A Multimodal Multidimensional (4D) Map of the Mouse Brain

A Multimodal Multidimensional (4D) Map of the Mouse Brain

Project Overview

This is a project to develop a detailed multidimensional digital atlas of the mouse nervous system. It will, for the first time, enable a unified framework for representing brain maps and gene expression maps. It will provide the ability to chart the anatomy of gene expression in the brain of adult and developing mice. It will establish the linkage between genotype and phenotype. It will enable the comparison of gene expression maps from different animals, laboratories and strains.

Our goals are to collect data from multiple modalities (MRI, PET, blockface imaging and histology), reconstruct these data, place them in a defined coordinate system, delineate and describe the anatomy and develop the appropriate informatics tools to interact with these data. We will do this for adults and developing animals at various maturational stages. We will use the C57BL/6J mouse strain. The product of these efforts will be a series of comprehensive digital atlases describing the probabilistic neuroanatomy of the mouse, a set of tools to import images of gene expression into the atlas and tools for interacting with the atlas statistically and visually in 4D.

This is an enormous undertaking. We are cognizant of the technical, scientific and labor intensive difficulties associated with this project. However, we are experienced in all the appropriate neuroscientific and neuroinformatics disciplines. We have performed preliminary experiments for every aspect of the research plan. We have previously developed atlases of other species along with useable and distributable software that demonstrates our ability to deliver mature products to the community.

The research plan includes three performance sites; UCLA, USC and CALTECH. Each was chosen because of the participant’s ability to provide significant expertise in each of the requisite elements of this proposal. Each complements the other resulting in a powerful team of investigators with unique and relevant resources and experience necessary for this project. We have already established productive working relationships.

Our plans also call for the use of workshops to help coordinate the research and development between our efforts and those of other grantees as well as the gene mapping community at large.

The structure of this proposal is of an integrated and unified project. In spite of the participation from multiple institutions, the research plan describes the work without identifying the responsible investigator. The budget and its justification provide details regarding the allocation of funds and the specific activities supported.

Specific Aims

The overall goals of this project are to develop and implement a probabilistic atlas of the adult and developing C57BL/6J mouse. These atlases will describe the mouse nervous system in detail within a well defined coordinate system. Since the atlas will be based on multiple subjects, it will describe the morphological variability within this strain as well as its development at maturational stages from in utero to adult. The data will be comprised of in vivo and post-mortem imagery. Tools for visualizing, measuring, spatially normalizing (deformation correction) and mapping gene expression data will be created and validated.

In addition to these design driven goals, we will test several hypotheses using the aforementioned atlases and tools. The product of this effort will be an atlas system for the C57BL/6J mouse, tools for using it and evidence that it enables the linkage between brain mapping and gene mapping.

Goals

1. To develop and implement an anatomic framework to map gene expression in the brain. This framework will be comprised of novel imaging data including mMRI, mPET, blockface imaging and histology.

2.  To create a set of tools for colocalizing data from different markers, animals and laboratories.

3.  To collect multimodality data describing the brain of the C57BL/6J mouse strain.

4.  Dissemination of the map (and requisite interactive tools) enabling – output (ability for others to use information/data) and input (ability to incorporate data from other sources).

Hypotheses

1.  There is a relationship between gene expression and morphology.

2. Patterns of gene expression co-vary with morphological changes during development.

3. Anatomy from histological delineations accurately represents in vivo morphology.

4. Within strain morphometric variability will be less than between strains.

The experiments necessary to test these hypotheses will utilize the atlases and associated tools along with gene expression maps (GEMS) collected as part of this study. In this way we can begin to test the utility and validity of our multimodality, multidimensional mouse atlas.

Background and Significance

Mapping the genome

The mouse is a living encyclopedia of known gene functions and a repository for the unknown gene functions that produce a developing, metabolizing, reproducing mammal. Consisting of up to one hundred thousand genes, the task of sequencing and cataloging the entire mouse genome is one of enormous scope and complexity. However, as mouse genome project advances, there has been a great demand for the structural, functional, and anatomic correlates of gene expression. Genetic maps have localized genes to specific sites on chromosomes, but their pattern of expression has only begun to be touched upon. Even so, there exists no coherent framework for the cataloging and comparison of gene expression. What is required is a Spatial and Temporal Atlas of Gene Expression (STAGE) to coordinate the collection and analysis of the enormous amount of data generated by the genome project.

