Daniel H. Turnbull - US grants

magnetic resonance imaging; nuclear magnetic resonance spectroscopy; biomedical equipment purchase;

DESCRIPTION (Adapted from the applicant's abstract): The availability of genetic analysis and transgenic techniques in the mouse have led to its widespread acceptance as the preferred animal model for studying mammalian development and many human diseases. The basic limitation for studying development in the mouse, or any other mammal, is the inaccessibility of the embryos which are encased in the maternal uterus. Recently, several groups have developed methods to inject cells and lineage-tracing viruses into the lateral forebrain ventricles of rat and mouse embryos at relatively late stages of embryogenesis (mouse gestational age 12.5 days, E12.5 or later) but these injections are not targeted to specific regions since the cells and viruses are free to move throughout embryonic ventricles before integrating in the brain. Furthermore, much of embryonic brain development has been completed before the agents can be injected. This laboratory has developed a high resolution UBM to visualize mouse embryos, in vivo. Preliminary studies have demonstrated that 40-50 MHZ UBM can be used as a guidance system, allowing targeting injections of cells and retrovirus into the embryonic muse brain and other organs at stages as early as E9.5. The overall goal of this project is to develop instrumentation to improve both the visualization and delivery capability injection system, enabling more accurate targeted injections into live mouse embryos in utero at all stages of development between E8.5 and E13.5 The specific aims are: 1) to produce a set of transducers which optimize the imaging and guidance performance of the UBM for a range of mouse embryonic stages between E8.5 and E13.5; 2) to develop 50 MHz multi- element annular array transducers to improve the UBM image characteristic over a wide depth of field; 3) to implement robust 3 dimensional (3-D) UBM imaging protocols, including real-time 3-D image manipulation, in order to improve the accuracy of site-specific ultrasound-guide injections; and 3) to determine the feasibility of using contrast agents to improve the localization of the infection site. The combination of recent breakthroughs in mouse genetics, together with this state-of-the-art UBM technology, will provide powerful new tools for studying mammalian development and human disease models in the mouse.

DESCRIPTION: The overall goal of this proposal is to develop a means of visualizing cells injected into the early mouse brain embryo and other tissues with high resoution MR micro-imaging, in vivo, in order to study the integration and proliferation patterns of the transplanted neural progenitors. The applicants propose to make use of a 16 amino-acid peptide, the third helix of the Drosophila Antennapedia homeodomain, which has recently been shown to b capable of translocating through cell membranes with large molecular weight molecules attached to the peptide. The specific aims of the proposed research are the following. 1) Synthesize novel peptide conjugates with gadolinium-base MR contrast agents and the 16 peptide homeodomain. 2) Evaluate the peptide-Gd conjugate for its potential to be internalized by cultured neural cell lines and primary neural cells, and quantitate the resulting MR contrast enhancement of cells in vitro. 3) Evaluate the utility of these MR-labeled cells for studying in vivo integration and proliferation patterns of neural progenitors. MR-labeled cells will be injected into specific target regions in the brains o mouse embryos staged between gestational ages 9.5 to 13.5 days, and 3D MR imag data will be collected and analyzed from individual embryos at selected timepoints up to 72 hours after injection.

