Small regulatory RNAs carry out diverse functions in both bacterial and eukaryotic cell physiology. Despite a known role for sRNAs in ribosome biogenesis in eukaryotes, the potential role for sRNAs in bacterial ribosome biogenesis and/or (pre-) ribosome degradation and storage under stress conditions is unknown. Based on their functional properties and versatilities I hypothesize that small RNAs are involved in bacterial ribosome biogenesis. Using a combination of bioinformatic, cell biological and biochemical techniques in vitro and in vivo, I aim to identify small RNAs with physiologically relevant functions in ribosome assembly either as assembly co-factors or as regulators of ribosome biogenesis in response to environmental changes. Moreover, I will investigate at which conditions the identified sRNAs regulate ribosome biogenesis, which interaction partners are involved, which mechanisms are employed and which consequences result from sRNA function. To determine the influence of sRNAs on folding and processing of rRNA, I will use an in-vitro co-transcriptional assembly system.
In parallel, I aim to identify artificial small RNAs that could be used as effectors of ribosome biogenesis or as probes to monitor co-transcriptional folding of ribosomal RNA and thus the kinetics of ribosome assembly in mechanistic detail. The work will provide physiological and mechanistic insight into the function and regulation by sRNAs in ribosome biogenesis as well as into co-transcriptional rRNA folding.

While the fundamental functions of sleep remain unknown, deficits in learning and memory after sleep loss are shared between humans and genetic model systems, including the fruit fly. My past research has found that sleep strongly modulates the consolidation of recently learned associations into long-term memories - sleep deprivation after learning prevents memory consolidation, and acutely inducing sleep enhances the consolidation of newly acquired memories that would typically be forgotten. While these data demonstrate an important role for sleep in memory processing, the molecular processes that occur during sleep to support memory consolidation remain unknown. To date, the complexity and parallel structures of memory-encoding and sleep control circuits in mammalian systems prevent researchers from properly investigating these mechanisms. In the fruit fly, however, memory-encoding circuits in the Mushroom Body and sleep control neurons in the dorsal Fan-shaped Body are accessible for in vivo whole cell recordings and genetically addressable at the resolution of individual neurons. The proposed project will 1) use in vivo patch clamp recordings to examine the neural circuits that promote sleep during memory consolidation, and 2) profile actively translated mRNAs in memory-encoding circuits to identify proteins that facilitate memory consolidation during sleep. Understanding and controlling these mechanisms will not only provide insight into the neural functions of sleep, but support the development of tools to offset the cognitive consequences of sleep loss and to influence whether recently acquired memories are retained or forgotten.

Viral entry into its host cell may results in either successful or abortive infection. Abortive infection of permissive cells, which are able to support viral replication, is rarely discussed but is quite abundant. What makes one cell resist to infection while its neighbor succumb to it is the subject of this research proposal. This is important as it could lead to the discovery of novel cellular anti-viral modalities and deepen our understanding of host-virus interactions.
My goal is to characterize the anti-viral response to viral infections in single cells, its variability among genetically identical cells and how this relates to infection outcome (successful or abortive).
To do so, I will learn and implement new technologies developed by the Tay lab. The success of the project relies on using a combination of live-cell imaging with in-lab fabricated microfluidics devices that allow the retrieval of single cells with known fates for further analyses, such as single-cell RNA-sequencing. By using viruses that express fluorescent markers I will be able to monitor infection in living cells in real-time, extract and molecularly profile cells in specific time points, for example in the process of aborting viral infection (when fluorescent proteins level stop raising or begin to drop). The results from this screening approach will be validated using standard cell-biology and virology techniques. I also plan to use this technology to investigate the response of the known anti-viral pathways.
I expect to reveal new molecular participants in the anti-viral response as well as to explore the variability in the response among single cells and how it affects infection outcome.

