Hynek Wichterle, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Motor function depends on specification of hundreds of distinct motor neuron (MN) subtypes within the developing neural tube. The motor neuron subtype identity is determined by anteroposterior (AP) patterning signals that subdivide the otherwise uniform neural tube into molecularly and functionally distinct segments. In comparison to the detailed understanding of dorsoventral patterning and specification of the generic MN identity, relatively little is known about the molecular mechanisms underlying anteroposterior patterning of the spinal cord and acquisition of MN subtype identity. Here we take advantage of our ability to direct differentiation of ES cells into motor neurons acquiring distinct subtype identities in vitro. We will use this system to study the genetic cascade underlying the acquisition of anteroposterior identity: 1) we will define extrinsic signals that regulate AP patterning; 2) we will define the time point during which AP identity is consolidated and 3) we will define molecular programs that underlie this process. Gaining insight into the process of acquisition of specific neuronal segmental and subtype identities during ES cell differentiation will have an impact on the utilization of ES cells to study and treat neurodegenerative diseases. The differential susceptibility of individual motor neuron subtypes to degeneration might reveal new potential targets for therapeutic intervention. In addition, our ability to direct MN subtype identity will be essential for effective implementation of MN cell replacement therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Transcriptional programming of cell identity is gaining importance at both basic developmental and clinical level. While the phenomenology of cell programming and reprogramming by forced expression of transcription factors is well described, the mechanism of action of programming factors or the sequence of regulatory events resulting in a cell adopting a new identity are largely unknown. We propose to combine the strengths of stem cell biology with genomic and computational approaches to characterize the process of transcriptional programming of motor neuron (MN) identity. We developed efficient methods for the induction of MN identity in differentiating embryonic stem cells (ESCs) by the expression of defined transcription factors. Using the system we will combine biochemical, genomic and computational analysis to address following questions: i) whether programming factors directly regulate terminal motor neuron effector genes or initiate a cascade of intermediate transcription programs; ii) whether recruitment of programming factors to DNA is cooperative and which factors determine the specificity of DNA binding; iii) whether identified MN enhancers are inaccessible for programming factor binding in cell types refractory to MN programming; iv) whether identified secondary binding motifs for Onecut and Ebf transcription factors contribute to productive regulation of MN specific gene expression; v) whether additional secondary motifs recruit ancillary transcription factors to NIL bound enhancers that contribute to productive regulation of MN specific gene expression. Together these studies will provide fundamental insight into the developmental processes underlying specification of defined cell identity and will provide novel and efficient source of motor neurons for disease modeling, study and drug discovery. PUBLIC HEALTH RELEVANCE: We propose to characterize the process of transcriptional programming of motor neuron identity. Understanding the process through which sets of transcription factors control gene expression will inform novel and efficient ways to generate clinically relevant cell types from pluripotent stem cells.

DESCRIPTION (provided by applicant): Death of spinal motor neurons is a hallmark of devastating and currently untreatable neurodegenerative diseases. However, little is currently known about the reasons why motor neurons are particularly sensitive to mutations affecting broadly expressed genes, such as SOD1 in amyotrophic lateral sclerosis or SMN in spinal muscular atrophy. It is assumed that cell intrinsic differences in motor neurons underlie the disease susceptibility, but nature of such modifiers on neuronal survival is currently poorly understood. Here we propose to study a recently generated mutation of a mir-17~92 cluster of micro RNAs. The deletion of this cluster results in a striking loss of limb muscle innervating spinal motor neurons, while other spinal neuronal classes appear unaffected. We propose to: first, study the cellular pathology of mir-17~92 null animals to determine whether any other cells besides motor neurons are dying in mir-17~92 null spinal cords and whether individual motor neuron subtypes exhibit different degree of degeneration. We will examine whether motor neuron cell death is due to cell autonomous function of miRNAs or due to the loss of miRNAs in other cells that might impinge on motor neuron survival in a non-cell autonomous fashion. Finally, we will determine whether miRNAs are required both in progenitors and postmitotic neurons to exert their pro-survival function. Second, we propose to dissect which specific miRNAs from the mir-17~92 cluster are involved in motor neuron death and which genes are deregulated in the absence of the miRNAs. We will test whether previously identified pro-apoptotic targets are involved in motor neuron degeneration. Furthermore, we will perform unbiased expression screen and test the function of selected identified biochemical pathways in motor neuron survival. As a lead into future studies we propose to determine whether overexpression of the miRNA cluster might save motor neurons from naturally occurring programmed cell death. We expect that these studies will define cellular pathologies in the developing spinal cord connected to the loss of mir-17 ~ 92 clusters and identify relevant molecular pathways linking the miRNAs and motor neuron survival. Understanding miRNA controlled cell type specific survival pathways might provide new targets for slowing down or arresting progression of motor neuron loss in motor neuron diseases.

