraction with purified weaver mouse astroglia. To analyze the influence of normal neurons on the proliferation rate of transformed astroglia, we will add normal neurons to clones of rodent C6, G26 and human U251 glioma cell lines and quantitate the proliferation rate of the glioma cells.
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0.954 |
1985 — 1990 |
Hatten, Mary Elizabeth |
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. |
Biochemistry of Granule Cell Migration
Cell surface glycoproteins would be isolated from embryonic mouse cerebellum at different developmental stages and characterized by affinity binding to plant lectins immobilized on Sepharose. These studies should determine the fraction of cell surface glycoproteins under developmental regulation. The importance of both cell surface carbohydrates and carbohydrate-binding proteins (lectins) to cell migrations and interactions would be assessed in vitro with monosaccharides and lectins immobilized on the surface of microwell tissue culture dishes. Detailed analysis of cell migration on derivatized substrata would be made with a time-lapse television system adapted for use with an inverted phase-optics microscope. Cerebellar cell types would be identified by electron microscopy and by specific cell markers. Cell separation following cell agglutination in the presence of plant lectins and sedimentation on a ficoll-isopague gradient would also be assessed by electron microscopy.
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0.954 |
1991 — 2007 |
Hatten, Mary Elizabeth |
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. |
Molecular Mechanisms of Granule Cell Migration
DESCRIPTION (from applicant's abstract): The overall goal of the proposed research is to understand the mechanism of neuronal migration in the vertebrate brain. Two broad goals are to discover the genes required for the migration of CNS neurons on glial substrates and to develop an experimental approach to imaging large cohorts of migrating neurons, in situ, in real time. In prior cycles of this grant, the investigator discovered a gene astrotactin (Astn), that functions as a neuronal ligand for granule cell migration along glial guides. The predicted protein structure of Astn has an extra cellular region containing two potential glycosylation sites, and regions of homology to adhesion molecules of the fibronectin III family and the EGF receptor. The first aim of the proposed research is to carry out a quantitative analysis of a targeted deletion of Astn, examining the size of primary structures, and the development of the two principal cerebellar neurons, the granule cell and the Purkinje cell. In preliminary studies, they discovered an Astn2 and are searching for other family members. In another group of experiments, they will use biochemical methods to isolate the glial receptor for ASTN. Finally, they will extend their long-standing interest in migration to the in vivo setting, using newly developed computer-based methods to follow the migration of large cohorts of granule cells, labeled with retroviral transfer of the GFP marker or with GFP-Astn "knock-in" mice. The new imaging experiments should provide the first information on the movements of granule cells in vivo, using 3D time-lapse recordings of large cohorts of cells. Together these experiments will generate information on the neuron-glial ligand astrotactin, and its role in migration. As the discovery of a three more astrotactin genes demonstrates that Astn is part of a gene family, they will examine the redundancy in function of these genes. The discovery of the glial receptor will provide fundamental information on mechanisms of glial differentiation, information critical to both brain development and brain trauma. Finally, being able to view the movements of granule cells in real time in situ will inform about the relationship between axon (parallel fiber) extension and the locomotion of the granule neuron down the Bergmann glial fiber.
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1 |
1992 — 1995 |
Hatten, Mary Elizabeth |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Control of Neural Cell Identity in Developng Cns
The overall goal of this program is to provide information on early phases of vertebrate CNS development. The proposed program will focus on two major issues: the development of midline structures that establish the pattern of axonal projections in the embryonic CNS and the mechanisms that control neural fate determination in brain. The aim of the first two subprojects will be to examine the early development of two midline structures that guide developing axons, the floor plate of the spinal cord and the optic chiasm of the visual system. In Project 1, Dr. Dodd will use PCR-based assays for floor plate specific genes to examine the control of floor plate induction by axial mesoderm. In Project 2, Dr. Mason will use neuroanatomical approaches to examine the specification of cell type in the developing optic chiasm. A second general aim will be the control of neural fate and differentiation in mammalian CNS. In Project 3, Dr. Jessell will use molecular approaches to examine the role of floor plate-specific genes, and proteins encoded by these novel genes, in neural differentiation. In Project 4, Dr. Hatten will use a novel in vitro system to examine the role of extrinsic signals in the specification of cerebellar granule cell identity. In Project 5, Dr. Heintz will use molecular biological approaches to identify novel genes that show regulated expression during cerebellar granule cell neurogenesis and differentiation.