9728287 TURNBULL A persistent question in developmental neurobiology is when and how neuronal cells become committed to become particular cell types. Transplantation of cells and tissues from one region of the embryonic brain to another provides a method to test whether neural cells take on the characteristics of their new environment, or maintain the characteristics of their region of origin. In the past, these methods have been used in chick embryos, which are relatively easily accessible at embryonic stages. These studies have shown that tissue grafts from the mid-hindbrain, or early stage brainstem region maintain characteristics of the mid-hindbrain even after transplantation to far removed regions of the embryonic forebrain. Testing the commitment of mammalian neural cells through transplantation has been more difficult, because of the inaccessibility of embryos encased in the maternal uterus. Dr. Turnbull has recently developed a high resolution ultrasound imaging system which allows in utero image-guided transplantation of early stage neural cells between specific regions of the embryonic mouse brain. Using this system to transplant cells between embryonic mid-hindbrain and forebrain regions, Dr. Turnbull's laboratory has determined that a subpopulation of mouse mid-hindbrain cells are capable of taking on characteristics of the forebrain after transplantation, at embryonic stages significantly later than would have been predicted from previous experiments in the chick. Dr. Turnbull and his coworkers will now use this approach to characterize the embryonic stages at which cells from the mouse midbrain and cerebellum become committed to a mid-hindbrain fate. In addition, transplantation experiments will be conducted using neural cells from mouse mutant strains, in which alterations in specific genes are known to disrupt normal development of the early mid-hindbrain region. By comparing ultimate fates of transplanted normal cells versus cells obtained from defined mutant m ouse strains, these investigators will determine the role of several genes in committing mid-hindbrain cells to the fate of their region of origin. The experimental techniques being developed in this project, combining direct embryonic manipulations with the extensive genetic information and defined mutant strains available in the mouse, will provide a powerful new approach for studying developmental processes and genetic interactions in the mammalian nervous system.

The availability of transgenic and gene targeting methods, and the large number of spontaneous and induced mutant mice with altered neural development, have led to new insights into the genetic pathways involved in patterning events and cell differentiation in the mouse CNS. The same genetic techniques also are being used increasingly to produce mouse models of human degenerative brain diseases. Lacking are effective, non- invasive methods of analyzing the dynamic, 3-D processes involved in mouse neural development and brain diseases. Lacking are effective, non- invasive methods of analyzing the dynamic, 3-D processes involved in mouse neural development and brain disease. Clinical evaluation of human brain development and disease relies heavily on medical imaging technologies, with ultrasound imaging providing the means for detecting developmental defects, and magnetic resonance (MR) imaging techniques being widely used for non-invasive imaging of the anatomical structure and function of the postnatal human brain. We are developing high resolution in vivo ultrasound and MR micro-imaging methods for visualizing the developing mouse brain. The broad goals of this project are to develop a unique combination of ultrasound and MR micro-imaging techniques to analyze neural development and degeneration in the mouse from early embryonic to adult stages. The specific aims are: 1) To develop ultrasound instrumentation to optimize resolution for imaging early stage mouse embryos. 2) To develop robust 3-D ultrasound imaging methods for in utero mouse embryo imaging. 3) To develop optimal rf coils for 7 Tesla MR micro-imaging of mouse brain development. 4) To optimize a set of MR pulse sequences for anatomical and functional imaging of the mouse brain. The combination of recent breakthroughs in mouse genetics together with this high resolution ultrasound and MR micro-imaging technology will provide powerful new tools for studying mouse neural development and mouse models of human neurodegenerative disease, and for exploring in utero cell and gene therapy approaches.

DESCRIPTION (provided by applicant): The availability of transgenic and gene targeting methods, and the large number of spontaneous and induced mutant mice with altered neural development, have led to significant progress in dissecting the genetic pathways involved in patterning and cell specification in the mouse nervous system, and in producing many mouse models of human neurodegenerative diseases. Despite these advances, little is known currently about the relationship between genetic changes and altered brain function, in part because of the lack of readily available and efficient quantitative methods to analyze functional changes in the mouse brain. We have developed both ultrasound biomicroscopy (UBM) and magnetic resonance micro-imaging (muMRI) approaches to analyze anatomical and functional neural development and disease in the mouse from early embryonic to adult stages. UBM-guided injections have also provided the means to perform rapid, direct gain-of-function genetic studies in the embryonic mouse CNS. The combination of UBM and muMRI approaches being developed can provide in vivo assessment of brain function in normal and disease model mice. The broad goals of this project are to develop in vivo micro-imaging approaches enabling analysis of normal and abnormal function in the developing mouse brain, and the relationships between genetic changes and altered brain function from embryonic to adult stages. The specific aims are: 1) To develop and test two new direct methods for gain-of-function studies in the embryonic mouse CNS. 2) To test whether transgenic, CNS-specific over-expression of iron transport and storage proteins can be used for in vivo imaging of gene expression with gMRI. 3) To develop UBM and pMRI methods to quantify blood volume and flow in the developing mouse brain. 4) To quantify changes in manganese-enhanced MRI resulting from stimulation of neuronal activity in the early postnatal to adult mouse brain. The combination of genetic engineering approaches in the mouse with advanced ultrasound and magnetic resonance micro-imaging technology will provide powerful new tools for analyzing mouse developmental neurobiology, and leading to new insights into mammalian brain development and neurodegenerative diseases.

