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Setting
Priorities for Molecular Neuroanatomy
in the Postgenomic Era
Summary and recommendations from a workshop
organized under the auspices of the NIMH, NIDA, and NINDS at Laguna Beach,
CA in January 2002
Marc Tessier-Lavigne and Lubert Stryer, organizers
Part I: Overview and Summary
- Opportunities and Challenges
The brain is the most complex organ in our body,
responsible for perception, behavior, cognition, memory, and
consciousness. It is comprised of about a trillion nerve cells, or
neurons, whose intricate and precisely wired connections underlie all
of these functions. But the neurons are not all the same: many
thousands of different classes of neurons, defined by a variety of
criteria such as morphology, patterns of connectivity, and expression
of particular neurotransmitters and receptors, serve as the cellular
building blocks of the brain. Each of these neuronal types has a
specific physiological role in brain function. Neurological and
psychiatric diseases are diseases of particular neurons or neural
circuits.
The complexity of brain cell types and circuits
is reflected in the complexity of gene expression patterns in the
brain. It is believed that perhaps a third to half of all genes are
largely or exclusively dedicated to directing the development,
maintenance and functioning of the brain. With more than 30,000 genes
in the human genome, the task of mapping all genes to the many
thousands of neuronal classes and neuronal circuits Ð what is being
called molecular neuroanatomy Ð might seem beyond reach. In fact,
this mapping is taking place. It is proving to be remarkably
informative and illuminating despite being performed on a relatively
small scale thus far, with about a thousand genes mapped, and some
mapped only in particular subregions of the brain. Such studies have
shown that analysis of gene expression in neurons can yield essential
information on neural development and function, such as the identity
of neurons involved in responses to particular drugs, or the genes
that control the development of particular classes of neurons. Such
analysis has also defined molecular markers of particular neuronal
cell types, helping with the taxonomy of brain cell types Ð the
division of known neuronal classes into further subclasses. Especially
important, the availability of cell type-specific molecular markers
for particular neuronal classes has provided tools to deliver genes
and gene products to those neurons (in ways discussed below),
dramatically facilitating the analysis of their development,
connectivity, function, and dysfunction.
The potential medical benefits that will derive
from this knowledge are immense. The identification of genes expressed
in particular classes of neurons linked to specific diseases provides
new drug targets for the treatment of a wide range of ailments
including stroke, spinal cord injury, neurodegenerative diseases like
Parkinson's disease, brain tumors, schizophrenia, depression, anxiety
disorders, and addiction. Neuronal cell type-specific markers provide
a means for developing gene therapies that involved changing gene
expression in particular neurons. They also make it possible to
visualize neural circuits in their normal and abnormal states, which
is likely to have a large impact on the diagnosis of disease and the
evaluation of the effectiveness of therapy. The identification of
transcription factors that control cell fate and connectivity in the
brain will accelerate the development of therapies to regenerate
nervous tissue.
In the 1990s, several Institutes of the National
Institutes of Health, recognizing the importance and promise of
molecular neuroanatomy for public health, launched the Brain Molecular
Anatomy Project (BMAP), a series of funding initiatives to map gene
and gene product expression to neuronal cell types to create a
Molecular Brain Map. In January 2002, a workshop was convened by NIMH,
NINDS, and NIDA to bring together experts in the field (Table 1) to
take stock of existing efforts, formulate recommendations for upcoming
work in this field, and help establish scientific priorities,
especially in light of the exceptional opportunities created by the
completion of the Human Genome Project and the identification of
nearly all genes in the genome.
- A need to generate rapidly a Molecular Brain Map
A consensus view of the working group,
reiterating the premise of BMAP, is that enormous benefit will derive
from a systematic, large-scale, and organized effort to generate a
Molecular Brain Map for humans and the mouse. The utility of a
systematic effort is well illustrated by the Human Genome Project.
Prior to the Project, the genome was already being sequenced in a
piecemeal fashion by thousands of investigators world-wide, but these
laboratory-based initiatives involved considerable redundancy as well
as inefficiencies because of their small scale. An organized effort to
sequence the human genome Ð and that of other species Ð made it
possible to achieve an enormous economy of scale and to complete the
sequencing of genomes much more rapidly, thereby empowering the entire
biomedical community and greatly accelerating the pace of discovery of
new knowledge and novel therapeutics. In the same way, the creation of
a Molecular Brain Map would eventually result from the independent
activities of individual investigators, but an economy of scale can be
achieved through more systematic efforts, like those already supported
by the NIH (see below). The acceleration of this process will in turn
accelerate the pace of discovery in the neurosciences, neurology, and
psychiatry.
- Summary of recommendations
The working group achieved consensus on the
following principles, many of which have already been incorporated
into existing efforts at the National Institutes of Health.
- It affirmed the importance of driving to
completion the generation of a Molecular Brain Map, a tool that
will revolutionize the study of both normal brain function and
development, as well as neurological and psychiatric disease.
- An impressive start has been made in
initiating several approaches to accomplish this goal, but the
pace needs to be markedly accelerated. At the next stage, the
scope should also be broadened: It is essential that functional
circuits as well as individual neurons be mapped, i.e. the Gene
Expression Map needs to be supplemented with a Connectivity Map.
Both efforts would benefit at this stage from the simultaneous
pursuit of a broad survey of expression of all genes, and an
in-depth focus on particular model neural circuits.
- It affirmed the need to set standards for the
generation, acquisition, mining and sharing of data in this Map,
to permit its efficient construction and utilization.
- It catalogued a set of enabling reagents,
datasets, and complementary technologies that need to be developed
to construct this Map efficiently and to exploit it to the fullest
for the study of brain function and dysfunction (Tables 2 and 3).
- It affirmed the importance of full public
access to the information, reagents, and technologies that will be
generated in this initiative, to leverage these resources for the
advancement of knowledge and for biomedical progress.
In Parts II-IV of this report, we provide
background information on molecular neuroanatomy, including a
description of existing large scale initiatives, and discuss emerging
opportunities. In part V, we discuss in detail the recommendations and
priorities formulated by the participants that were summarized above.