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1 |
1993 — 1997 |
Hatten, Mary E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Advanced Microscopy
light microscopy; electron microscopy; biomedical facility; confocal scanning microscopy; microscopy; computer data analysis; video recording system; photography;
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1 |
1993 — 1997 |
Hatten, Mary E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Animal
animal breeding; biomedical facility; laboratory mouse; animal colony; germ free condition; genetically modified animals;
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1 |
1993 — 1997 |
Hatten, Mary E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Neurogenesis in the Mammalian Cerebellum
The overall goal of this project is to provide information on the mechanisms that control the specification of granule cell fate in the cerebellar cortex. In the proposed research, we will examine, at the cellular and molecular levels, the signals that control the differentiation of precursor cells derived from the external germinal layer (EGL) of the mouse cerebellar cortex, using an in vitro model system developed in our laboratory. Two issues will be addressed. First, we will determine whether growth factors, in particular those with high levels of expression in developing cerebellum (bFGF, NT3 and BDNF) regulate granule cell specification. Second, to provide a genetic analysis of granule cell determination, we will analyze EGL precursor cells from the neurological mutant mouse weaver, an animal with phenotypic defects in granule cell differentiation, including a failure of neurite outgrowth and glial-guided migration.
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1 |
1995 — 1997 |
Hatten, Mary Elizabeth |
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. |
Weaver Locus and Cerebellar Development
The study of neurological mutations offers a powerful approach to the identification of genes that regulate mammalian brain development. Work in our laboratory has established that the weaver gene regulates a critical step in neuronal differentiation, blocking the progression of immature granule neurons through the early steps of differentiation, including neurite production and migration. The proposed research will combine the tools of cell and molecular biology to further characterize the weaver gene. To understand earlier events in the development of weaver granule cells that map near the weaver locus, genetic markers will be used to identify weaver embryos by a simple PCR analysis, allowing developmental analysis of the midline aspect of the mid/hindbrain territory. In the proposed program, we will examine whether weaver cells show a deficit in the expression of genes encoding DNA binding proteins that mark the cerebellar primordium, and the generation of specific classes of cerebellar neurons by expression of cellular antigen markers within the midline region of the cerebellar anlage. To identify the ligand revealed by the weaver deficit, we will pursue a biochemical approach, fractionating membrane material from GC-B6 cells and using in vitro assays to identify the activity. Peptide sequence for candidate proteins will be obtained in order to obtain cDNAs encoding these ligands from granule cell cDNA libraries. Finally, the proposed research will use in vitro assays to clone the weaver gene. To refine the chromosomal location of weaver, new inter-species backcrosses will be carried out, after which a contiguous set of yeast artificial chromosomes (YACs) containing DNA from the region containing the weaver locus will be constructed. Transfer of DNAs from these YACs to immortalized granule cells will provide a reagent for the rapid location of the weaver gene on the YAC contig, by assaying the ability of transfected cells to rescue weaver granule cell differentiation in complementation assays. Candidate genes will be isolated and screened for the weaver mutation. The proof that the candidate cDNA encodes the weaver gene will be tested in the in vitro complementation assays and ultimately in transgenic mice.
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1 |
1996 — 2002 |
Hatten, Mary Elizabeth |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Control of Neural Cell Identity in Developing Cns
The overall goal of this program is to provide information on early phases of vertebrate CNS development. The proposed program will focus on two major issues: the development of midline structures that establish the pattern of axonal projections in the embryonic CNS and the mechanisms that control neural fate determination in brain. The aim of the first two subprojects will be to examine the early development of two midline structures that guide developing axons, the floor plate of the spinal cord and the optic chiasm of the visual system. In Project 1, Dr. Dodd will use PCR-based assays for floor plate specific genes to examine the control of floor plate induction by axial mesoderm. In Project 2, Dr. Mason will use neuroanatomical approaches to examine the specification of cell type in the developing optic chiasm. A second general aim will be the control of neural fate and differentiation in mammalian CNS. In Project 3, Dr. Jessell will use molecular approaches to examine the role of floor plate-specific genes, and proteins encoded by these novel genes, in neural differentiation. In Project 4, Dr. Hatten will use a novel in vitro system to examine the role of extrinsic signals in the specification of cerebellar granule cell identity. In Project 5, Dr. Heintz will use molecular biological approaches to identify novel genes that show regulated expression during cerebellar granule cell neurogenesis and differentiation.