DESCRIPTION (provided by applicant): Primary brain tumors are among the most aggressive and fatal human neoplasms, often affecting children and younger adults. The availability of transgenic and gene targeting methods, and the large number of spontaneous and induced mutant mouse strains, have led to the increased use of the mouse as a key animal model system to study cancer. In the past several years, the use of mouse genetics has led to significant progress in understanding the genetic pathways and molecular events involved in the formation and progression of both medulloblastoma and glioma, the most common pediatric and adult brain tumors, respectively. In order to realize the full potential of these genetically engineered mouse models, it is imperative to develop in vivo microscopic imaging approaches, allowing longitudinal analysis of tumor progression and response to novel therapeutic agents. Angiogenesis is thought to be a critical prerequisite for tumor progression, and brain tumor malignancy is intimately related to angiogenesis and vascular density, especially in gliomas. The broad goals of this project are to develop both functional and molecular magnetic resonance micro-imaging (mu MRI) approaches to detect and quantify angiogenesis in mouse brain tumors. We have recently used in utero retrovirus injection to induce medulloblastomas in the postnatal mouse cerebellum. We propose to use a similar retrovirus injection, a measure of angiogenesis to induce gliomas, and will also investigate a transgenic mouse glioma model. We are developing contrast-enhanced perfusion mu MRI techniques to measure cerebral blood volume (CBV) approach in normal mouse brain and brain tumors. A direct, in vivo molecular targeting approach will also be developed to assess angiogenesis. We will generate transgenic mice that overexpress cell surface receptors in neovascular endothelial cells, and image the brains of these mice after intravenous injection of a superparamagnetic MRI contrast agent-tagged ligand. Brain tumors will be induced in these transgenic mice and imaged with mu MRI to quantify vascular density in the developing brain tumors. The functional and molecular mu MRI approaches will be developed during an exploratory, feasibility phase (R21) and later used in careful studies of tumor progression and response to anti-angiogenic therapies in a development phase (R33). The new technologies developed under this project are of critical importance for understanding angiogenesis and brain tumor progression, and will revolutionize tumor biology in genetically accurate mouse models of cancer.

DESCRIPTION (provided by applicant): Over the past decade, investigations using genetically-engineered mice have led to new insights into the genetic control of embryonic vascular development, which has also had a major impact on our understanding of neovascularization in many human diseases including cancer, atherosclerosis and diabetes. Micro-imaging methods such as ultrasound biomicroscopy (UBM) and magnetic resonance micro-imaging (micro-MRI) can play an important role in this research, enabling direct in utero visualization of the developing mouse embryo. To date, there has been relatively little progress in the area of molecular imaging with ultrasound and MRI, especially in the area of vascular development. UBM is a real time imaging method enabling noninvasive in vivo analysis of mouse embryonic cardiovascular anatomy and hemodynamics, and can also be applied for image-guided intravascular injection of contrast agents. Micro-MRI provides better 3D resolution and more flexibility than UBM in manipulating cellular/tissue contrast, including more available contrast agents and approaches for cell-targeted imaging, but requires longer acquisition times, and has only recently been demonstrated for effective in utero imaging of mouse embryos. Several reports have recently demonstrated that biotinylation of cell surfaces can be achieved, allowing cell-targeted imaging with avidin-conjugated contrast agents, which are now available for both ultrasound and MRI. This is an attractive option for imaging vascular endothelial cells (VECs), since contrast agents can be delivered to the cells of interest via intravascular injection, even at embryonic stages of development. Moreover, the binding between avidin and biotin is the strongest found in nature, which should make it possible to label vascular cells even in the face of high wall shear rates associated with arterial blood flow. The specific aims of this project are: 1) To optimize the micro-MRI protocols required for in utero analysis of cardiovascular development;2) To produce transgenic mice designed for targeted imaging of VECs with UBM and micro-MRI;and 3) To establish VEC-targeted micro-MRI approaches for improved analyses of embryonic vasculature. The approaches developed in this project will provide powerful new tools for direct analysis of vascular development in living mouse embryos. Significantly, these new molecular imaging methods will provide, for the first time, the ability to detect vascular gene expression in utero in normal and genetically-engineered mice. PUBLIC HEALTH RELEVANCE: This project aims to move ultrasound and MRI vascular micro-imaging methods beyond measures of anatomy and function, to the level of in vivo molecular imaging in genetically-engineered mouse models of a wide range of cardiovascular disease. These new molecular imaging approaches will revolutionize mouse genetics, enabling new studies linking gene expression to vascular morphology and physiological function in the best characterized mammalian model of human development and disease.