? DESCRIPTION (provided by applicant): The hallmark of multicellular organism development is concerted differentiation of pluripotent cells into diverse postmitotic cell types. This process relies on transcriptional programs that in a coordinated manner activate expression of terminal effector genes and repress genes expressed in other lineages. While progress has been made in identifying transcriptional activators and mechanisms through which they establish new cell identities, little is currently known about the process underlying progressive repression of alternative developmental programs. Specifically, many transcriptional repressors that control differentiation of neural cells are expressed only transiently during early patterning of neuroepithelium and it has not been determined whether these repressors act solely on genes expressed in neural progenitors or whether they can silence enhancers in a persistent fashion to prospectively modify gene expression even following their downregualtion. Here we will examine effects of a transiently expressed motor neuron repressor Nkx2.2 on chromatin modifications of enhancers it binds and on patterns of gene expression in mouse motor neuron progenitors (while Nkx2.2 is ectopically expressed) and following its downregulation. Function of Nkx2.2 in enhancers that are stably silenced will be experimentally tested by deletion of Nkx2.2 binding motif. Together these studies will reveal fundamental mechanisms underlying stable silencing of poised or active enhancers during terminal differentiation of cells in multicellular organisms. As silenced enhancers need to be reverted into an active or poised state during cell fate reprogramming, our findings might be instrumental for the development of more efficient patient cell reprogramming strategies for diagnostic and therapeutic purposes.

Transcriptional programming of cell identity is gaining importance at both the basic developmental and the clinical levels. While the phenomenology of cell programming and reprogramming by forced expression of transcription factors is well described, the mechanisms of action of programming factors or the sequence of regulatory events resulting in a cell adopting a new identity are largely unknown. We are combining the strengths of stem cell biology with genomic and computational approaches to map the process of transcriptional programming of spinal motor neuron (MN) identity at a deep molecular level. We have developed efficient methods for the induction of MN identity in differentiating embryonic stem cells (ESCs) by the expression of programming transcription factors. Using this system, we combine biochemical, genomic, and computational analysis to address following questions: i) how is Isl1 recruited to transient enhancers in postmitotic motor neurons; ii) does Isl1 control enhancer activation; iii) are Klf factors bound to MN enhancers important for mediator and cohesin recruitment; iv) what motifs and factors coordinate interactions between distal and proximal MN-specific enhancers; v) can we infer the mechanisms controlling MN subtype specification and maturation by studying cell type and cell stage-specific regulatory regions in primary MNs. Together these studies will provide fundamental insight into the developmental processes underlying the specification of defined cell identity in the complex vertebrate nervous system and will provide a novel and efficient source of MNs for disease modeling, functional analysis, and drug discovery.

Mutations causing late-onset neurodegenerative diseases often remain phenotypically latent for decades before the first symptoms of neurodegeneration are detected. The subtle effects of the mutations on neuronal function, combined with cell-line-to-cell line variability makes modeling diseases like ALS extremely challenging. To increase the robustness of in vitro ALS models, we will generate a set of 20-60 cell lines derived from an ALS iPSC line carrying a highly penetrant and aggressive FUSP525L mutation. In half of these cell lines we will correct the disease-causing mutation using a CRISPR-mediated homologous recombination strategy. Additionally, we will introduce a unique genomic barcode into each of the isogenic cell lines. These barcodes will be used to distinguished between corrected and ALS cells, and will also be used as a means of motor neuron quantification through barcode sequencing. We will use this system to examine whether FUS mutant ALS motor neurons exhibit basal decreases in their fitness, and whether stressors that mirror known ALS phenotypes by inducing ER stress, misfolded protein accumulation, or mitochondrial dysfunction can selectively potentiate the degeneration of ALS motor neurons over their isogenic wild-type counterparts. The proposed platform could then easily be adapted to screen full libraries of bioactive compounds for additional stressors that might illuminate novel degenerative pathways or point to new therapeutic targets. Finally, this platform will be invaluable for the discovery of new compounds that decrease ALS motor neuron degeneration in response to stressors, and may contribute to the discovery of new therapeutic agents for a disease where there are currently very few options for treatment.

Our ability to produce neurons from pluripotent stem cells holds great promise for studying the assembly, function, and dysfunction of the nervous system in experimentally accessible settings. While there has been a dramatic advance in the development of new protocols for differentiation of stem cells into increasingly diverse types of neurons, stem cell-derived neurons fail to acquire a fully mature neuronal identity. In order to study the function of mature nerve cells and to model adult onset neurodegenerative disorders in a more relevant context, it is paramount to develop methodologies that yield nerve cells more closely resembling those found in the adult CNS. Towards this goal, we will perform a longitudinal study of gene expression and chromatin changes in primary motor neurons in the mouse spinal cord. We will identify enhancers that control mature gene expression programs and develop methods for reprograming immature stem cell-derived motor neurons to a mature state. This work will impact three important areas: 1) it will provide the first detailed analysis of gene expression changes associated with motor neuron maturation in vivo, 2) it will define new transcriptional regulators controlling neuronal maturation, and 3) it will yield a new effective method for reprogramming immature stem cell-derived motor neurons to a more mature state that will serve as a better model for adult-onset neurodegenerative diseases, such as amyotrophic lateral sclerosis.