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1 |
1998 — 2002 |
Hatten, Mary Elizabeth |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Control of Granule Cell Specification in the Cerebellum
The overall goal of this project is to examine the control of granule neuron specification and differentiation in embryonic stages of vertebrate cerebellar development. In the first group of experiments, expression of the zinc finger transcription factor RU49 will be used to map the earliest granule precursors in the midbrain/hindbrain region of mouse embryos ib embryonic days 9.5 through 13.5. The second set of experiments will localize expression of the TGFbeta family of growth factors (BMP6, BMP7 or GDF7) in the midbrain/hindbrain region of the neural tube, and test the role of these factors in induction of RU49. In a third set of experiments, the role of the basic helix loop helix transcription factor Math-1 (atonal) in EGL cell specification and differentiation will be studied Expression of Math-1 will be defined in situ hybridization of mRNAs in vivo between E8.0 and p15, as well as in cultures of purified progenitor cells and the function of Math-1 will be investigated by over-expression of the gene and by expression of a dominant negative form of the gene. In a fourth group of experiments, video microscopy will be used to define the mitotic activity and mode of movement of progenitor cells within the rhombic lip cells during the initial steps of the formation of the external germinal layer (EGL). The relationship between cell division and movement of cells onto the surface of the cerebellar anlage will be examined by tracing dye-labeled cells in explants of embronic midbrain/hindbrain tissue. The behavior of cells in the rhombic lip will be compared among three vertebrates: Mus musculis, Gallus domesticus and Xenopus laevis.
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1 |
2000 — 2004 |
Hatten, Mary Elizabeth |
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. |
Genetics of Cerebellar Axon Formation
DESCRIPTION (Verbatim From the Applicant's Abstract): The cerebellar circuitry develops through the coordinated outgrowth of the axons of the two principal classes of neurons, the granule cell and the Purkinje cell, and the formation of interconnections among them. The proposed research will study the genetic control of cerebellar neurite extension. To identify genes that function in the signal cascades required for axon outgrowth, we have cloned two mouse homologues of the C. elegans gene UNC51 that encodes a protein serine/threonine kinase that functions in axon formation. Preliminary experiments demonstrate that one of the murine homologues, Unc51.1 is expressed in cerebellar granule neurons and functions in granule cell neurite extension in vitro and in vivo. The proposed research will use a combination of in vitro primary neuron culture, molecular cloning, biochemistry and mouse molecular genetics to test the hypothesis that Unc51.1 and Unc51.2 have essential roles in axon formation in the developing cerebellar cortex. The Research Plan has three main aims. The first group of experiments will characterize the expression and function of Unc51.2 in developing and adult brain. The function of the gene in granule cells or Purkinje cells will be tested by expressing a kinase deficient form of the gene and assaying neurite outgrowth in vitro and in vivo. In a second group of experiments, we will use the yeast two-hybrid system to identify signaling pathways for murine Unc51.1 and Unc51.2. Finally, we will generate in vivo models of Unc51.1 and Unc51.2 to test for protein function by making null alleles of each of the genes using conventional gene targeting approach. In mice with null alleles, we will be able to examine the function of the genes in neurite extension in all of the CNS neurons in which the gene is expressed. In the BAC transgenic animals we can examine the effect of kinase-deficient neurons on the development of the other cerebellar neurons and glia.