Table 1: Workshop Participants
David J. Anderson, Ph.D.
California Institute of Technology |
Carolee Barlow, M.D.,
Ph.D.
The Salk Institute for Biological Studies |
Sydney Brenner, Ph.D.
The Salk Institute for Biological Studies |
Catherine Dulac, Ph.D.
Harvard University |
Gregor Eichele, Ph.D.
Baylor College of Medicine |
Scott Fraser, Ph.D.
California Institute of Technology |
Jeffrey M. Friedman,
M.D., Ph.D.
The Rockefeller University |
Fred H. Gage, Ph.D.
The Salk Institute for Biological Studies |
Paul Greengard, Ph.D.
The Rockefeller University |
Bruce Hamilton, Ph.D.
University of California, San Diego, School of Medicine |
Mary-Beth Hatten, Ph.D.
The Rockefeller University |
Nathaniel Heintz, Ph.D.
The Rockefeller University |
Ali Hemmati-Brivanlou,
Ph.D.
The Rockefeller University |
RenŽ Hen, Ph.D.
Columbia University College of Physicians and Surgeons |
Tom Jessell, Ph.D.
Columbia University College of Physicians and Surgeons |
Alexandra L. Joyner,
Ph.D.
New York University Medical Center |
Eric Kandel, M.D.
Columbia University College of Physicians and Surgeons |
Larry Katz, Ph.D.
Duke University Medical Center |
Stuart Kim, Ph.D.
Stanford University School Of Medicine |
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Alex Kolodkin, Ph.D.
Johns Hopkins University
School of Medicine |
George Lake, Ph.D.
Institute for Systems Biology, Seattle |
Pat R. Levitt, Ph.D.
University of Pittsburgh School of Medicine |
David Lockhart, Ph.D.
The Salk Institute for Biological Studies |
Robert C. Malenka, M.D.
Ph.D.
Stanford University School of Medicine |
Susan K. McConnell,
Ph.D.
Stanford University |
Dennis OÕLeary, Ph.D.
The Salk Institute for Biological Studies |
John Rubenstein, M.D.,
Ph.D.
University of California, San Francisco |
Edward Scolnick, M.D.
Merck Research Laboratories |
Lubert Stryer, M.D.
Stanford University |
Michael P. Stryker,
Ph.D.
University of California, San Francisco |
Joseph S. Takahashni,
Ph.D.
Northwestern University |
Marc Tessier-Lavigne,
Ph.D.
Stanford University |
Roger Y. Tsien, Ph.D.
University of California, San Diego |
Arthur Toga, Ph.D.
University of California, Los Angeles |
Kamil Ugurbil, Ph.D.
University of Minnesota |
Chris A. Walsh, M.D.,
Ph.D.
Harvard Medical School
BIDMC Room 816
Harvard Institutes of Medicine |
Richard Woychick, Ph.D.
Lynx Therapeutics, Inc. |
Charles Zuker, Ph.D.
University of California, San Diego |
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NIH Program Staff |
James Battey, M.D.,
Ph.D.
National Institute of Deafness and Other Communication
Disorders |
Robert Baughman, Ph.D.
National Institute of Neurological Disorders and Stroke |
Hemin Chin, Ph.D.
National Institute of Mental Health |
Stephen Foote, Ph.D.
National Institute of Mental Health |
Glen Hanson, Ph.D.,
D.D.S.
National Institute on Drug Abuse |
Michael Huerta, Ph.D.
National Institute of Mental Health |
Michael Iadorola, Ph.D.
National Institute on Dental and Craniofacial Research |
Gabrielle Leblanc, Ph.D.
National Institute of Neurological Disorders and Stroke |
Steven O. Moldin, Ph.D.
National Institute of Mental Health |
Bret Peterson, Ph.D.
National Center for Research Resources |
Jonathan Pollock, Ph.D.
National Institute on Drug Abuse |
Brad Wise, Ph.D.
National Institute on Aging |
Graeme Wistow, Ph.D.
National Eye Institute |
Observer |
John H. Williams, Ph.D.
The Wellcome Trust |
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Table 2: Enabling Reagents and Datasets
- A dataset of full-length
transcripts/cDNAs expressed in the nervous system;
this dataset is well on its way to completion.
- A bank of Bacterial Artificial
Chromosomes that permit transgenic labeling and
manipulation of major neuronal populations in the
mouse brain; only a few hundred of these exist today.
- A bank of short promoter elements
for extending such manipulations to primates and other
non-genetic systems; few of these exist at present.
- A set of antibody probes (for
immunohistochemistry) that help extend and leverage
the Molecular Brain Map. A priority is the generation
of antibodies to the ~1,500 transcription factors
encoded in the genome, to help identify neuronal cell
types and stem cells; only a small fraction (~10%) of
these exist at present.
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Table 3: Complementary Technologies
Short term:
- Improved methods for isolation of
high-quality mRNA from small numbers of neurons,
including laser-capturing of mRNA from neurons
identified through expression of a transgenic reporter
(such as GFP), and for faithful linear amplification
of cDNA from the mRNA for gene expression analysis
Medium term and longer term:
- Improved methods for mapping
neuronal circuits (identifying all inputs to each
neuron and all outputs from each neuron)
- Methods for detecting electrical
activity in mammalian neurons by optical recording
using genetically-encoded reporters
- Methods for controlling activity
in defined neurons, in particular genetically-encoded
modulators of electrical activity that can be
activated with specific signals such as
pharmacological agents or light
- Methods for detecting neuronal
activity in deep brain structures using such
genetically-encoded reporters
- Methods for mapping patterns of
neuronal activity onto patterns of gene expression and
neuronal interconnectivity
- Methods for persistent labeling
of neurons over time, to follow plastic changes in
morphology, connections, or function
- Methods for visualizing changes
in neuronal connections, such as pulse-chase labeling
of synapses
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Part II: Background: Gene-Based and
Cell-Based Approaches to Creating a Map
- Historical background
Using histological tools like the Golgi staining
method, neuroanatomists in the late 19th and early 20th centuries
defined hundreds of different neuronal cell types based on their
location in the brain, morphology, and connectivity. Throughout the
20th century, this number grew, and neuroscientists were also able to
assign particular physiological functions to a large number of these
neuronal types and many of the circuits linking these cells. At
present, the number of distinct neuronal cell types is not precisely
known, but it is estimated that there must be at least several
thousand. This estimate is supported by the finding of novel
subclasses whenever a particular population of neurons is studied in
detail. For example, a recent assessment of amacrine cells subtypes in
the retina, previously thought to number about a dozen, revealed the
existence of more than thirty easily definable subtypes. These
thousands of cell types, through their specific patterns of
interconnections, direct the functioning of the brain.