The purpose of this proposal is to recreate an ancient living system, an organism that uses RNA catalysts, ribozymes, as part of its translation apparatus. We will achieve this grand goal by progressively reintroducing ribozymes into cellular metabolism, ultimately replacing protein enzymes. To this end, Hiroaki Suga will develop novel ribozymes, Flexizymes, that can charge tRNAs with amino acids. Andy Ellington will adapt Flexizymes for use in cells, and replace cognate aminoacyl-tRNA synthetases. Michael Jewett will adapt Flexizyme charging to an orthogonal ribosome that can specifically utilize Flexizymes. Both Ellington and Jewett will hand organisms containing Flexizymes and orthogonal ribosomes to Philippe Marliere for high-throughput evolutionary adaptation and optimization. Ultimately, this will result in the creation of an organism that serves as a doppelganger for ancient living systems in transition from an RNA to a protein world. This ribo-organism will be unique in that it will have two translation apparatuses operating side-by-side, one of which has the normal complement of cellular machinery, and one of which has Flexizymes, orthogonal tRNAs, an orthogonal ribosome, and a new genetic code. We will initially carry out our goals by altering the machinery for the incorporation of histidine, but eventually expand the genetic code to unnatural amino acid analogues of histidine, 1,2,4-triazole-3-alanine (T3A) and alpha-hydroxy histidine (AHH), in order to highlight potential biotechnology applications. The incorporation of these new amino acids into the genetic code, both as 21st amino acids (via suppression) and in competition with an existing amino acid, histidine (via missense incorporation), should serve as a modern experimental surrogate for the ancient establishment of the genetic code. This project is also notable for its broad interdisciplinary flavor, in that it spans from a chemist (Suga) to a biochemist (Ellington) to a bioengineer (Jewett) to an evolutionary biologist (Marliere). Each individual field touches on the other, but the overall arc traverses a much broader swath of science and technology than would otherwise be possible. The intermingling of distinct cultures is also apparent in the scope of the work, which for the first time attempts multi-scale evolutionary optimization of an entire cellular sub-system, translation.

Mammals have the ability to generate an infinite variety of motor behaviors, from simple actions such as walking to highly complex movements like object manipulation or speech. The flexibility of the motor repertoire is essential for animal survival because it enables the generation of goal-directed movements adapted to environmental constrains. Some motor patterns are present at birth while new motor skills can be acquired through training and experience. Not surprisingly, communication among numerous brain areas is needed to ensure accurate acquisition and execution of motor programs. The classical model for motor learning proposes that only a subset of structures along the motor command pathway within forebrain and cerebellum are subjected to activity-dependent adjustments and become reorganized during acquisition of a new skill. In contrast, downstream motor regions located in the brainstem are considered to be simple executive centers for stereotyped motor behaviors. Here I challenge this model and propose that learning-induced plasticity in brainstem motor circuits is essential to adapt the executive motor signal to drive new skills. To evaluate my hypothesis I will combine cutting-edge viral and mouse genetic tools together with in vivo and ex vivo electrophysiology to characterize how learning-induced signals are encoded in the brainstem. This proposal supports a new conceptual framework in which plasticity mechanisms along the entire motor command pathway including brainstem circuits, underlie the formation of motor memories.

The segmented body pattern of vertebrates is established in the early development through the periodic formation of somites, the vertebrae precursors, from the presomitic mesoderm (PSM). The rhythmic formation of somites is under control of a molecular oscillator, the segmentation clock, which controls the cyclic transcription of a group of genes within the PSM.
Previous microarray analyses and candidate gene approaches identify many cyclic genes in the PSM, mostly genes associated to the Wnt, Notch and FGF-signaling pathways. Yet, a comprehensive, dynamic and quantitative analysis of the transcriptional dynamics and regulation of cyclic genes is still lacking, mostly due to the challenge to obtain samples with unequivocal time resolution. By using a microfluidic system that allows controlling the rhythm and, thus, synchronizing samples in relation to an external reference time, we are now able to obtain nearly unlimited PSM cells accurately staged. With a combination of high-throughput sequencing methods, we plan to identify unknown cyclic genes and regulatory elements (enhancers/promoter) with a role in somitogenesis. We aim to establish a temporal relationship between genes and their regulatory regions, and identify molecular mechanisms that control the rhythmic formation of somites. For example we may discover that cyclic expression relies on the periodic interplay of multiple enhancers. Or, that it is caused by cyclic epigenomic modification within regulatory elements. Additionally, by identifying sequence motifs within these elements we will be able to connect signaling pathways to oscillatory gene expression.

