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We propose to further our understanding of the molecular mechanisms that direct
stem cells during neural development with the ultimate goal of enabling stem
cell based regenerative medicine for neurodegenerative diseases. We will study
the etiology of Spinal Muscular Atrophy (SMA) in the context of the mechanisms
that we elucidate with the goal of developing clues to potential therapeutic
targets for this developmental disease. To understand how external cues direct
development, we will elucidate the transcriptional regulatory networks
underlying neural development and represent this understanding in predictive
computational models. Our studies will begin with undifferentiated embryonic
stem (ES) cells, and using protocols that we have pioneered, we will elucidate
the mechanism of ES cell development and fate commitment in specific neuron
subtypes. Our work is structured into three projects. Collaborators at Columbia
University will identify the transcription factors potentially involved in motor neuron
identity, iteratively define transcriptional networks, and characterize the
transcriptional consequences of SMA. Drawing upon these results, collaborators at
the Whitehead Institute will discover how key transcriptional and chromatin regulators
control the gene
expression programs of mouse and human embryonic stem cells and discover how
this regulatory circuitry changes upon differentiation into spinal progenitor
cells and then specific classes of central nervous system cells such as motor
neurons. Using data from both of these projects, collaborators at the
Massachusetts Institute of Technology will build a model
of transcriptional regulation during neural development that integrates
expression data, factor binding data, chromatin data, shRNA knock down data, and
genome sequence in both human and mouse, examine the gene expression
consequences of our SMA model in the context of the deduced regulatory networks,
and explore the validity of the mouse model for human ES cell differentiation.
Collaborators at both Columbia University and the Whitehead Institute will test
the models produced by collaborators at MIT.
Columbia University
Studying the way in which specific groups of motor neurons in the
spinal cord develop to innervate specific muscles is central to
understanding how precise control of breathing and movement is
achieved. These studies will provide clues for understanding why
specific groups of motor neurons degenerate and die in patients
with diseases such as spinal muscular atrophy (SMA). This project
will identify molecular mechanisms involved both in normal
development and pathologic degeneration of this important
neuronal population.
Considerable progress has been made in defining the transcriptional
events that control the stepwise differentiation of unspecified neural
precursors into motor neurons that innervate specific muscle targets.
Despite these advances, much remains to be learned of the transcriptional
regulatory network that subtends this process. Based on the ability
to generate homogeneous preparations of specific motor neuron subtypes
from ES cells, this project will take a global approach to defining
transcriptional differences between motor neurons and other spinal neurons.
Transcription factors identified will then be used to identify target
genes and thereby iteratively define transcriptional networks. The work
is structured around three aims.
- Aim 1 will use transcription factors known to determine motor neuron
or dorsal interneuron fate to drive ES cell differentiation, in
order to identify factors potentially involved in the acquisition of
generic motor neuron identity.
- Aim 2 will use intrinsic and extrinsic factors that drive the
differentiation of motor neurons characteristic of specific columns
or pools from mouse ES cells, to define the transcrptional logic that
controls motor neuon subtype identity.
- These basic advances will be applied in Aim 3. to the study of
Spinal Muscular Atrophy (SMA),
a developmental disease that results from reduction in levels of
a protein, SMN, that is required for motor neuron survival.
New ES cell lines will be derived from motor neurons expressing
normal and reduced levels of SMN, obtained from mouse models for
SMA. These disease-specific ES cells will be screened for
differences in transcriptional repertoire.
Functional testing of genes identified as potential effectors in these
screens will be performed using a panel of in vitro and in vivo test
systems focused on motor neuron differentiation, survival and axon
growth. The project should also provide new approaches to potential
therapeutic targets in SMA.
Whitehead Institute
Our goal is to discover how key transcriptional and chromatin
regulators control the gene expression programs of mouse and human
embryonic stem cells and to discover how this regulatory circuitry
changes upon differentiation into spinal progenitor cells and then
specific classes of central nervous system cells such as motor
neurons. This knowledge will enable us to define the transcriptional
regulatory processes that control a cells progress to its terminally
differentiated state, and may provide new insights into the means by which
embryonic stem (ES) cells can be programmed for therapeutic purposes. To
accomplish this, the specific aims of the proposal are:
- To identify the genomic targets of key transcriptional regulators
and the transcription apparatus in mouse embryonic stem cells,
spinal progenitor cells and motor neurons Genome-wide location analysis,
a powerful technique that identifies the set of genes that are bound
by specific regulatory proteins in living cells, will be used to
identify the target genes of key transcriptional regulators.
- To identify the location of the MLL1 and polycomb chromatin
regulators genome - wide in mouse embryonic stem cells, spinal
progenitor cells and motor neurons. Genome-wide location analysis
will be used to identify the portion of the genome that is silenced
by PRC2 and the portion of the genome that is actively maintained by
MLL1 in each cell type. This analysis will be complemented with
analysis of the histone modifications (histone trimethyl H3K27 and
trimethyl H3K4) generated by these regulators.
- To identify the targets of key transcriptional regulators and
chromatin structure in human ES cells that have been directed towards
neural differentiation. In vitro differentiation of ES cells gives
us the unique opportunity to apply genome wide analyses to the
earliest steps of human neurogenesis as well as a variety of
mature neuronal and glial subtypes such as spinal motor neurons,
dopaminergic midbrain neurons and oligodendrocytes. Having
complementary mouse and human data will allow us to map conserved
gene targets and chromatin structure, and will help us model the
consequences of SMN deficiency in human.
Massachusetts Institute of Technology
We will build predictive regulatory models of neural development to
help us understand what goes wrong in neural pathologies. Using our
models we will explore why motor neurons die in Spinal Muscular
Atrophy (SMA), develop clues for therapeutic targets that might help
individuals with SMA, and study how stem cells respond to external
cues that could lead to methods of programming stem cells for therapies.
We will build models of transcriptional regulation that identify at
the molecular level how cells become committed to a particular neural
fate and explore how embryonic stem (ES) cells can be programmed for
therapeutic purposes. We will examine the effect of Survival of
Motor Neuron (SMN) protein deficiency on the resulting regulatory
models and the corresponding etiology of Spinal Muscular Atrophy (SMA).
We will build our models by discovering key elements of the
transcriptional regulatory network that underlie the development of
mouse ES cells into spinal progenitor cells and finally into specific
classes of central nervous system cells such as motor neurons. We
will also examine the same neuronal developmental process in human
ES cells and develop models for studying the conservation between
the mouse and human transcriptional networks in this system.
Our work is structured around three aims.
- Aim 1 will map the active genome in mouse ES cells, spinal
progenitor cells, and motor neurons using high-resolution models of
transcription factor binding, chromatin structure and RNA
Polymerase II activity.
- Aim 2 will discover key aspects of the transcriptional regulatory
network that underlies neuronal fate specification in mouse ES
cells and the etiology of SMA in the context of this network.
We will use likelihood based methods to build a consensus model of
transcriptional regulation during neural development that explains
our high -resolution mouse transcription factor binding data, chromatin
data, corresponding expression data, and DNA motifs.
- Aim 3 will determine whether the regulatory network that we
discover in Aims 1 and 2 also controls the neuronal differentiation
of human ES cells. This comparison of mouse and human ES cell
programs will test the validity of the mouse model for human ES
cell differentiation and open the door to further manipulation
of human ES cells.
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