Subpopulations of neurons are increasingly being
distinguished based on their pattern of gene expression. Historically,
molecular neuroanatomy started with cellular pharmacology - the
definition of the neurotransmitters and neurotransmitter receptors
made by particular neurons, using a variety of physiological, in
situ radioligand binding, and histochemical methods. The
development of the technique of immunohistochemistry accelerated the
mapping of proteins (such as neuropeptides) and other epitopes (such
as specific carbohydrate moieties) to particular neurons in cases
where a suitable antibody was available. However, it is the
development of sensitive and reproducible mRNA in situ
hybridization techniques that unleashed the systematic analysis of
gene expression in neurons, because this approach can be readily
applied to all genes without requiring the labor-intensive development
of specific detection reagents (such as antibodies); in all cases, a
small gene fragment is sufficient to develop an appropriate in situ
hybridization probe. In situ hybridization methods have been
supplemented by transgenic (promoter-based, and BAC-based) and
knock-in approaches, which make it possible to visualize the pattern
of expression of particular genes using genetically encoded reporters
driven from the gene locus in transgenic mouse lines. (These
approaches are explained further below).
- Gene- (and Gene Product-) Based vs. Cell- (and
Region-) Based Approaches to Gene Expression Profiling
Existing powerful methods for mapping gene and
gene product expression are summarized in Table 4, and are divided
into gene- (and gene product) based and cell- (and region) based
approaches. In situ hybridization is an example of a gene-based
method for gene expression analysis. Such methods make it possible to
detect a single gene product (e.g. a particular mRNA) in a very large
number of cells in brain slices.
A complementary approach is, however, provided
by cell- (and region-) based methods of analysis. Such methods
involve isolating small brain regions or, in the limit, specific
neuronal populations or even single cells, extracting mRNA from these
cells, and subjecting them to gene expression analysis using DNA
arrays or other methods (such as direct cDNA library sequencing).
These methods make it possible to detect a very large number
(thousands) of gene products in a small number of cells.
Table 4: Methods to Map Genes and Gene Products
to Neurons
A. Gene- (and Gene-Product) Based:
Method |
Limitations/Limiting
Steps |
Current
Applicability |
Histochemical
Radioligand binding
Immunohistochemistry
Transgenic: promoter-based |
Requires
optimized stain
Requires optimized ligand
Requires antibodies
Requires promoters |
Current
Applicability |
Transgenic:
BAC-based or knock-in |
Limited
by generation of constructs, ES cells, mice |
Hundreds
to thousands of genes |
In
situ hybridization |
Limited
by tissue sectioning |
Thousands
to tens of thousands of genes |
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B. Cell- (and Region) Based:
Methods for
isolating cells and mRNA:
Dissection (for small or large region)
Cell marking and purification (e.g. by Fluorescence
Activated Cell Sorting)
Single cell picking
Laser-capture microdissection
Methods for analyzing gene expression
DNA arrays
cDNA library sequencing
Other (SAGE, MPSS, etcÉ) |
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Gene-based and cell-based approaches are
complementary, with different advantages and disadvantages.
- Gene- (and gene-product) based approaches
have the advantage of providing a broad overview of any particular
gene throughout the brain. Of these methods, in situ
hybridization has the highest throughput. Its disadvantage can be
limited cellular resolution, particularly since only the cell body
(or part of the cell body) is labeled. In some instances, it is
possible to tell within different brain regions exactly which
cells are expressing the gene, for example when the gene is found
in cells with a particular morphological feature (such as a large
cell body) that makes it possible to distinguish them from other
cells in the vicinity. In many cases, however, it is not possible
to assign the expressed gene to a specific cell. As a consequence,
when comparing two different genes expressed in the same region,
it is often not possible to know whether the two are expressed by
the same or different cells, unless the expression analysis is
performed simultaneously with two probes that can be visualized
independently (e.g. using two fluorescent tags). Such
double-labeling technologies are being improved but are still
limiting at present. Better cellular resolution can be obtained by
other gene- or gene-product based methods that result in labeling
of the neuron's processes, since shape and connectivity often
identify particular neurons. This can be achieved, for instance,
with transgenic approaches discussed in the next section. Thus,
although such approaches have a lower-throughput than in situ hybridization,
they help provide greater resolution.
- Cell- (and region-) based approaches
have a complementary set of advantages and disadvantages. In these
approaches, the entire pattern of genes expressed by a particular
cell (or population of like cells) can be identified by extracting
mRNA from the cell(s) and subjecting it to analysis using DNA
arrays (or through some other method such as direct cDNA library
sequencing). These technologies make it possible to determine the
expression profile of a small number of cells, and in some cases,
even a single cell. What limits these approaches is the difficulty
of obtaining mRNA from just the cells of interest. In experimental
animals such as mice, specific neuronal populations can often be
isolated and purified (see below) and used as a source of mRNA,
but this approach is of limited utility in the human brain. An
alternative method is laser capture microdissection, in which mRNA
is directly isolated from specific cells identified in fixed brain
sections. Laser-capture is thus more applicable to human tissue,
but the method is still being perfected (for instance, the
captured mRNA can be degraded, a technical problem that is likely
to be resolved before long). It is also limited by the ability to
identify the neurons of interest in tissue sections (discussed in
more detail below).