Although the basis of nucleotide binding and oligomerization domain (Nod) receptors signaling has been discussed for several years, the molecular details that underlie this process are still being uncovered. Moreover its presence on brain neurons hasn’t been described yet. The role of Nod2 receptor goes beyond simply sensing the presence of a potential pathogen and activating the immune system. Its activation has been linked to stem cells cytoprotection, analgesic effects, protection from cerebral ischemia-reperfusion damage and altered sleeping patterns. Moreover, mutations on this gene have been associated with bipolar disorder and schizophrenia in humans. The cytosolic Nod2 receptor can be activated by muramyl dipeptides a product of the degradation of peptidoglycan, a polymer present in almost all bacteria cell wall and increasing evidence demonstrate that endogenous ligands like damage-associated molecular patters, can also activate Nod receptors. Particularly, not much is known about the impact of Nod2 activation on behavior or the mechanisms and the specific effects on brain physiology. Understanding the role of Nod2 on brain neurons can lead to better understanding of many neurological disorders. Preliminary data from Dr. Lledo lab have shown, the presence of Nod2 receptors in neurons from the striatal and thalamic areas of the brain. They also observed that Nod2 knock-outs show less anxiety, more social aggressiveness and lack of discrimination of olfactory cues in social interactions and that Nod2 signaling can selectively disrupt paradoxical sleep. Therefore the purpose of this proposal is to assess the impact of Nod2 signaling on brain activity and behavior.

Polyploid cells, which contain more than two pairs of homologous chromosomes, are widely present across protozoa, plants and animals. Polyploidization occurs in many cell and tissue types as part of a developmental program, and is crucial for increases in metabolic output, cell mass and cell size. Two cell cycle variations, endoreplication and endomitosis, are known to give rise to polyploid cells. Although the function and molecular regulation of endoreplication has been extensively studied, the molecular factors that control endomitosis are largely unknown. Specifically, it is unclear how endomitotic cycles are initiated and executed, and what the advantages are of endomitosis over endoreplication. The C. elegans intestine provides an ideal system to study these questions, as transitions between canonical, endomitotic and endoreplicative cycles all take place at defined moments during development. In this study, we will identify the molecular regulators of endomitosis in the C. elegans intestine by performing single-cell sequencing and RNA Tomography sequencing (Tomo-seq) of intestinal cells at specific cell-cycle stages. Moreover, we will uncover the physiological importance of endomitosis and the functional difference between endomitosis and endoreplication by generating tissue-specific C. elegans mutants with alterations in the intestinal cell cycles and analyzing their intestinal function. Together, these approaches will provide an in-depth analysis of endomitosis mechanism and function, providing insights into how tissues specify cell-cycle programs and what the importance is of specific forms of polyploidization.

While cancer has been shown to rely on the accumulation of multiple mutations, conditions such as injury have been also shown to trigger cancer. Consistently, a recurrent mutation in Squamous Cell Carcinoma (SCC) in the Ras oncogene is not sufficient to trigger SCC unless either injury or a second mutation occurs. Yet, it remains elusive how wound-triggered behaviors lead to SCC. This proposal aims to identify wound-induced epithelial and niche cell behaviors that are required for cancer initiation. My post-doctoral lab has recently established an imaging approach to study how epithelial stem cell behaviors are coordinated to close a wound in live mice. I will build on this understanding and combine the study of wound repair in the context of a mouse model expressing activated-Ras mutation in epithelial stem cells. Specifically, I will determine what changes in cell behaviors, such as proliferation and migration, occur in the Ras model and I will use drug-based and genetic manipulation models to determine the functional impact of examined mutant behaviors towards SCC initiation. Furthermore, I will interrogate and manipulate the immune response, a central component of both the wound repair process and SCC, in the epithelial Ras mutant model to dissect the immune cell role during SCC insurgence. Our innovative live imaging approaches to track my populations of interest in combination with manipulative approaches will allow me to identify the relevant cells and behaviors towards cancer initiation. All together my project will provide new insights into how the physiological process of wound repair can be hijacked into a process of malignant transformation.

My goal is to understand how the cadherin-catenin complex, a force-responsive protein assembly that links neighboring cells, transduces extracellular forces into intracellular signaling events. Thus far, absolute force magnitude has been considered the main control parameter in mechanotransduction; but because mechanical cues in tissues and organs are subject to tight temporal control, it is likely that force timescales complement force magnitude to modulate junctional remodeling and downstream signal transduction. For example, during Drosophila embryogenesis regular contraction forces mediate the formation of the ventral furrow and gastrulation. In this project, I will characterize, for the first time to my knowledge, the response of intercellular adhesions to not only force amplitude, but also its temporal variation. Specifically, I will combine single-molecule force spectroscopy and single-molecule fluorescence microscopy techniques to directly quantify the kinetics of individual cadherin-catenins complexes in cells upon temporally controlled mechanical stimuli. Using this approach, I will determine how the cadherin-catenin complex integrates temporal cues into the mechanotransduction process. As a working model, I propose that cadherin clustering responds both the force magnitude and timescale, and that clustering is the key event that drives downstream signal transduction. My long-term goal as an independent investigator is to use the approaches outlined in this proposal to understand how macromolecular complexes produce, detect and respond to forces to accomplish biological processes such as mitosis, cell migration and mechanotransduction.

