Project Stem Cell to Neuron

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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 specific groups of 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 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.

  1. 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.
  2. 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.
  3. 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:

  1. 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.
  2. 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.
  3. 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.

  1. 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.
  2. 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.
  3. 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.