Thus, gene-based approaches give broad coverage
of all brain regions but often with more limited cellular resolution.
Cell-based approaches can provide high cellular resolution and give
information on the complete set of transcripts expressed by a cell,
but their throughput is lower and they are more difficult to apply to
the human brain. Both types of approaches are needed.
- Tools to Deliver Genes to Particular Neurons
Facilitate Cell-Based Approaches
The utility of cell-based approaches is limited
by the ability to isolate and to purify particular neuronal
populations (for approaches based on cell isolation), or to recognize
particular populations in tissue sections (in the case of laser
capture microdissection). Some select populations of neurons can be
purified based on physical characteristics, such as size, through the
use of probes (such as antibodies) directed against particular cell
surface epitopes, or by using fluorescent markers injected into the
termination sites of the neurons' axons, which are taken up by the
axons and retrogradely transported. However, these approaches are
currently applicable only to small numbers of neurons.
More general approaches to isolating particular
populations of neurons involve transgenic approaches in which
expression of a genetically-encoded reporter, such as the Green
Fluorescent Protein (GFP) or some other molecular tag is driven in the
neurons of interest in transgenic mice. The neurons can then be
recognized and purified by some other method that makes use of the
reporter tag, e.g. by Fluorescence Activated Cell Sorting using GFP
fluorescence. (In principle, the molecular tagging of neurons should
also permit their identification in tissue sections for laser-capture
microdissection; current laser-capture methods are not readily
compatible with such molecular tagging, although it can be expected
that this technical problem will be solved before long).
The transgenic labeling approaches require the
ability to drive reporter expression in the cells. This is usually
done by first identifying a particular marker gene expressed in the
neurons of interest; the marker must be specific for those cells - at
least in a particular region of brain that can be isolated through
dissection from any other regions where the marker is expressed. Once
an adequate marker gene is identified, the generation of a transgenic
mouse in which the reporter is expressed from the marker gene locus is
currently achieved primarily by three methods.
- Short promoters. In the case of some
marker genes, it is possible to isolate a promoter element
(stretch of DNA) that directs expression of the reporter. One
disadvantage of this approach is that it is often labor-intensive
and difficult to identify the promoter. Another is that it can
often be difficult to isolate a transgenic mouse line in which the
reporter, driven by the promoter, gives faithful expression,
because of position effects on integration of the reporter
construct in the host genome. One advantage, however, is that
small promoters can be used in viral delivery systems, which can
be used more readily in organisms other than mice (including
primates).
- Bacterial Artificial Chromosomes (BACs).
As an alternative, large (>50kB) bacterial artificial
chromosomes (BACs) containing the gene of interest as well as its
control regions can be modified to drive expression of a reporter
construct in a pattern that mirrors that of the starting gene when
reintroduced into transgenic animals. BACs have the disadvantage
that they are too large for use in viral vectors. However, in
transgenic mice, BACs have some advantages over small promoter
elements: they are readily isolated, can contain all the gene
regulatory elements, and because of their size are usually less
subject to position effects on integration. In principle, BACs can
also be used in other species that are amenable to the generation
of transgenic animals (such as rats).
- Knock-ins. Another means of delivering
reporters to particular cell types is through knock-in technology
in mice. In this approach, a reporter is introduced into a
particular marker gene locus by homologous recombination in
embryonic stem (ES) cells, and transgenic mice are generated from
the ES cells. This approach is applicable only in species where
knock-ins are possible, primarily mice. One advantage of this over
other transgenic approaches is that the marker gene can be
inactivated during the procedure, so that gene function can be
assessed. Traditionally, a disadvantage has been that making
knock-ins has been labor intensive, but this has been improving.
The ability to deliver constructs selectively to
particular neuronal populations is so important to generating and
exploiting a Molecular Brain Map that a high priority should be
assigned to generating the tools (such as BACs and promoter elements)
that will make this possible (Table 2).
- Tools to deliver genes to particular neuronal
populations facilitate tracing neuronal connections, and monitoring
and manipulating neuronal function
In addition to allowing the marking and
isolation of particular populations of neurons, these tools make it
possible to deliver other transgenic constructs to the neurons, which
facilitates identifying patterns of neuronal connections, and
monitoring and manipulating electrical activity, and manipulating
neuronal function.
- Mapping connectivity. Traditionally,
the connections a neuron makes have been identified using
electrophysiological recordings, from histochemical techniques
(such as the Golgi technique), or through the use of anterograde
or retrograde tracers (such as Horseradish Peroxidase (HRP)) that
make it possible visualize the patterns of projections of
individual neurons. These methods are laborious and in many cases
applicable only to small populations of cells. The ability to
drive expression of reporter genes in particular neuronal
populations makes it possible to readily trace their connections
by delivering genetically encoded reporters of neuronal
projections provided by proteins such as beta-galactosidase,
alkaline phosphatase or GFP (or modified versions of such proteins
that are readily transported down axons), which allow simple
histochemical or fluorescent labeling of axons of neurons making
the reporter. In addition, much effort is currently being devoted
to devising genetically-encoded tracers that are transported
across synapses and hence permit transsynaptic tracing of
connections, both anterogradely and retrogradely, allowing
patterns of connectivity to be defined.
- Monitoring and manipulating neuronal
activity. The labeling of specific neuronal populations with
genetically encoded fluorescent markers like GFP makes it possible
to identify these neurons in slices or cultures of brain tissue,
allowing for electrical recordings with microelectrodes on these
neurons. Potentially even more powerful are genetically encoded
reporters of electrical activity. At present, indirect measures of
electrical activity can be obtained with genetically encoded
reporters of cellular properties like intracellular calcium
concentration that can serve as indirect measures of electrical
activity. Proteins whose optical properties change with membrane
potential have been devised that can be used to monitor electrical
activity of model cells such as frog oocytes; the extension of
this technology to create similar proteins that can function in
mammalian neurons would make it possible to monitor electrical
activity of neurons through optical recording after delivery to
these neurons using the approaches described above. Similarly,
genetically encoded modulators of neuronal activity, such as
particular ion channels that can be opened or closed through use
of pharmacological agents or other means (e.g. by light
stimulation), when delivered to particular neuronal populations,
provide tools to control neuronal activity and thereby to assess
neuronal function.