DESCRIPTION (provided by applicant): Functional neuroimaging methods for mapping neural activity in the mouse brain are required to analyze altered brain function in genetically-engineered mice with defined neuroanatomical and/or neurological defects. In larger rodents and primates, including humans, functional magnetic resonance imaging (fMRI) has been used increasingly to map brain activity in the major sensory systems. However, effective fMRI approaches have been difficult to implement in mice because of the high spatial resolution requirements and the presence of significant susceptibility artifacts at the high magnetic fields used for mouse MRI. Among sensory brain regions, the auditory system has been difficult to study with fMRI in mice or larger mammals because of severe susceptibility artifacts in the lower auditory centers and because of the inherent difficulties in uncoupling the auditory response to a defined acoustic stimulus from the response to the background noise produced by the MRI gradient coils. We have recently investigated a Manganese (Mn)-Enhanced MRI (MEMRI) method with the potential to provide high resolution maps of auditory brain activity induced by defined acoustic stimulation in awake behaving mice outside the magnet. Our preliminary results show that sound-induced neural activity in auditory centers of the mouse brain can be detected with MEMRI, and that altered patterns of enhancement/activity can be quantified in mice with conductive hearing loss. The broad goals of this project are to further develop these approaches, optimizing MEMRI protocols for mapping auditory neural activity in normal and deafened mice, enabling in vivo brain imaging studies of the development and plasticity of the auditory mouse brain. The specific aims are: 1) To establish an optimal MEMRI protocol to detect neural activity in the mouse auditory midbrain. 2) To establish the spatial / frequency resolution of MEMRI for auditory brain mapping in the mouse. 3) To define and characterize altered patterns of MEMRI enhancement in unilaterally deaf mice. These studies will form the basis for future brain mapping investigations in normal and genetically-engineered mice, providing much needed new neuroimaging tools for studying the genetic pathways underlying normal hearing and hearing disorders.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Over the past decade, investigations using genetically-engineered mice have led to new insights into the genetic control of embryonic vascular development, which has also had a major impact on our understanding of neovascularization in diseases such as cancer and diabetic retinopathy. Noninvasive micro-imaging methods such as ultrasound biomicroscopy (UBM) and high-resolution magnetic resonance imaging (MRI) can play an important role in this research, enabling direct in utero visualization of the developing mouse embryo. We have pioneered the use of UBM and UBM-guided Doppler ultrasound for in vivo anatomical and functional cardiovascular development in normal and mutant mouse embryos. MRI of live embryos has been more difficult, but several groups have shown the utility of contrast-perfusion for high resolution MRI analysis of vascular structures in fixed mouse embryos. Lacking in these studies has been the ability to image molecular targets directly, to better understand and correlate gene expression patterns with morphological and functional data provided by UBM and MRI. Recent advances indicate that cell-specific vascular imaging should be possible using ultrasound and MRI contrast agents targeted to specific endothelial cell receptors. This strategy is likely to be successful first in transgenic mice that over-express defined receptors from endothelial cell-specific promoters, although similar approaches may enable future imaging of endogenous receptors in mice and men. Targeted ultrasound contrast agents have the additional advantage of potentiating in vivo cell-specific gene delivery, via ultrasound-mediated transfection, or sonoporation. [unreadable] The specific aims are: [unreadable] [unreadable] 1) To produce transgenic mice over-expressing Transferrin Receptor (TfR) from endothelial promoters. [unreadable] 2) To determine optimal conditions for sonoporation in the mouse embryonic cardiovascular system. [unreadable] 3) To develop and test TfR-specific contrast agents for in utero targeted UBM imaging and sonoporation of the mouse embryonic cardiovascular system. [unreadable] 4) To develop and test TfR-specific contrast agents for targeted vascular MRI in fixed mouse embryos. [unreadable] [unreadable] The approaches developed in this project will provide powerful new tools for direct analysis of vascular development in living mouse embryos. Significantly, these new molecular imaging methods will provide, for the first time, the ability to detect gene expression in utero in normal and genetically-engineered mice. (End of Abstract) [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Genetic manipulation in the mouse, using gene targeting and transgene insertions, is now a prevalent method for studying developmental processes and for modeling human diseases. While techniques to alter the genetic makeup of the mouse continue to be refined and used more widely, our ability to rapidly and effectively analyze mutant mouse phenotypes has become the limiting step in many investigations. The Mouse Imaging Facility at NYU School of Medicine employs both ultrasound and magnetic resonance (MR) micro-imaging approaches to analyze anatomical, functional and molecular changes in a wide variety of mouse models, from early embryonic to adult stages of development. There is a long history of support for the Mouse Imaging Facility at NYU School of Medicine, under the joint sponsorship of the Skirball Institute of Biomolecular Medicine and the NYU Cancer Institute. In this application, we request funds to cover the costs of purchasing a new state-of-the-art MR console, bringing our 7-Tesla MR Micro-imaging system up to current standards, and allowing us to continue to support a broad set of NIH-funded projects requiring mouse MRI. This upgrade is well justified, since the console and ancillary equipment are over seven years old, and most of the subsystems are obsolete and difficult or impossible to repair/replace. The research projects currently utilizing MR micro-imaging include analysis of developmental and functional abnormalities in mice with defects in heart and brain development, detection of Multiple Sclerosis lesions in a transgenic mouse model, analysis of tumor progression in mouse models using perfusion MRI and MR spectroscopy, and the development and application of MR contrast reagents targeted to amyloid for plaque detection and monitoring in transgenic mouse models of Alzheimer's Disease. The upgrade of the MR micro-imaging console in the Mouse Imaging Facility will ensure that these innovative and critical applications of mouse MRI will continue to be available to this strong group of NIH-funded investigators.

