Plant development, growth and responses to abiotic and biotic stresses are regulated by hormone-mediated signaling processes. Recently the role of peptides as plant hormonal signals has become appreciated. Among these peptide signals are the Plant Elicitor Peptides (Peps); 23 amino acid peptides derived from larger precursor proteins (PROPEPs). Biochemically isolated from Arabidopsis, Peps are endogenous regulators of Arabidopsis thaliana innate immunity and bind two receptors, PEPR1 and PEPR2, to induce transcription of defence-related genes. Constitutive expression of PROPEP encoding genes results in enhanced pathogen resistance. Pep homologs also induce disease resistance in maize, implicating this family of peptide hormones as regulators of immunity throughout the plant kingdom. Despite their widespread role, little is known about biogenesis of active Pep signal from inactive precursor proteins. Production of Peps requires proteolytic processing, and although Peps interact with extracellular receptors, PROPEP precursors have no traditional signal sequence for export out of the cell. Neither processing nor export of active signal is understood. I propose to begin characterization of the maturation pathway and transportation of Peps by first: examining localization and transport of the Peps in planta, and second: investigating the production and maturation process of Peps in cell. The proposed studies will utilize molecular biology and biochemical methods to delineate spatial and temporal production of active signal. Definition of molecular mechanisms regulating Pep activation of immune responses will aid in their application to enhancing disease resistance in crops.

Autism spectrum disorders (ASD) are severe neurodevelopmental disorders. Recent epidemiologic studies reveal that ASD affects 1 in every 110 children in the United States. The etiology of ASD remains unknown, and is believed to involve a complex interaction between genetic, neurobiological and environmental factors. Mounting evidence now supports a role for the immune system in autism. Studies suggest that atypical maternal immune responses in the prenatal stage may play a crucial role in the pathogenesis of ASD. Mothers of autistic children harbor anti-fetal brain antibodies; transfer of these antibodies to pregnant mice can induce ASD-like behaviors in offspring. Intriguingly, a significant subset of ASD patients have been reported to display ‘leaky gut’ syndrome, which involves permeability of the gut barrier and exposure of the immune system to the gut microbiota. As many bacterial antigens resemble human molecules, the immune response generated to microbial antigens may cross-react with self proteins (a process known as molecular mimicry). Molecular mimicry has been hypothesized to play a role in neuro-pathologies such as multiple sclerosis. I propose to use the maternal immune activation mouse model of autism to test the influence of gut microbiota in the etiology of autism and specifically examine the role of molecular mimicry in this disease. Furthermore, I will test the beneficial effects of a probiotic bacterium in ameliorating gut & brain pathology and ASD-like behaviors. Understanding how immune responses to gut bacteria promote disease in a preclinical model may result in transformative advances in the treatment of autism.

Schizophrenia is a developmental neurological disorder accompanied by strong psychological phenotypes such as hallucinations and depression. The origin of this disease likely lies in epigenetic factors combined with adverse environmental influences. However, the genes that might be responsible, and their regulation by epigenetic factors, have yet to be identified. Here, we will focus on the role of the epigenetic factor microRNA-137 (miR-137) in the regulation of two putative target genes (CACNA1C, CSMD1) and its impact on the morphology and function of neuronal connections during development and adulthood. MIR-137, CACNA1C, and CSMD1 have been identified in genome-wide association studies to contain single-nucleotide-polymorphisms (SNPs) that are significantly associated with a risk for schizophrenia. CACNA1C encodes the alpha 1 subunit of the L-type calcium channel (CaV1.2), which has previously been implicated in bipolar disorder. CSMD1 encodes the CUB and Sushi domain multiple domain 1 protein, which plays a role in the complement system and prevents host cells from undergoing apoptosis. Both these membrane-localized proteins appear to be crucial for neuronal development, function, and survival. We will examine the role of these proteins in development, and assess the consequences of schizophrenia-associated SNPs in or near these genes using induced neurons derived from human patient fibroblasts. The results of this study will give us an insight into how the potential misregulation of these factors, which may cause altered intracellular calcium concentration, cell apoptosis, and synapse modifications, contributes to schizophrenia phenotypes.

At daylight levels where visual perception is dominated by cone photoreceptors, signal propagation through the retina is largely limited by noise originating in cones. But, the impact of cone noise on ganglion cell sensitivity (retinal output) is relatively unknown. Noise inherent in the phototransduction cascade or synaptic noise resulting from statistical variations in neurotransmitter release from cone terminals has been proposed to limit cone vision. However, the relative contribution of cone noise to retinal output in comparison to noise introduced by downstream retinal neurons in the cone pathway is unresolved. My project will delineate these noise sources in the unique midget circuit of the central primate retina, which lacks convergence (one-to-one connection from cone to bipolar to ganglion cell), and thus allows precise measurements of noise at each anatomical stage of visual processing.