There are significant limitations on existing
genetically encoded constructs for tracing connections and monitoring
and manipulating neuronal activity, so that exploiting the Molecular
Brain Map fully will require improving both sets of technologies
(Table 3).
Part III: Requirements of a
Molecular Brain Map
With this background, we now discuss what needs to
be discovered.
- A Molecular Map of the Adult Mouse Brain
We first discuss the challenges involved in
establishing a Molecular Brain Map for the normal adult mouse brain.
We focus on the mouse because its brain is highly analogous in
structure and organization to the human brain, but at the same time it
is readily amenable to manipulation of gene activity and function in a
manner not yet possible in species more closely related to humans.
These properties have made the mouse the key model organism for
molecular studies of brain function and dysfunction, and there was
consensus at the workshop on the need to give highest priority to
generating a mouse Map.
A comprehensive Molecular Brain Map should
provide the pattern of expression of all genes in all neurons
throughout the brain, and involves the following component parts.
- Mapping all genes and splice variants.
The existing Genome Projects are in the process of completing the
cataloguing of the more than 30,000 genes in the genomes of the
human and the mouse. In the first instance, it will be important to
describe the expression of all these genes. A further complexity
arises from the fact that many genes are subject to alternative
splicing, which can often alter the function of gene products. This
splicing is often poorly understood. Ultimately, a comprehensive Map
should incorporate information on the specific splice variants for
each gene that are expressed by each neuron.
- Devising an Atlas of the brain onto which
information can be mapped. The enormous amount of information
generated through expression analysis will be of general utility
only if it can be organized in a way that makes it easily
accessible by the community of researchers. In practice, this
means that the information must be mapped onto a standard brain
atlas, in which neuronal populations are assigned particular
coordinates. There are of course variations in brain size and
structure among individuals in any given species, so that any
atlas will be at best an idealization. It will be the most
accurate in the case of inbred strains of animals, such as mice,
that show the least variation between individuals. Efforts have
been made in the past decade to develop such atlases for mice and
other species, including humans.
- Integrating information obtained from
gene-based and cell- (or region-) based approaches.
As discussed above, gene-based approaches such as in situ
hybridization permit the mapping of all genes, but the degree of
cellular resolution is limited. Cell-based approaches make it
possible to define for particular cells the full complete of genes
that are expressed, but this must be done on a laborious
cell-by-cell basis. The two approaches are complementary, making
it desirable to use both, and to cross-reference the information
obtained by the two approaches.
- Mapping circuits as well as cells.
Understanding the pattern of interconnections of specific neural
cells is essential to understanding their roles in nervous system
function and dysfunction. Therefore, a comprehensive Brain Map
should include not just information about the patterns of gene
expression of cells, but also about the connections those cells
make with other neural cells. The identification of genes that can
serve as markers for particular cell types, and whose promoters
can be used to drive cell-type specific expression of transgenes,
can help trace the connections of these cells, through the
transgenic expression in the cells of genetically encoded markers
of neural connectivity. A powerful variation on this theme,
recently developed, involves driving in the cells not the
genetically encoded marker itself, but rather a recombinase like cre
that makes it possible to activate a genetically encoded
reporter delivered to the cells by other means (e.g. using a
virus). Technologies like these have the potential to dramatically
facilitate the tracing of connections in the complex environment
of the brain. While this type of tracing has been initiated in an
investigator-initiated way in several laboratories, there is at
present no systematic effort to accelerate the development of a
Connectivity Map; this should be given priority.
- Mapping proteins not just mRNAs.
For technical reasons mentioned above, the analysis of mRNA
expression patterns is considerably more straightforward than the
analysis of the expression patterns of their protein products, so
that highest priority is being given to high-throughput mRNA
expression analysis. Ultimately, however, it will be necessary to
know cell type-specific expression patterns of protein products
and their subcellular localization on particular portions of
neurons or in specific intracellular compartments. The improvement
of proteomic methods is the focus of intense activity in all areas
of biomedical research, and these methods should be incorporated
into the generation of Molecular Brain Maps in an ongoing way as
they are developed.
One specific initiative in proteomics deserves
mention and high priority at this time: the generation of antibodies
to transcription factors. It is estimated that there are approximately
1,500 transcription factors encoded in the genome. The identity of
particular neural cell types is controlled by combinatorial expression
of specific transcription factors, and the availability of antibody
probes to detect transcription factors in histological sections of the
brain will accelerate attempts to identify neural cells, including
neural stem cells, and to devise means to alter the development and
fate of these cells for neural repair. Furthermore, transcription
factors as a class have proven in general to be readily amenable to
the generation antibodies that work in immunohistochemistry, whereas
many other important classes of proteins (e.g. G protein-coupled
receptors) are often more refractory. The combination of importance
and ease justifies giving priority to the generation of antibodies to
transcription factors (Table 2).
From this description, it is evident that the
development of a comprehensive Molecular Brain Map will be an
iterative process, as information from gene-based and cell-based
approaches accumulates and is incorporated into an ever more refined
Atlas incorporating information not just about the locations of cells
but also about their interconnectivity and function. In particular, it
can be expected that in many cases what is thought of as a single
homogeneous class of neurons defined by expression of a particular
maker gene will, on further analysis, be discovered to comprise two or
more subpopulations. Such refinements, subdivisions and
reinterpretations are expected, and will be facilitated by the
integration of information obtained from gene-based and cell-based
approaches, and the integration of that information with other
anatomical and functional data. As part of this refinement process,
more accurate cell-type specific markers are likely to be defined that
will permit gene manipulation in particular populations of neurons
with high selectivity.
- Three axes: species, developmental stage, and
disease condition
A single Molecular Brain Map of the adult mouse
brain would already provide an invaluable tool, dramatically
accelerating the pace of discovery by freeing individual investigators
of the need to derive the information in a piecemeal and inefficient
way. However, the full utility of the Map will be evident only as a
variety of Maps are generated to document gene expression in different
species, developmental stages, and disease conditions.