The Mouse Imaging Core of the New York University Cancer Institute (NYUCI) provides services to members utilizing two micro-imaging methods in the living mouse: 1) magnetic resonance micro-imaging (micro-MRI);and 2) ultrasound biomicroscopy (UBM). The focus of this imaging core on mice has been motivated by the increasing use of genetically engineered mice as model systems for studying cancer. Magnetic resonance and ultrasound imaging are indispensable tools used in the clinical diagnosis and staging of human cancer. To realize the full potential of mouse models of cancer, it is imperative to develop n vivo microscopic imaging approaches, allowing analysis, of disease progression and response to therapeutic agents in mice. The Mouse Imaging Core includes a 30-55 MHz UBM scanner and a 7 Tesla micro-MRI system, both situated in the Skirball SPF Mouse Facility, and available for noninvasive microimaging, functional analysis of blood flow and perfusion, as well as UBM-guided manipulation in mice from early embryonic through adult stages of development. The development of instrumentation and imaging approaches to manipulate developmental processes, to detect tumors and to analyze angiogenesis, tumor morphology, progression, regression and metastases has the potential to revolutionize cancer research. In combination with transgenic and gene targeting approaches in the mouse, in vivo microscopic imaging methods provide powerful and efficient new tools for studying the molecular and genetic mechanisms underlying oncogenesis.