The fidelity with which cones encode light relies on efficient discrimination of changes in transmitter release from noise induced by the probabilistic nature of release. Although, this probabilistic, Poisson model of release is known for central synapses with low release probability, it has not been tested for specialized ribbon-type synapses present at photoreceptor and bipolar terminals. This study will directly measure the statistics of quantal responses from cone bipolar cells and from ganglion cells in the midget circuit to test whether they obey Poisson statistics or alternative models of release. This will further illustrate how these synapses function at low noise to account for the striking sensitivity of cone vision.

One of the fundamental unsolved questions in neuroscience is: How is the same cortical circuitry used for different behaviors? The neocortex is organized into functionally distinct areas. During behavior, these cortical areas exhibit changes in spiking activity and connectivity. My ultimate goal is to better understand the cellular and synaptic neurophysiological mechanisms that underlie different functional areas working in concert to drive behavior.

The model system I will use is the mouse visual cortex. Primary visual cortex is surrounded by many higher visual areas. Each of these areas has specialized functions, with some areas being more selective for form, and others motion. It remains unknown how these multiple visual pathways are regulated.

Here I will develop a new form of two photon microscopy with an unprecedented field of view that will enable imaging of two cortical areas simultaneously at single neuron resolution. This type of imaging has been lacking, and its development would transform our ability to explore neural circuitry at the systems level. This wide field of view two photon microscope will be used for calcium imaging and electrophysiology, in some cases combined with optogenetic methods for controlling neural activity. This system will enable me to activate and inhibit different components of neural circuitry with millisecond precision to determine the effect on neuronal activity and interactions between distinct cortical areas, all at unprecedented subcellular resolution.

It has long been appreciated that the sense of hearing is critically important to many animals, mediating behaviors as diverse as prey capture in bats, courtship in frogs, and territorial defense in birds. Over the past decade, there has been a growing interest in vocalizations produced by mice, spurred on in part by the recent explosion in molecular techniques available for studying neural circuits and behavior. Past work has demonstrated that newborn mouse pups emit vocalizations when they are separated from their nest, and that these pup calls attract the mother and result in her retrieval of the pup. Little is known, however, about how pup call is represented in the mouse auditory system, or how the detection of a pup call stimulus ultimately leads to the generation of a behavioral response. To gain insight into how the mouse auditory system represents information about pup calls, as well as how this representation may underlie behavior, we will perform two-photon calcium imaging and targeted patch clamp recordings from neurons in the auditory cortex of awake, head-fixed mice, as well as optogenetic manipulation of pup call-responsive neurons in freely behaving mother mice. We will use these techniques to answer questions such as: Is there a region of mouse auditory cortex that is preferentially activated by pup call? Do call-responsive neurons also respond to non-pup call stimuli? How do the receptive fields of these neurons arise? And which areas of auditory cortex are able to mediate this pup call-evoked behavior? Our results will provide an important step towards understanding how perceptions and behaviors arise from the activity of circuits in the brain.

The activation of leukocytes plays an important role in immunity to control pathogen invasion. For instance, in macrophages, Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and induce macrophage activation and functioning, whereas in T-cells, the T-cell receptor (TCR) recognizes foreign antigens and initiates T-cell activation. However, beyond TLR and TCR signaling, relatively little is known how macrophages or T-cells are activated. Recent studies have now indicated the involvement of G-protein signaling in this process.
In the proposed study, I will analyze how G-protein coupled receptor (GPCR) signaling is involved in the activation of leukocytes. Using macrophages and T-cells, I will analyze which G-protein pathways are stimulated upon leukocyte activation. After determining the involved pathway(s), I will molecularly dissect these pathways using specific inhibitors and reporter systems. Finally, I will screen for the involved GPCRs in non-leukocyte cells expressing the complement C3 receptor and different GPCRs.
Together, to our knowledge, this study aims for the first time to systematically analyze the involvement of GPCRs in leukocyte activation. Thereby, this work will not only advance our understanding of leukocyte functioning and activation, but may also lead to novel drug targets against infectious and autoimmune diseases.