Mice provide many advantages as a model system for mammalian genetics. Short generation time, large litters, and relatively low cost of care, make it a pragmatic choice. The existence of inbred strains of mice, where every individual (of the same sex) is genetically identical, enhances the information content of STAGEs, allowing for the collection of data from multiple individuals, secure in the knowledge that there will be no variation due to genetic factors, something not readily accomplished or impossible in other (outbred) species. The comparison across different strains of mice (i.e. C57BL/6 and Spret/Ei) will allow for analysis of discrete genetic differences which may already be cataloged (Lyon, 1996). In addition, the generation of transgenic (gain-of-function) and targeted knock-out (loss-of-function) mice represent a new kind of mapping directed toward the elucidation of gene and genome function by the time-honored genetic approach of comparing mutant and normal phenotypes. Combining genetic remodeling, genetic maps, banks of genes and emerging methods for assessing gene expression on a whole tissue level, the true potential of the mouse as a mammalian model is beginning to emerge.

Gene expression can be studied in a number of ways. Measurements of mRNA within a tissue can be done by traditional electrophoretic techniques such as Northern blotting and RT-PCR. Both are technically simple, but limited by the crudity of harvesting tissue manually. Cytochemical methods such as in situ hybridization can also measure the expression of a specific mRNA, but in turn are limited by their qualitative nature. Furthermore, gene expression can also be measured by the level of protein expressed. These measurements can be made either electrophoretically by Western blot or by cytochemical methods such as immunohistochemistry. In both cases protein levels are measured by the binding of an antibody specific for the protein itself, making this perhaps the best indicator of gene expression. (Should microPET be mentioned here?)YES AND POINT TO PRELIM RESULTS

Atlases and Maps

Atlases of normal mouse development have immense pedagogical value and provide researchers studying normal, mutant, and transgenic mice a standard against which specific examples may be compared and contrasted. Standard methods of atlas construction typically involve sacrificing, fixing, sectioning, staining, then recording photomicrographs of individual sections. Photographic plates are the raw material of most atlases that contain three additional critical elements: 1) annotation in the form of graphical reconstructions highlighting important detail; 2) nomenclature in the form of descriptions and names of discrete structures; and 3) imposition of a 3D coordinate system so that anatomy can be referred to using a standardized atlas. Atlases of this type for the mouse have been presented by Rugh (1990), Theiler (1989) and Kaufman (1992). The advent of powerful inexpensive computers coupled with the ability to conveniently transport large amounts of data (via CD-ROM or over the Internet) are bringing about changes in the way atlases are constructed and in the ways they can be used. When in book form, the intrinsically 3D animal must be viewed as a series of 2D sections. Moreover, the orientations available to the viewer are limited to samples of standard planes of section (e.g. sagittal, coronal, axial). These restrictions make it difficult to follow complex 3D structures and hinder comparison of one's own 'oblique' sections with the 'perpendicular' sections found in the atlases. Digital atlases have the potential to obviate both of these vexing problems (Williams & Doyle, 1996; Kaufman, et al., 1997; Gibaud et al., 1997 and Toga et al., 1995). With the section data reconstructed into three dimensions, highlighting complex structures and computationally sectioning at arbitrary angles becomes possible. Quantitative morphological measurements (volumes, distances, angles) can be accomplished and maps can be generated that amalgamate data from various experimental techniques. Temporal and spatial gene and protein expression patterns, axonal trajectories, patterns of vasculature, and specific neuronal responses to stimuli can all be combined to obtain a canonical organism or system. Such a data set could potentially embody all quantitative information known about the animal in a concise framework.

Informatics

Motivated by such benefits, several efforts are underway to generate digital atlases. There is at least one commercially available CD-ROM rat atlas (Paxinos and Watson, 1991) and other less ambitious CD-ROM undertakings (Ghosh, et al., 1994; Smith, et al., 1996). A number of World Wide Web sites present a variety of two dimensional data (www.rodents) and some aim towards being three dimensional atlases (www_atlases; Toga et al., 1995). Based upon the atlas of the developing mouse (Kaufman, 1992) the Edinburgh group has embarked on a significant effort to create a database to house gene expression (Ringwald et al., 1994). In our own laboratories, the ICBM effort at mapping the human brain, is based upon a digital 3D representation of a population’s anatomy. The spatial normalization, warping, morphometrics, visualization, databasing and related informatics efforts included in brain mapping have made enormous progress in the last few years (Koslow & Huerta, 1998; Toga & Mazziotta, 1996,1999; Toga, 1999). Many of these advancements have direct relevance to the present project and specific examples are provided below.