DESCRIPTION (provided by applicant): A promising therapeutic approach for brain injury and many neurological diseases is to harness the endogenous neural stem cells (NSCs) to replenish damaged neurons and glia. To study the response of NSCs to brain injury in mouse models, we are developing micro-MRI methods to label and track cells originating in the subventricular zone (SVZ) of the lateral ventricles, a site of persistent neurogenesis in the neonatal to adult forebrain in which NSCs generate highly proliferative neuroblasts (NBs) that migrate long distances to the olfactory bulb in the normal brain, and to lesion sites during brain injury. Specifically, we will analyze NB migration after excitotoxic neuronal injury, which is highly relevant to many human neurological and neuro-degenerative diseases. By performing in vivo neuroimaging experiments at different developmental stages, these studies will provide critical new information on the stage-dependent differences in the potential of endogenous NSCs to mediate repair, which should have direct implications for the responsiveness of patients experiencing brain injury at different ages. All of the micro-MRI results will be validated with histology, including immunohistochemistry to determine the final fates of the NBs after migration into injury sites. We will also begin to analyze the effects of specific growth factors on NSC and NB behavior. The specific aims of the project are: 1) to establish the temporal and spatial characteristics of NB cell migration in the mouse RMS from neonatal to adult stages of development, using in situ magnetic cell labeling and in vivo micro-MRI;2) to analyze stage-dependent changes in NB cell migration after brain injury, with and without administration of growth factors known to induce NSC proliferation;and 3) to determine the final distributions and fates of the magnetically labeled NBs after migration into the olfactory bulb and injury sites, using immunohistochemistry on histological sections taken after micro-MRI. The ability to perform the imaging studies in mice will enable important future studies of NSC behaviors in genetically-engineered mouse models of many human brain diseases, and to use this vast resource of mouse models for testing drugs designed to enhance the therapeutic effects of the endogenous NSCs. PUBLIC HEALTH RELEVANCE: We are developing magnetic resonance micro-imaging approaches to label endogenous neural stem cells (NSCs) in the mouse brain, and to track their migrations at different developmental stages, from neonatal to adult, as well as in mice with brain injury, with and without administration of growth factors to enhance the response of the NSCs. The excitotoxic neuronal injury model is highly relevant to stroke and many human neurodevelopmental and neurodegenerative diseases. The ability to perform the imaging studies in mice will enable important future studies of NSC behaviors in genetically- engineered mouse models of many human brain diseases, and to use this vast resource of mouse models for testing drugs designed to enhance the therapeutic effects of the endogenous NSCs.

Genetically-engineered mice are currently being developed for in vivo studies of brain development and a wide range of neurodevelopmental diseases. Indeed, defined mutant mice have been critical for identifying the affected genetic pathways and addressing the underlying cellular and molecular basis of developmental brain diseases. Lacking in these efforts have been effective in vivo imaging methods that can be used to study mouse models of neurodevelopmental disorders, especially during early postnatal stages when disease is first manifested, and the greatest changes in brain structure and function are likely to occur. A major challenge is therefore to develop and validate in vivo imaging techniques that can detect and monitor early changes in brain structure and function in the developing mouse brain. We have established quantitative, in vivo manganese (Mn)-enhanced MRI (MEMRI) approaches for analyzing the early postnatal mouse brain, showing that MEMRI provides an exquisitely sensitive method for revealing multiple nuclei and axonal tracts in the early postnatal mouse brain. Results from our laboratory and others have already proven the utility of MEMRI for assessing neural activity and connectivity. These new findings now point to the potential of MEMRI for in vivo detection and quantitative analysis of functional circuits in the developing mouse brain. We have also discovered that the Divalent Metal Transporter, DMT1 can be utilized as an effective reporter gene for MEMRI. We now propose to develop and test a combination of DMT1 expression with MEMRI to provide a precise in vivo approach to analyze functional connectivity in the mouse brain, starting from critical neonatal stages when the circuitry is first established. We will test this new imaging technology in mice with mutations in the mid-hindbrain (MHB) genes engrailed (En1 and En2) and Fgf17, which have morphological and functional cerebellum and midbrain phenotypes. Recent evidence also suggests that both En and Fgf17 mutant mice have defects in MHB circuitry. We will therefore use DMT1-MEMRI to study MHB circuitry in these mice during the critical postnatal period of brain development. The specific aims of the project are: 1) Determine the normal stage-dependent MEMRI intensities in defined nuclei in wildtype (WT), En and Fgf17 mutant mice; 2) Utilize DMT1 to genetically label defined nuclei for MEMRI analysis of midbrain and cerebellar circuits; and 3) Analyze differences in functional circuitry between WT, En and Fgf17 mouse mutants using DMT1-MEMRI. This research has high potential to establish an innovative, genetically-controlled form of MEMRI for in vivo analysis of circuits in the developing mouse brain, providing critical new tools for analyzing mouse models of a wide variety of neurodevelopmental disorders, including cerebellum hypoplasia syndromes (e.g., Joubert and Dandy-Walker syndromes), autism spectrum disorders, and schizophrenia.