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1 |
2003 — 2007 |
Hatten, Mary Elizabeth |
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. |
Embryonic Development of the Cerebellum
DESCRIPTION (provided by applicant): Our previous work has focused on the specification of granule cells in the embryonic cerebellum. Here we wish to turn our attention to the other principal neuron of the cerebellum, the Purkinje cell and to the deep neurons. The overall aim of this project is to determine the topographic expression of four classes of transcription factors, LIM homeodomain genes (Lhx1, Lhx3, Lhx5, and Lhx9), basic helix-loop-helix genes (Math1, Mash1, Ngn1, Ngn2), Paired box containing genes (Pax2) and Even-Skipped genes (Evx1,2) in Purkinje and deep neuron progenitor cell populations in the embryonic cerebellar primordium, and to use these markers to examine factors that shape cell fate specification. The action of local inductive factors, BMPs on gene expression and cell specification patterning will be tested in gene expression studies in the chick embryo and in the Dreher mutant mouse.First, we will define patterns of gene expression in murine and avian embryos focusing on the time periods when deep neurons and Purkinje cells are generated. We will further refine our analysis by studying BAC transgenic mice for Lhx1, Lhx3, Lhx5, Lhx9, Math1, Mash1, Ngn1, Ngn2 and Evx1 generated in another project. Second, we will study the Dreher mutant mouse, because it suffers a loss of BMPs, and use in ovo electroporation in avian embryos to define the role of BMP6, BMP7 and GDF7 in transcription factor expression patterns. Finally, we will study the function of these transcription factors in cerebellar cell differentiation by analyzing mice with targeted null mutations and by transfecting morpholino antisense oligonucleotides into avian embryos.
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1 |
2006 — 2010 |
Hatten, Mary Elizabeth |
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. |
Role of Mpar6 Polarity Cns Neuronal Migration
The migration of young neurons from sites where they are generated into the positions where they establish the circuitry of the adult brain is a critical step in development. Defects in migration cause a host of human birth defects, ranging from severe mental retardation to subtle learning disabilities, as well as a large number of the epilepsies. Our lab has focused on understanding the genes that control migration, in the hope that insights into this key step in normal development. We use the migration of the cerebellar granule neuron as a model system to examine the molecular control of glial-guided neuronal migration. The establishment of neuronal polarity is a key step in initiating neuronal migration along the glial guide. Screens for genes that function in granule neuron migration revealed high levels of expression of the polarity signaling complex mPar6a neurons exiting the cycle and establishing polarity. In C. elegans, a set of 6 PAR proteins establish anterior/posterior asymmetries and control subsequent asymmetric cell divisions. PAR proteins are conserved throughout evolution. In Preliminary Studies (Solecki et al, 2004), we discovered that the mParGa signaling complex is localized in the centrosome of migrating cerebellar granule neurons, where it coordinates the movement of the centrosome and the nucleus as the neuron migrates along the glial fiber. In the proposed research, we will study the other components of the mParGa complex, aPKC^ and Par3, in the polarity of migrating granule neurons. The role of mPartxx in cell division suggests that targeted loss of function mutants and shRNA experiments will not be feasible. We will therefore use a novel method developed by Roger Tsien to incorporate a genetic tag (TC) which binds the dye ReAshS, into mParGq and use chromophore-assisted light inactivation with a monochromatic laser to inactivate mParGa in the centrosome. For those experiments, we will generate TC-mPar6a BAG transgenic mice, enabling studies on granule cells and cortical neurons. In a final group of experiments, we will study a receptor/ligand system expressed in granule cells which interacts with the mParGa complex, the EphB ligands ephrin-B1 and ephrin-B2. Together, these experiments will provide novel information on the regulation of neuronal migration in cortical regions of developing brain.