Name /

Reference

/ Features

Muritech

UT Memphis
Edinburgh
Chemoarchitectonic Atlas of the Developing Mouse Brain / Jacobowitz & Abbott, 1997 / Calretinin, calbindin, serotonin, tyrosine hydroxylase, AChE, 336 dpi/20 mm
Atlas of the Prenatal Mouse Brain / Shambra, Lauder & Silver, 1992 / H & E; 10mm

Technology

High resolution MR. The use of magnetic resonance imaging has revolutionized the noninvasive investigation of neuroanatomy and function, and is an integral component of digital atlasing. Recent reports of the observation of subtle intracortical structure such as the stria of Gennari (Clark et al., 1992), the histological confirmation of the normal MR distribution of the corticospinal tract (Yagishita et al, 1994) and the ability to selectively image myelin (MacKay et al, 1994) support efforts to apply MR techniques to the imaging of anatomy. The combination of higher field magnets and post processing techniques for image enhancement (Kui Ying et al, 1996; Holmes et al, 1997) has also recently revealed structures as fine as thalamic nuclei, the origin of the thalamocortical tracts, and there is evidence that we can discriminate in vivo cortical architectonic regions. These technical innovations permit the macroscopic observation of fascicles through the in vivo and cranially-intact post mortem brain. Even greater strides can be taken by using extremely high field instruments in the smaller primate species, using microscopic MRI.

Microscopic MRI. The notion of using MRI at microscopic resolutions arose early in the development of this technique (Lauterbur 1973). The spatial resolution in biological samples is typically limited by line-width broadening (T2 effects), diffusion, signal-to-noise ratio (S/N), factors whose physical limits have been discussed in detail by Callaghan (Callaghan 1991) and others (House 1984; Cho et al. 1988; Kuhn 1990; Blumich and Kuhn 1992; Zhou and Lauterbur 1992). Estimates of the theoretical limits of resolution in the MR image range from 2 to 0.5 micron (Cho et al. 1988; Kuhn 1990; Callaghan 1991). By judicious choice of experimental conditions (e.g. bandwidth and gradient strength), deterioration in resolution due to the combination of these effects can be compensated to a degree, resulting in a practical spatial resolution, currently limited by the amount of time available to acquire the image. The quality and usable resolution of these MRI images can be enhanced in several ways, such as: increasing the main magnetic field strength to 12T ; optimizing the radio frequency (RF) coil for small samples; employing 3D volume imaging rather than slice imaging; and using fast imaging pulse sequences (e.g. DEFT, FLASH, EPI). Indeed, several groups have achieved spatial resolutions of 10 micron or less (Blumich and Kuhn 1992), and MRI of rodent eyes and xenographs correlates well with subsequent histological examinations of the same tissues. Aguayo and coworkers (1987) resolved cell clusters and structures as small as basement membranes.

Features and Benefits

The creation of a comprehensive framework capable of encompassing diverse information about the mouse holds tremendous promise for integrating the genotype and phenotype of this animal. Genetic information is expressed in complex and ever-changing patterns throughout the development of the animal. A comprehensive description of these patterns and how they relate to the emerging morphology is crucial to our understanding of the interactions that underlie the processes of development, normal structure and function, disease and evolution.

Studies of gene expression are rapidly producing a vast amount of information relating to these complex patterns. It is currently impossible to adequately compare results from different animals, investigators and laboratories. It is also difficult to make comparisons between the expression of different genes in order to assess the possibility of complex networks of genetic interaction. These problems cannot be addressed by conventional means of publication, but require the development of an electronic database, together with tools for cross-modality correlation. Moreover, text descriptions of gene expression are of limited value due the spatial complexity of the patterns and partly because domains of gene expression do not necessarily correspond to named anatomical structures. The proposed multimodality, multidimensional atlas will address these limitations.