DESCRIPTION (provided by applicant): Genetically-engineered mice have been utilized widely for in vivo studies of vascular development and for studying vascular changes underlying pathogenesis in a wide range of human diseases such as stroke, cancer and ischemic heart disease. As a result, there is a critical need for in vivo methods to analyze dynamic changes in three-dimensional (3D) vascular morphology and gene expression patterns during development and disease in mouse models. To progress in this area, reporter mice are required for vascular imaging with more penetration than conventional optical microscopy. During the previous funding period, we established two novel reporter systems for ultrasound and magnetic resonance imaging (MRI), in vivo methods that can be used for high-resolution, 3D vascular imaging in developing and adult mice. Specifically, we developed a Biotag transgene for cell surface biotinylation, and generated Tie2-Biotag transgenic mice for targeted imaging of vascular endothelial cells (VECs) using avidinated contrast agents for both ultrasound and MRI. We also discovered that the Divalent Metal Transporter, DMT1 can be utilized as a highly effective reporter gene for Mn-enhanced MRI (MEMRI), an in vivo imaging method that has the potential for labeling both VECs and smooth muscle cells (SMCs), the two major vascular cell types. We now propose to take full advantage of these breakthroughs in molecular imaging technology, and to generate and validate the next generation universal reporter mice for imaging vascular morphologies and gene expression patterns from embryonic to adult stages. These reporter mice will be used to establish in vivo approaches for molecular imaging of the developing vasculature in wild type (WT) mouse embryos, and in Gli2-/- mutants, which we showed have patterning defects in the cerebral arteries. We will also utilize in vivo models of adult angiogenesis to test and validate the universal reporter mice. The specific aims of the project are: 1) Optimize ultrasound and MRI protocols for vascular imaging from embryonic to adult stages; 2) Establish a universal Biotag reporter mouse for in vivo, multi-modality expression imaging of a variety of VEC genes; and 3) Establish a universal DMT1 reporter mouse for in vivo MEMRI imaging of both VECs and SMCs. This research will generate and validate universal reporter mice for in vivo vascular imaging with MRI and ultrasound, enabling unprecedented studies of dynamic changes in vascular morphologies and gene expression patterns, from embryonic to adult stages.