- Maps in different species.
Just as important as the creation of a Molecular Brain Map for the
mouse is the creation of such a map for the adult human brain. A
number of approaches feasible in the mouse are not possible in the
human (e.g. those of a transgenic nature), but in situ
hybridization (gene-based) and laser-capture microdissection
(cell-based) are both possible. Thus, it should be possible to
generate such a Map for humans, though these limitations in mapping
technologies appropriate for the human brain, as well as the
variation in brain size and structure alluded to above, will result
in the generation of a lower-resolution Map, at least in the first
instance. After humans, a Molecular Map for the brain of one or more
non-human primates (whose genomes have been sequenced) will be
desirable, as primates can be excellent models for a variety of
higher brain functions that are either not present or not easily
studied in the mouse.
- Maps at different developmental stages.
Understanding how the adult brain is generated during development
will require creating Maps at different stages in embryonic, fetal,
and early postnatal life. One or more Embryonic and Fetal Maps will
help identify the genes responsible for the generation and
differentiation of neuronal cells, as well as the genes important in
establishing the initial connectivity of the brain. One or more
Postnatal Maps will help elucidate mechanisms through which early
sensory experience helps shape the ultimate pattern of neuronal
connections. High resolution Maps at these stages will be most
useful.
- Maps in different states (disease,
drug-treatment, injury, or diverse normal physiological states)
and strains.
Important clues to the
causes of disease will come from generating Maps in different
disease states in both animal models such as the mouse, and also in
humans. Comparing Maps in different strains of the same animal (e.g.
different mouse or rat strains) that show significant behavioral or
physiological differences can similarly be expected to shed light on
the causes of these differences. Similarly, insight into both drug
mechanisms and some disease states will be obtained by generating
Maps following drug treatment, especially for those drugs, such as
anti-depressants, that require considerable time to achieve their
effects (and which therefore likely involve changes in gene
expression). Maps generated following injury or trauma to the
nervous system will provide information on the brain's response to
these insults, including on the behavior of stem cells, and changes
in gene expression that may either facilitate or impede regenerative
responses. Finally, even in the normal, uninjured brain, the
generation of different Maps is likely to be useful, for example in
discerning changes in gene expression that occur in particular
learning paradigms. In general, for each of these stages it is
anticipated that low resolution Maps might be generated in the first
instance, with subsequent high resolution mapping of particular
brain regions fingered by the first-pass analysis.
Part IV: Existing Large Scale
Efforts Funded by the NIH
The NIH recognized the need for a Molecular Brain
Map in the 1990s. Many pilot efforts have been funded to further the
development of this Map, and are not described here because of space
constraints. Three large-scale efforts funded at high levels by various
Institutes of the NIH are, however, discussed in detail in Appendix 1 and
introduced briefly here.
- Creating a dataset of all transcripts expressed
in the adult and developing mouse brain, and a physical collection of
cDNA probes for each of these transcripts.
This resource and dataset, created by Dr. Bento
Soares (University of Iowa) complements the Genome Projects in helping
identify transcripts and splice-variants of genes expressed in the
brain, and in generating cDNA libraries and full-length cDNA/EST
probes that can facilitate analysis of these genes.
- Creating a database of gene expression patterns
in the nervous system
The GENSAT project, initiated by Drs. Gabrielle
Leblanc, Bob Baughman, and colleagues at NINDS, aims to systematically
map the expression patterns of thousands of genes in histological
sections of the mouse brain and spinal cord in the adult and at three
stages of development (E10.5, E15.5, and P7). The gene expression data
are being collected by two groups of investigators, one led by Dr.
Gregor Eichele (Baylor College of Medicine and Max Planck Institute,
Hannover), and another led by Drs. Nathaniel Heintz, Mary-Beth Hatten
(Rockefeller University) and Alexandra Joyner (New York University).
Dr. Eichele's group is collecting data using high throughput in situ
hybridization, whereas Dr. HeintzÕs group is using BAC (bacterial
artificial chromosome) transgenic technology. In the latter approach,
transgenic mouse lines are generated in which expression of a
reporter, Green Fluorescent Protein (GFP) is driven in the same
patterns as selected genes. The gene expression data from both efforts
is to be placed in a public database at the National Center for
Biotechnology Information (NCBI). The project is collecting data for
300 genes in its first year, and aims to ramp up to at least a
thousand genes per year in future years.
- Creating Atlases of the mouse and human brains
Dr. Arthur Toga of the University of California
at Los Angeles and his colleagues have been developing computerized
brain Atlases, and tools to map data obtained from gene expression
analysis or other approaches (e.g. functional MRI) onto those Atlases.
In the case of the mouse, the result is a formal computerized
representation of the mouse brain, providing coordinates that define
the diverse anatomical structures and landmarks.
These projects are helping set the groundwork
for accelerating the generation of comprehensive Molecular Brain Maps
for the human and the mouse.
Part V: Goals and Priorities
The working group applauded existing large-scale
efforts for the establishment of a Molecular Brain Map, and achieved broad
consensus on the following priorities going forward, building on those
initial efforts.
- A two-pronged approach: breadth and depth
- Breadth: A first priority is to
accelerate efforts like those just mentioned to provide a broad
but comprehensive survey of expression of all genes in the adult
mouse and human brains. Achieving this goal involves several
challenges.
- Increased throughput. The current
throughput of existing efforts in the mouse is on the order of
many hundreds of genes a year. To deal with the more than
30,000 genes in the mammalian genome in a matter of years
rather than decades, it will therefore be necessary to
increase this throughput by an order of magnitude at
least.