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0.915 |
2007 |
Hatten, Mary Elizabeth |
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. |
Role of Mpar6 Polarity in Cns Neuronal Migration
[unreadable] DESCRIPTION (provided by applicant): The migration of young neurons from sites where they are generated into the positions where they establish the circuitry of the adult brain is a critical step in development. Defects in migration cause a host of human birth defects, ranging from severe mental retardation to subtle learning disabilities, as well as a large number of the epilepsies. Our lab has focused on understanding the genes that control migration, in the hope that insights into this key step in normal development. We use the migration of the cerebellar granule neuron as a model system to examine the molecular control of glial-guided neuronal migration. The establishment of neuronal polarity is a key step in initiating neuronal migration along the glial guide. Screens for genes that function in granule neuron migration revealed high levels of expression of the polarity signaling complex mPar6a neurons exiting the cycle and establishing polarity. In C. elegans, a set of 6 PAR proteins establish anterior/posterior asymmetries and control subsequent asymmetric cell divisions. PAR proteins are conserved throughout evolution. In Preliminary Studies (Solecki et al, 2004), we discovered that the mPar6a signaling complex is localized in the centrosome of migrating cerebellar granule neurons, where it coordinates the movement of the centrosome and the nucleus as the neuron migrates along the glial fiber. In the proposed research, we will study the other components of the mPar6a complex, aPKCzeta and Par3, in the polarity of migrating granule neurons. The role of mPar6a in cell division suggests that targeted loss of function mutants and shRNA experiments will not be feasible. We will therefore use a novel method developed by Roger Tsien to incorporate a genetic tag (TC) which binds the dye ReAshS, into mParGq and use chromophore-assisted light inactivation with a monochromatic laser to inactivate mParGa in the centrosome. For those experiments, we will generate TC-mPar6a BAC transgenic mice, enabling studies on granule cells and cortical neurons. In a final group of experiments, we will study a receptor/ligand system expressed in granule cells which interacts with the mParGa complex, the EphB ligands ephrin-B1 and ephrin-B2. Together, these experiments will provide novel information on the regulation of neuronal migration in cortical regions of developing brain. [unreadable] [unreadable]
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0.915 |
2011 — 2015 |
Hatten, Mary Elizabeth |
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. |
Role of Cdc42 and Par6 Polarity Complex in Cns Neuronal Migration
DESCRIPTION (provided by applicant): The development of the mammalian brain depends on the migrations of neuronal precursors from germinal zones, where they are generated, and their assembly into neuronal laminae, where synaptic connections form. Since CNS migration disorders are associated with a number of cortical malformations, and are a major cause of disease in the developing human brain, including mental retardation and epilepsy, a clearer understanding of the molecular control of CNS neuronal migration could be relevant to the diagnosis and treatment of human developmental brain disorders. Neuronal migration critically depends on the polarization of the neuron in the direction of moment, and with support from this grant, we previously established that the conserved mPar61 polarity complex localizes to the centrosome and coordinates the forward movement of the centrosome and soma, by a mechanism that includes activation of acto-myosin contractile motors in the proximal region of the leading process, in migrating cerebellar granule neurons. The overall goal of the proposed research is to define the relative contributions of the master polarity regulator Cdc42 and the three Par6 isoforms to critical steps in CNS migration, including the formation and maintenance of a highly polarized leading process, and to define their role(s) in receptor trafficking of neuronal adhesion proteins. Although a clear role for Cdc42 has been established in the migration of many non-neuronal cells, and in dendritic arborization and axon guidance, the roles of Cdc42 and the relative role of the Par6 isoforms have not been analyzed in high-resolution time-lapse imaging of live, migrating CNS neurons. Given the importance of glial-guided migration to the formation of neuronal layers in cortical regions of brain, we will focus on this highly specialized migration system, using cerebellar granule neurons migrating on glia as our model system. In Aim 1, we will study a conditional loss of function of Cdc42;a similar plan will be implemented in Aim 3 for each of the three Par6 isoforms expressed in granule cell progenitors (GCPs). As a complementary approach, in Aims 2 &3, we will use siRNAs and shRNAs to knockdown Cdc42 and Par6 isoform levels and compare knockdown phenotypes with conditional loss of function phenotypes. If Cdc42 is in the same genetic pathway with any or all of the Par6 isoforms, we would expect to see similar phenotypes for Cdc42 and Par6 isoform loss of function. We will also use Raichu probes for Cdc42, kindly provided by Dr. Miki Matsuda, which are FRET-based probes that monitor Cdc42 activation in localized regions of a cell. These probes will enable us to evaluate the spatiotemporal localization of Cdc42 activation relative to the Par6 isoforms in migrating GCPs. The discovery of changing patterns of Par6 expression during cerebellar development is an exciting opportunity to understand their relative contributions to neuronal migration and whether they act within a Cdc42 signaling pathway. PUBLIC HEALTH RELEVANCE: The proposed research will examine the molecular pathways that control cell polarity during neuronal migration in the developing brain. We will test the role of wild type and conditional loss of function of the polarity regulator Cdc42 and three isoforms of the Par6 polarity proteins on two critical steps in glial-guided neuronal migration: (1) the extension of a leading process in the direction of migration, and (2) the localization of adhesion proteins that bind the neuron to the glial fiber. These studies will be important for understanding the mechanisms of diseases that affect normal brain development, including mental retardation, autism and epilepsy.