Life involves a myriad of inter-related chemical processes, almost none of which would proceed at adequate rates without the assistance of enzymes. Enzymes are “Nature’s catalysts”, accelerating the rates of these reactions by up to ~20 orders of magnitude, thus making life as we know it possible. No consensus has yet emerged about the ultimate origin of the "catalytic power" of enzymes, with a rather wide variety of hypotheses put forward over the years. In addition, the limitations in our understanding of enzyme catalysis are highlighted by our inability to reproduce the catalytic power of the best naturally occurring enzymes in any human made catalyst (including de novo designed enzymes); as Feynman famously pointed out, "What I cannot create, I do not understand". This lack of a definitive understanding in the origins of enzyme catalysis is most likely related to the fact that natural enzymes are the complex outcome of natural selection operating over vast expanses of time in an evolutionary process that may be determined to some extent by contingency. Here, we posit that an in-depth understanding of the catalytic power of enzymes will only be possible when we have at our disposal a procedure that routinely reproduces the emergence of new functions from non-catalytic (or minimally catalytic) scaffolds in the laboratory. Therefore, during the course of this project, we will:
1) use phylogenetic analysis to reconstruct sequences of ancestral proteins from a structure having high potential to serve as a scaffold for generating novel functionalities;
2) prepare the encoded proteins in the lab and test them not just experimentally but also computationally for the biophysical and biochemical properties that confer high evolvability on a protein scaffold;
3) synthesize very large libraries of up to ten trillion (10E13) variants using highly evolvable ancestral proteins as scaffolds;
4) use ultra-high-throughput in vitro and in vivo methodologies to screen these libraries for non-natural functions;
5) use both experimental and multiscale modeling tools to characterize the resulting de novo enzymes for the biophysical, biochemical and structural features relevant for efficient catalysis;
6) use the above characterization to test different hypotheses about the evolutionary and molecular origin of enzyme catalysis.

While there is growing realization that sleep is important for memory consolidation, most evidence remains correlative and the causal role of specific sleep events remains unclear. Moreover, there is a substantial gap between detailed electrophysiological animal research and non-invasive cognitive human research – where recording neural activity in the medial temporal lobe is particularly challenging. The proposed research aims to understand the causal role that sleep plays in memory consolidation. Upon informed consent, epilepsy patients at UCLA implanted with intracranial electrodes for clinical monitoring will participate in learning/memory paradigms combined with recordings and intracranial electrical stimulation during sleep. I will address two specific aims: (1) Determine which sleep activities correlate best with learning and memory improvements: I hypothesize that for declarative memory, NREM sleep will be best correlated with learning, and that coordinated coupling between neocortical slow waves, sleep spindles and hippocampal ripples, co-occurring with reactivation of neuronal ensembles that were selectively engaged in the learning task, will be maximally correlated with successful learning. (2) Elucidate the causal mechanisms through intracranial electrical stimulation. I hypothesize that electrical stimulation in the neocortex that mimics or locks to endogenous slow wave up-states and hippocampal ripples will be effective in promoting memory consolidation. This proposal has the potential to generate a breakthrough in our understanding of the coordination between neocortex and hippocampus during sleep and how sleep causally aids human memory consolidation.

Cell motility plays an important role in a multitude of biological processes, including embryogenesis, wound healing, and cancer metastasis. As cells move, they exert forces onto their environment and become increasingly polarized, resulting in an elongated shape and signaling and cytoskeletal components that become localized in the anterior or posterior of the cell. It is currently unclear how force generation is correlated with the distribution of key signaling components and how cells regulate their polarity. To address these questions requires a careful and precise control of external cues and the ability to link molecular events to the cell's macroscopic behavior and the generation of forces. In this project, I will quantify the establishment and reversal of cell polarity during chemotaxis using innovative microfluidic devices together with traction force microscopy (TFM) and confocal microscopy. Specifically, I will expose unpolarized Dictyostelium discoideum cells to carefully controlled chemoattractant gradients generated in microfluidic devices. I will quantify the kinetics of several key signaling components and of traction forces using confocal microscopy and TFM, respectively. Furthermore, I will examine how polarity is reversed by rapidly changing the gradient experienced by cells that migrate in specially designed 1D micropatterned tracks. By correlating the dynamics of the markers with the observed traction force patterns, I will be able to determine how cell polarity is established and reversed. Our experiments should result in a more comprehensive understanding of cell polarity, a crucial component of cell motility in many eukaryotic cell types.