Project Summary/Abstract The goal of this proposal is to phenotype early- to mid-gestational mouse embryos by segmenting select organ systems in 3D data sets acquired in utero with high-frequency ultrasound (HFU). The International Mouse Phe- notyping Consortium (IMPC), which includes the NIH Knockout (KO) Mouse Phenotyping Program (KOMP2), will generate 20,000 mouse strains in the next decade, including many important models of human structural birth defects and congenital diseases. The development of phenotyping methods that provide for ef?cient pipeline analyses of defects in embryonic growth in the KO mouse strains is a high priority for this effort. An in utero 3D imaging approach, enabling volumetric and longitudinal analyses of a variety of organ systems over a range of early- to mid-gestational stage mouse embryos, would provide added bene?t and critical additional in vivo data not currently available. Commercial HFU systems are widely available in many research centers largely thanks to the NIH-funded Small Animal Imaging Research Programs and Shared Instrumentation Programs. HFU is therefore an excellent candidate modality to provide in utero 3D image data that can be quantitatively analyzed and archived to support the KOMP2/IMPC embryonic lethal phenotyping pipeline and future phenotyping efforts. We propose to develop and validate in utero 3D HFU image-acquisition protocols and image-processing meth- ods that permit noninvasive, longitudinal studies of embryonic development and, in particular, the detection and characterization of KO phenotypes. Volumetric HFU data will be collected in utero from mouse embryos staged between E9.5 to 15.5 in order to establish a database of normal development. Algorithms will be developed to segment 3D regions and extract parameters that quantify embryonic stage and identify regional changes between normal and KO embryos. We will acquire data with a custom, annular-array system and with a VisualSonics Vevo 2100. The ?ne-resolution annular-array data will be used to initially develop the image-processing algorithms and then the algorithms will be adapted for Vevo 2100 data. We will compare the quantitative parameters derived from the segmentation results obtained from the two scanners to ensure that the Vevo 2100 is able to provide equivalent mutant detection and quanti?cation. Initial testing will be undertaken using wild-type and En1 and Gli2 mutants that have known defects. Finally, the acquisition and processing protocols will be applied to 3D Vevo 2100 data from 5-10 KOMP2 KO mouse lines with embryonic defects in a variety of organ systems to validate the HFU methods for detecting and characterizing phenotypes in these mutant embryos.

Abstract: Brain development is a highly dynamic yet precisely orchestrated process. Using genetically modified mouse models, we are in the process of unveiling the complex mechanisms that control critical cellular events in the developing brain. High-throughput imaging tools will greatly benefit studies in this area by charactering brain phenotypes at the macroscopic/mesoscopic levels and directing subsequent examinations at the cellular and molecular levels. In this project, multiple novel magnetic resonance imaging (MRI) techniques will be developed to non-invasively exam a wide range of phenotypes in the developing mouse brain from mid- embryonic stage to adolescence. The target phenotypes include macroscopic brain morphology and structural connectivity, microstructural organization, neuronal migration and differentiation, and postnatal brain activity. The proposed techniques include fast imaging sequences, novel image contrasts, optimized imaging coils/holder, and image analysis tools, many of which stem from on our existing expertise. In Aim 1, we will develop imaging tools to achieve high-throughput in vivo multi-contrast MRI of the developing mouse brain. We will collect multi-contrast MRI data to construct an in vivo MRI atlas of the developing mouse brain to assist mouse brain phenotype analysis and use the sas4-/- mouse, a model of microcephaly, to test the performance of the technique. In Aim 2, we will use novel diffusion MRI techniques to characterize macroscopic morphology, connectivity, and microstructural organization in the developing brain. In particular, high angular resolution diffusion imaging (HARDI) will be used to resolve complex tissue microstructural organization and reconstruct connectivity between major brain regions, and the new oscillating gradient diffusion MRI technique will be used to exam changes in cellularity in the developing cortex associated with neuronal migration. Detailed examination of the relationships between diffusion MRI-based markers and specific histological markers will determine their sensitivity to the underlying developmental processes. In Aim 3, we will use novel Manganese (Mn2+)-enhanced MRI as another tissue contrast, which reflects postnatal brain activity and potentially neuronal differentiation in the embryonic brain, to examine the developing mouse brain. We will examine the contrast patterns of Mn2+-enhanced MRI in the embryonic and neonatal mouse brain with the patterns of neuronal differentiation observed in histological data to determine the sensitivity of Mn2+-enhanced MRI to neuronal differentiation. In addition, we will investigate potential toxic effects of Mn2+ on brain development, and establish protocols that minimize these effects. In Aims 2 and 3, the techniques will also be used to characterize three mutant mouse models with abnormal brain phenotypes resulting from defects in neuronal migration and differentiation. The imaging techniques and knowledge gained in this project will greatly enhance our ability to quantitatively characterize the phenotypes of mutant mouse models in order to achieve a deep understanding of brain development and disorders.