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0.915 |
2015 — 2016 |
Hatten, Mary Elizabeth |
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.) |
Development of a Model System to Study Human Cerebellar Neurons
? DESCRIPTION (provided by applicant): The cerebellum has a critical role in motor coordination, balance and controlling eye sacchades, with recent evidence highlighting a role in feed-forward learning, visuo-spatial memory, attention, language, and other higher cognitive functions. Importantly, cerebellar pathology and dysfunction have been linked to developmental diseases such as autism and ADHD. While mouse models of such complex disorders have provided critical insights, mouse genetic models do not always model human disease phenotypes. There is therefore a critical need for a human model system to study cerebellar development and dysfunction. Excitingly, it is now possible to create human model systems of the central nervous system through the use of human pluripotent stem cells (hPSCs). While hPSC-based human model systems have been developed for disorders such as Parkinson's disease and Amyotrophic Lateral Sclerosis through the differentiation of dopaminergic or motor neuron subtypes, protocols for the generation of specific cerebellar neurons are lacking. The proposed research aims to develop methods to differentiate hPSCs into the two primary neurons of the cerebellum, the granule cell (GC) and the Purkinje cell (PC), and thoroughly characterize resulting cells. To assess gene expression, a novel genetic tool, the bacTRAP, will be employed to isolate translating mRNA specifically from EGFP-tagged GCs or PCs within a heterogeneous culture. Following RNA sequencing, results will be compared to datasets of various developmental stages of native mouse GCs and PCs already obtained in the lab. To assess physiology, basic membrane properties as well as GC and PC specific currents will be measured in vitro. To assess the ability to integrate into the cerebellar circuit, we will adapt methods we reported for mES cells to implant hPSC-derived GCs and PCs into the neonatal mouse cerebellum. Clarity or ClearT2 tissue clearing methods and novel whole brain imaging techniques will allow imaging of the development and integration of implanted neurons within the mouse cerebellar circuit. These assays will provide a detailed analysis of hPSC-derived GC and PC gene expression and functional capacity, against which patient-hPSC derived cerebellar neurons, as well as other neural subtypes, can be assessed. The Hatten lab has carried out seminal studies on cerebellar development and neuronal migration. In preliminary work, the Hatten lab has generated protocols for the differentiation of mouse ES cells into cerebellar neurons and utilized bacTRAP to obtain gene expression datasets of native mouse GCs and PCs. Importantly; we have adapted these differentiation protocols to hPSCs, generating definitive human GCs and PCs for the first time. The proposed research aims to refine these protocols to generate mature neurons, and to thoroughly characterize them through gene expression profiling, electrophysiology, and integration capacity into the mouse cerebellar circuit following implantation. These studies will create a critical new human model system of cerebellar development and dysfunction.