The muscular systems of humans and chimpanzees differ in a variety of traits, with human muscles built for dexterity and endurance, whereas chimpanzee muscles are built for strength. However, the genetic basis of this variation is little understood. Here, I propose to use a novel system of hybrid human-chimpanzee tetraploid induced pluripotent cells to study how the regulation of genes affecting muscle physiology has evolved along our lineage. This hybrid cell line offers a unique and unprecedented opportunity to tease out genetic from environmental factors, as well as cis- from trans-acting factors. I will first differentiate the parental and hybrid cells into skeletal myocytes, the main cells determining fitness capabilities. Then, I will identify allele-specific expression by comparing the gene expression levels of the hybrids to their parental cells. This will allow me to establish the first catalogue of cis- and trans-regulatory divergence in muscle cells. Finally, I offer to pick ten genes, where it is most probable to identify the variant the drives their diverged regulation, and to genetically edit them to investigate the phenotypic effect of each sequence change. This novel system will allow me to circumnavigate a key ethical barrier in great ape studies – obtaining great ape cells. Moreover, this system and pipeline could be used to study a variety of cells, thus establishing a novel approach to identify the regulatory variants that drove species-specific traits.

While hematopoietic stem cell (HSC) transplantation has become the standard of care for treating many hematologic diseases, wider use is restrained by immunologic incompatibility and inefficient reconstitution of hematopoiesis from allogeneic sources. Pluripotent stem cells (PSCs) promise an attractive alternative source of HSCs, but current PSC-derived HSCs display fetal phenotypes that lead to stem cell exhaustion and immature progeny function. We hypothesize that human PSC-derived definitive hemogenic endothelium (HE) can be efficiently converted to HSCs with adult-like functionality. To achieve this, we first propose to develop a robust reporter system that differentially marks fetal versus adult HSCs. By combining this with recently-developed technology to transgenically convert HE into fetal HSC-like cells, we can execute in vivo targeted and high-throughput genetic screens to identify TFs and pathways that regulate HSC maturity. We will subsequently find small molecules or morphogens that modulate activity of these regulatory factors to establish transgene-free conditions for the specification of adult-like HSCs. To ensure physiological relevance of the resultant cells, they will be assessed for long-term engraftment capability, functionality of their terminally-differentiated cells and appropriate response to stress conditions in vivo. This proposal will provide proof-of-concept that adult functionality can be conferred to PSC-derived cells, provide a sustainable platform for modeling adult hematologic diseases, and most importantly, bring us one step closer towards clinical grade PSC-derived HSCs for transplantation.

Tissue stem cells (SCs) depend upon their niche microenvironment, suggesting that spatially localized signals play key roles in determining SC identity and plasticity. Evidence from the hematopoietic system suggests that vasculature is a critical niche component. Vasculature also governs tumor-initiating SCs of squamous cell carcinomas. The spatial and temporal organization of capillary networks in healthy dermis suggests that vasculature also impacts normal hair follicle SC (HFSC) behavior, a notion further fueled by hair growth sensitivity to systemic changes. Despite several recent advances, the molecular basis by which a SC can sustain regeneration is still poorly understood. Here, I aim to perform the first spatiotemporal screening to dissect the molecular crosstalk between HFSCs and vasculature remodeling, and elucidate how crosstalk dictates ‘stemness’ identity to meet regenerative demands during normal hair cycling and wound-repair. Using mouse as model, I aim to tackle these objectives by: (1) Mapping and defining the spatial and temporal localization of the skin vasculature network relative to HFSCs during development and hair cycling. (2) Elucidate the molecular crosstalk between vasculature and HFSCs, and how it changes during capillary network remodeling. (3) Dissect mechanistically how vasculature-mediated signaling influences SC physiology during homeostasis and wound-repair by using in utero lentiviral delivery to skin epithelium of HFSC-specific activation of Cas9/guide gene silencing. By understanding how the vasculature dynamically redistributes and reshapes the niche, I hope to uncover new avenues for therapeutics in skin regeneration and wound-repair.