- Integrating and coordinating
gene-based and cell-based approaches. At present the
highest throughput is provided by in situ
hybridization (a gene-based approach). Because of its
limited cellular resolution, however, it is necessary to
pursue complementary approaches with equal vigor. This
includes transgenic approaches, such as BAC-mediated or
knock-in approaches, which are of lower throughput but provide
more detailed cellular resolution data on expression of
particular genes, and also provide tools to label particular
neuronal populations for cell-based approaches. Cell-based
approaches, in which mRNA is extracted from particular cells
and probed (e.g. using DNA arrays) to identify the
transcript profile of the cells, must also be pursued
vigorously. In particular, laser-capture microdissection
holds high promise, particularly for use on human tissue, but
the technique needs to be improved significantly (e.g. to
improve the quality of recovered mRNA, and to permit capture
from marked cells). These efforts will all benefit from coordination:
for instance, in selecting genes for in situ
hybridization and BAC transgenic analysis a priority should be
given to identifying regional- and cell-type specific
markers that can then be used for cell-based approaches.
- Broaden to include circuitry. An
essential part of a Molecular Brain Map will be information on
the pattern of connections made by individual neuronal
populations. Mapping connectivity has not been a focus
of large-scale efforts to date, and should become an integral
part of such efforts.
- Competition between centers and
technologies. Since the methods for most efficiently
collecting information about gene expression are still being
worked out, it was deemed essential to encourage competition
between multiple centers and multiple approaches.
- Depth: A second priority is to focus
in detail on a few selected brain regions and/or functional
circuits (such as the retina, spinal cord, or cerebellum). The
aim here is to characterize in detail all the neuronal subtypes in
the selected circuits, defining not just their full complement of
gene expression but also their interconnectivity. These data would
then be related to the functional properties of the system. The
reason for focusing on a few systems in depth is that this would
make it possible to discern whether any organizational or
analytic principles emerge relating gene expression patterns
to the structure and function of the circuit. Such principles
could then help guide and organize similar studies in other brain
regions.
- How many Maps?
- The adult mouse brain will continue to
be the first focus of efforts to create a Molecular Brain Map,
because of its relevance to the human brain, and the powerful
transgenic tools that facilitate Map generation. It was deemed
important, however, to initiate the creation of additional Maps.
- Priority should also be given to generating a
Molecular Brain Map for the adult human brain, despite the
difficulty of obtaining high quality post-mortem human brain
tissue for mapping studies, and the fact that transgenic
approaches are not possible. These limitations mean that the
generation of this Map will lag behind that of the mouse and have
lower resolution, at least initially. Nonetheless, given its
central position in the analysis of behavior and in biomedical
research, even a limited Map for the human brain will have broad
utility.
- It is likely to be desirable to generate
several other Maps. Strong arguments in the first instance can be
made for the broad importance of several Maps of the developing
mouse brain (to illuminate brain development and plasticity),
and a Map of another adult primate species (which will be
more experimentally tractable than the human brain, and closer in
structure and function than the mouse brain).
- Yet other Maps are likely be important, but
different constituencies of basic and clinical researchers are
likely to have different priorities. In many cases, partial
Maps may be sufficient. For example, illuminating some
neurological diseases may be achieved by focusing mapping efforts
on subregions of the brain or even particular neuronal populations
in either a mouse model of the disease or on post-mortem human
brain tissue.
- Need for a versatile repository for data
To be truly useful, the data generated by these
approaches must be organized into a central data repository that is
easily accessible to the community the "Genbank"
equivalent for brain gene expression. All data must be mapped onto a digital
brain Atlas, accessible through graphical interfaces, that can
integrate data from both quantitative gene expression analysis (such
as that provided from DNA arrays) with histochemical data (immunostaining,
in situ hybridization), and which is accessible for "where
is" and "what is in" queries. The format must be user-friendly
and make it straightforward for researchers who are focusing on a
particular brain region (for instance, researchers interested in a
particular region because of fMRI data implicating it in some
cognitive task) to obtain clues to the function of the region from the
data contained within the Map.
- Need for standards
The development of this database will require
the establishment of standards for the organization and display
of data, as well as standards for data collection that are
compatible with the digital atlas framework of the database. The
absence of such standards is severely limiting the utility of data
that are already being generated by existing large-scale efforts.
Various Institutes at the NIH have initiated discussions on setting
these standards, and they should be encouraged to drive this process
to completion as rapidly as possible, broadly involving the community
in the process.
- Public access is essential
The full impact of a Molecular Brain Map will be
felt only if all data in the database is accessible to the scientific
community at large. Standards for the timing of public release
of data therefore need to be defined and implemented. Early release
of data, for example on a quarterly basis or more frequently, is
essential both to ensure access and to ensure quality control by
the community. For example, data generated in large-scale efforts
funded by NIH and described in Part IV should be released to the
scientific community according to these standards.
- Need for tools to leverage and extend the
Molecular Brain Map
There was consensus on the need to develop novel
technologies to allow the Molecular Brain Map to be leveraged to its
fullest.
The first priority is to develop a bank of
specific promoters and modified BACs, to permit delivery of
transgenes to specific neuronal populations, and the simultaneous
improvement of efficient transgenic and/or viral delivery
methods for gene delivery in species other than mouse (including rats
and primates).
The impact of having this bank will be greatly
increased by the further development of genetically encoded
reporter and modulator constructs to allow: (i) the marking and
isolation of neurons, (ii) the tracing of all connections
made by a neuron, (iii) the persistent or time-lapse labeling of
connections, to help identify plastic changes in connections (iv)
the detection of electrical activity in neurons by optical and
other means, and (v) the controlled modulation of electrical
activity in specific neuronal populations (e.g. by expression of
an ion channel that can be gated by a specific pharmacological agent
or by light). The latter two applications in particular will help
define the function of particular neurons and neuronal circuits.
Finally, whereas the analysis of gene
transcripts (mRNAs) in neurons is amenable to high-throughput analysis
today and hence should be the initial focus of effects to construct
Molecular Brain Maps, the characterization of protein products and
their subcellular localization, i.e. the proteomic characterization
of neurons, is an essential longer term goal that will rely on
improvements in proteomic analysis methods throughout the biomedical
community. As discussed, one proteomic initiative that deserves
priority at this time is the generation of antibodies to
transcription factors.
These tools, reagents and methods are summarized
in Tables 2 and 3.