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0.915 |
2019 — 2020 |
Hatten, Mary Elizabeth |
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.) |
Chromatin Changes During Cns Migration and Circuit Formation
PROJECT SUMMARY/ABSTRACT! The proposed research will examine how chromatin regulates gene expression during CNS migration and circuit formation, using the cerebellar granule cell as a model. The work builds on our recent discovery that dramatic changes occur in the levels of chromatin remodeling genes during cerebellar development, including changes in histone modifying genes and in Tet genes, which oxidize 5-methyl- cytosine (5mC) into 5-hydroxymethyl-cytosine (5hmC), a brain-specific DNA modification. Our studies showed that increased levels of 5hmC correlated with increased levels of ion channel genes and of axon guidance (dendritic) genes in post-migratory neurons (Zhu et al, 2016). Moreover, RNAi-mediated knockdown of Tet genes in migrating cerebellar granule cells in ex vivo tissue slices blocked the transition from a bipolar, migrating cell into a multipolar cell that is extending dendrites and forming synaptic contacts with ingrowing afferent, mossy fibers (Zhu et al, 2016). These findings provided the first evidence for the idea that chromatin changes underlie the formation of the cerebellar circuitry.! In the proposed research, we will collaborate with our colleagues, Dr. David Allis and Dr. Erica Korb, who are authorities on chromatin biology, to characterize modifications in four basic histone methylation marks that regulate transcriptional activation and repression, histone 3 lysine residue 4 (H3K4), H3K9, H3K27 and H3K36, in a well-characterized CNS neuron, the cerebellar granule cell (GC), before, during and just after glial-guided migration, as the cerebellar circuitry forms. We will then identify changes in gene expression associated with these histone methylation changes. Finally, we will use RNAi methodology to study the function of histone methyltransferases and demethylases involved in generating these histone marks in migration, dendrite extension and synapse formation on ex vivo cerebellar slices. These studies will provide critical information on chromatin changes underlying CNS migration and circuit formation in the cerebellum.!
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2021 |
Hatten, Mary Elizabeth Hull, Court A (co-PI) [⬀] |
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. |
Role of Astn2 in Cerebellar Circuit Function and Asd-Related Behaviors
PROJECT SUMMARY The proposed research addresses a critically important question in autism spectrum disorder (ASD) research: how defects in cerebellar circuits contribute to ASD. In particular, it examines the role of the predominantly cerebellar gene ASTN2 in cerebellar circuit function and ASD-related behaviors. Copy number variations (CNVs) in ASTN2 have been identified as a significant risk factor for ASD (Lionel et al, 2014), suggesting that ASTN2 mutations such as those found in patients with ASTN2 CNVs, lead to altered cerebellar synaptic function. In addition, we recently reported a family with a paternally inherited intragenic ASTN2 duplication, which caused a heterozygous loss of function of ASTN2. The family manifested a range of neurodevelopmental disorders, including ASD, learning difficulties and speech and language delay (Behesti et al, 2018). Our cellular and molecular studies on mouse cerebellum show that ASTN2 binds to and regulates the trafficking of multiple synaptic proteins, including Neuroligins, which have been genetically linked to ASDs, and modulates cerebellar Purkinje cell (PC) synaptic activity (Behesti et al, 2018). To provide a genetic model to study cerebellar circuit function, we generated both a global loss of function Astn2 line and a floxed Astn2 line for conditional knockout experiments. New, preliminary evidence indicates that PCs in mice lacking Astn2 have a decrease in evoked excitation relative to inhibition in PCs and reduced PC dendritic spine density, suggesting specific cerebellar circuit defects. In addition, preliminary evidence shows mild motor deficits and defects in USVs and an open field assay, ASD-related behaviors. As other preliminary findings do not indicate major defects in cerebellar development, we hypothesize that the behavioral defects we observed relate to defects in the cerebellar circuitry with underlying changes in receptor trafficking. In the proposed research, we will 1) test how loss of Astn2 alters intrinsic excitability in PCs and the synaptic efficacy of their presynaptic inputs from GCs and molecular layer interneurons, 2) use proteomics to identify changes in the levels of synaptic proteins and live imaging to assess whether such changes relate to changes in the rate of endocytosis, 3) compare changes in PC dendritic branching as well as the regional distribution of PC spines in wild type and mutant animals to provide insight on whether there are changes in the organization of PC inputs during the establishment of the cerebellar circuitry, and 4) analyze changes in social behavior and ultrasonic vocalization in Astn2 wild type, heterozygous and mutant animals. Taken together, the proposed research will provide a new mouse model that allows us to link an ASD-related gene that is predominantly expressed in the cerebellum with specific cerebellar circuit function and molecular pathways.
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