Cell survival requires efficient adaptation to external changes. Bacteria use complex signaling networks to sense and respond to these changes, particularly nutrient availability. The current knowledge of bacterial responses to carbon starvation is based mainly on E. coli and involves small molecule second messengers like cAMP. However, intracellular responses to starvation and cAMP regulation may differ in other species. The alpha-proteobacterium Caulobacter crescentus is a powerful model for understanding how intracellular signals are transduced and impact cell-cycle progression in bacteria. Carbon starvation somehow prevents cell differentiation and a G1-S transition during the Caulobacter cell cycle. This differentiation step is normally triggered by the synthesis of a regulator called SpmX. I propose that the absence of a carbon source prevents SpmX synthesis through the inhibition of its transcriptional regulator, TacA. I will use ChIP-qPCR and in vivo phosphorylation assays to test this model and dissect the mechanisms that temporally regulate SpmX. Further, I hypothesize that, like E. coli, the main intracellular signal following carbon starvation in Caulobacter is cAMP, and that cAMP directly regulates TacA activation. I will measure cAMP intracellular levels using a FRET-based method and assess the impact of cAMP on TacA activity and SpmX synthesis. I will also use capture-compound mass spectrometry to identify the cAMP interactome in Caulobacter. I anticipate that this study will provide new insights into cAMP-dependent cellular responses and nutrient-sensing mechanisms in Caulobacter, which may differ significantly from what is known in E. coli.

During development, embryonic tissues develop into organs of stereotyped size and shape. Morphogens are secreted from discrete regions in developing organs and form spatial concentration gradients that guide gene activation, pattern formation and tissue growth. Morphogen gradients scale with tissue size, ensuring that morphological patterns remain proportionate in organs of different size. The mechanisms of morphogen dispersal and scaling are not yet clear. Two models for morphogen spreading attracted much attention in the field: i) fast extracellular diffusion versus ii) cycles of internalization and recycling (transcytosis). I will develop a generalized theory of transport that captures extracellular diffusion and transcytosis simultaneously: I will consider extracellular diffusion, morphogen/receptor binding/unbinding and various endocytic trafficking steps. This novel theoretical method will uncover the parameter space where either extracellular diffusion or transcytosis dominates the dynamics of dispersal. I will also explore whether both phenomena could contribute significantly to the length scale of the steady-state gradient. My model system will be the Decapentaplegic (Dpp) gradient in the fly wing. Using novel experimental endocytic assays from the host lab, I will parameterize Dpp trafficking dynamics to establish the prevalence of extracellular diffusion versus transcytosis. Unpublished work from the host lab shows that trafficking is spatially heterogeneous in the wing. I will upgrade my theory to a heterogeneous scenario with the potential to yield, as an emerging property, a novel mechanism of gradient scaling.

When we navigate and make decisions in familiar environments we rely on an internal, mental map of the features and general shape of the environment. Remarkably, work over the last four decades has revealed how different locations in space and different navigational plans are represented as patterns of activity in thousands of neurons in the brains of rodents. It stands to reason that this 'neural representation' of an environment should remain stable as long as an animal is using it, to allow the animal the reliably navigate. However, we found that the neural activity that makes up this map changes over time so that different neurons represent specific features of a familiar environment after several days. We do not have a theory to explain how the brain can build and use such a continually changing mental map, nor do we know why the map changes, or what purpose this continual change might serve. In this project we will build and experimentally test new theories and models of how the brain represents environments, potentially changing our understanding of how the brain works.

The random selection of an allele to be exclusively expressed affects gene expression and any possible phenotypes related to the allele. This may be one of the key mechanisms through which phenotypic variation is established. Estimates of the prevalence of autosomal random monoallelic expression (aRME) vary widely between different experiments and cell types and importantly, the frequencies of fixed and dynamic aRME have not been addressed in the same experiments. I aim to address the balance between fixed and dynamic aRME in an in vivo as well as in vitro model. By combining single cell, allele sensitive transcriptomics approaches, which have been developed in the Sandberg lab, combined with an inducible cell-lineage reporter system, I will be uniquely able to characterize the extent of both fixed and dynamic aRME. We hypothesize that the balance between fixed and dynamic aRME depends on the stabilization of expression on a given allele. According to that model, fixed as well as dynamic aRME is caused by stochastic expression that is then stabilized to different extents. By studying this in vivo we will be able to characterize the stability of aRME in different tissues where aRME stabilization may differ (eg. neurons vs. fibroblasts). The in vitro model on the other hand would give me the possibility to perturb the system and study the mechanisms that establish and stabilize aRME. Additionally, the data resulting from the experiments I propose will also provide a novel and unique insight into the relationship between gene expression and cell clonality in a wide range of mouse tissues and cell types.