- Need for organization and coordination
The successful development of a Molecular Brain
Map will require a concerted and large-scale effort. It is therefore
imperative that an ongoing Working Group be established,
comprising members of the scientific community as well as granting
agencies, to monitor developments and continually define and refine
priorities for molecular neuroanatomy.
- Need for both coordinated large-scale projects
and investigator-initiated projects
The collection of initiatives required to
generate and leverage a series of Molecular Brain Maps require a mix
of funding initiatives. Some aspects, such as the high throughput
generation, collection, and collation of gene expression data, will
benefit greatly from large-scale integrated funding initiatives.
Others, such as the development of tools to leverage the Molecular
Brain Maps, will continue to benefit from smaller-scale individual
investigator initiated projects. Both types of funding initiatives
should be supported by the NIH.
Appendix 1: Existing Large-Scale
Efforts
Various institutes at the NIH recognized the need
for a Molecular Brain Map in the 1990s. Many small-scale efforts have been
funded to further the development of this Map, and are not described here
in the interests of space. Several large-scale efforts funded at high
levels by various Institutes of the NIH are, however, discussed.
- Creating a dataset of all transcripts expressed
in the adult and developing mouse brain, and a physical collection of
cDNA probes for each of these transcripts.
Under contract to the NIMH, Dr. Bento Soares
(University of Iowa) has undertaken the generation of cDNA libraries
from mouse brain regions, and the direct sequencing of cDNAs from
these libraries, in an effort to identify all transcripts expressed in
the brain. In phase I, completed in the year 2000, a non-redundant
collection comprising approximately 30,000 brain and 9,000 retina
cDNAs/ESTs was identified, re-arrayed, sequence verified and made
publicly available. In phase II, initiated in September 2001, cDNA
libraries enriched in full-length transcripts are being generated from
whole brain and eyes at various developmental stages, and sequenced.
The identification of all transcripts has, of
course, been greatly accelerated by the sequencing of the human and
mouse genomes. Dr. Soares' project was initiated before it was clear
how long the sequencing of the human and mouse genomes would take. It
still remains complementary to the genome sequencing projects in
important ways. First, programs for identifying individual genes from
genomic sequence remain imperfect, so that cDNA sequencing efforts
continue to provide valuable information on the identity of individual
genes. Second, direct sequencing of cDNAs also provides important
information on the usage of alternative exons of particular genes
(alternative splicing and promoter usage) that is not always easily
inferred from genomic sequence. Finally, the project also provides a
physical series of cDNA probes for each of the genes that is
identified.
- Mapping gene expression
- Mapping gene expression through
high-throughput in situ hybridization
As part of NINDS's GENSAT project, Dr. Gregor Eichele (Baylor
College and Max Planck Institute, Hannover) and his colleagues have
undertaken an effort to map gene expression through high-throughput in
situ hybridization. This has involved the development of a robot
for performing reproducible in situ hybridization analysis on
sections of adult or embryonic brain, which will in the first
instance provide a collection of brain sections on microscope slides
on which the pattern of expression of individual genes is visualized
with a histochemical reaction product. The aim of the project, in
the first instance, is to map expression of over one thousand genes
per year to the adult mouse brain and the developing brain (at
E10.5, E15.5, and P7)
- Mapping gene expression through generation of
BAC transgenic mice
Also under the
auspices of NINDS's GENSAT project, a consortium of Dr. Nathaniel
Heinz, Dr. Mary-Beth Hatten and Dr. Alex Joyner (Rockefeller
University and New York University) and their colleagues has
undertaken to use BAC (bacterial artificial chromosome) transgenic
technology to map gene expression in the brain. In this approach,
for each gene a transgenic mouse is generated in which the reporter
GFP (green fluorescent protein) is expressed in a pattern mimicking
that of the starting gene. This is achieved by isolating a bacterial
artificial chromosome (BAC) containing the gene locus of interest
(including its regulatory regions), recombining a GFP cDNA into the
locus, and creating a transgenic mouse containing the modified BAC.
At high frequency, expression of GFP in such mice is representative
of expression of the endogenous gene. The aim of the project is to
ramp up to the production of about a thousand modified BACs per
year, which are then used to generate transgenic mouse lines,
followed by visualization of GFP in a select series of sections from
adult brains, and collection of images with this information. This
approach is complementary to the in situ hybridization
approach in (a). It is slower to make the modified BACs and
transgenic mouse lines than simply to perform in situ
hybridization. However, the GFP signal, unlike the in situ
hybridization signal, can often give more information on the
particular cell type expressing the gene because the morphology of
the cell and its pattern of projections often provides a unique
identifier of the cell. In addition, the modified BAC construct in
principle provides a valuable tool that can be used by other
investigators in transgenic mouse lines to mark cells expressing the
gene or to deliver other gene-modifying constructs to those cells
for functional studies in transgenic mice.
- Creation of Atlases of the mouse and human
brains
Under a grant supported by
NIMH, NINDS, NIDA, NIA, NIAAA, and NIDCD, Dr. Arthur Toga of the
University of California at Los Angeles, Dr. Russell Jacobs at the
California Institute of Technology, Dr. Larry Swanson of the
University of Southern California, and their colleagues, have been
developing computerized brain Atlases and tools to map data obtained
from gene expression analysis or other approaches (e.g. connectional
data, structural or functional MRI data, immunocytochemical data,
chemo- or cytoarchitectural data, etc. ) onto those Atlases. In the
case of the mouse, the result is a formal computerized
representation of the mouse brain, providing a systematic and
comprehensive digital space with links between a coordinate system
and systems of nomenclature for the structural subdivisions of the
nervous system. In addition, tools have been developed to map
information derived from brain sections onto this three-dimensional
Atlas, making it possible to correct for distortions of brain tissue
that occur during various histological procedures to visualize gene
expression. Such Atlases are being developed for adult mice and mice
of specific ages through development. Similarly, an Atlas of the
human brain has been devised, and software tools generated to import
information derived from multiple modalities (e.g. fMRI) and map it
onto the Atlas.
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