Protein degradation has two basic functions: (i) to eliminate abnormal proteins and (ii) to limit the lifetime of specific proteins and thus make them sensitive to regulation that can rapidly alter their activity levels. The latter function is often utilized during development. While previous studies have shown that the ubiquitin-proteasome system (UPS) is required for proper development, little is known about mechanism that controls UPS in response to developmental cues. To understand how these machineries are assembled and recognize specific substrates during development, I will utilize C. elegans oocyte-to-embryo transition system, in which the degradation of meiosis specific protein MEI-1 occurs in response to oocyte maturation. E3 ubiquitin ligase (E3) and posttranslational modification of Cullin regulates MEI-1 degradation. Interestingly, some of these components have both nuclear localization and export signals (NLS and NES), suggesting that they shuttle between the nucleus and cytoplasm. Moreover, the subcellular localization of E3 and MEI-1 are distinct, suggesting that the compartmentalization of these components is developmentally regulated. To analyze E3 assembly and substrate recognition, I will visualize these complexes and modified Cullins using Proximity Ligation Assay (PLA) to obtain spatial and temporal information that is not obtainable with methods used in previous studies. Concurrently, I will analyze the effects of NLS and/or NES depleted subunits on E3 assembly and function.

Analyzing the molecular mechanism of timely regulated E3 assembly and target recognition will contribute to a broader understanding of the developmental control of UPS.

Mental illnesses have been associated with immune responses. One well-documented example of this is the anxiety and depression found in Systemic Lupus Erythematosus (SLE) patients, referred to as autoimmunity associated behavioral syndrome (AABS) in the MRL/lpr and NZB/W mouse models of SLE. Although AABS has been characterized in these mouse models, underlying molecular mechanisms remain unknown due to the burden of backcrossing genetic deficiencies, from the C57BL/6 (B6) strain, onto these complex strains. The study of AABS on a B6 derived model of SLE would enable efficient study of required factors. We propose to study AABS in sanroque mice, a T cell driven B6 derived model of SLE. We will use the sucrose test, novelty-suppressed feeding test, marble-burying test, open field test, elevated plus maze test, and rotarod test. We will track elevation of anti-nuclear antibodies and cytokines in the serum of mice as this correlates with induction of AABS. We will then use flow cytometry and histology to characterize in more detail the lymphocytes infiltrating the CNS. Work by others suggests we should expect to find T cells producing interferon gamma (IFNg). Subsequently, we will test if IFNg is required for AABS during SLE using blocking antibodies. Finally, with extensive backcrossing or the availability of sanroque mice as a model, we will test genetically deficient mice, and use them to make standard and mixed bone marrow chimeras, to determine if and how AABS requires IFNg. This work will advance the field toward determining the precise molecular mechanisms of AABS and toward targeted therapies for SLE patients.

X-ray crystallography is one of the most powerful methods to reveal the structure and mechanism of biomacromolecules. However, some molecules, in particular many RNAs and ribonucleoproteins have properties unsuitable for crystallization: multiple conformation, structural flexibility as well as vulnerability to thermal or chemical denaturation. Engineering them for higher stability can improve their chances to be crystallized, but the engineering process is time-consuming, because it requires iterative expression and analysis of individual clones. We propose a synthetic biological approach to build a quick screen for crystallizable mutants. In this approach, the genotype and phenotype of a variant will be linked by co-segregation of its gene, encoded enzyme, and reaction system in a water-in-oil emulsion. This method would allow quick and efficient selection of variants with desired, high stability phenotype, which would then be iteratively screened for crystallization using high-throughput methods.

We will apply this approach to targets which contain RNA and have proven largely resistant to crystallization, 1) the RNA polymerase ribozyme, and 2) a ribonucleoprotein, telomerase. An artificial RNA polymerase ribozyme has been engineered by synthetic biological approach and is thought as a modern equivalent to the central ribozyme in the RNA world. Structural information of this ribozyme would greatly help to engineer it further, and to understand its function in mechanistic detail. Telomerase is a key enzyme of genome maintenance. A crystal structure of full-length human telomerase would serve as a basis for functional understanding of telomere synthesis and drug discovery

In the mammalian brain, dopamine (DA) transmission in the prefrontal cortex (PFC) is known to play an essential role in mediating executive functions such as decision making and working memory. How the DA signal exerts its influence differentially on distinct executive functions in the PFC, however, remains to be understood. In this project, I will tackle this question by applying optogenetic perturbation methods to mice performing a delayed response task that engages executive functions in the PFC. Although numerous studies have shed light on DA’s role in executive function in the PFC, the precise mechanisms by which DA modulates these functions remains poorly understood. By exploiting the high cell-type specificity and temporal/spatial precision of optogenetic methods, I will be able to avoid the issues such as poor cell-type specificity and/or the decreased temporal and spatial control of perturbations encountered in previous studies. In this project, I will first exploit the mouce’s superb capacity for olfaction by using a delayed-response task with olfactory cues. Second, I will be able to control neurons in the PFC and the ventral tegmental area (VTA) using channelrhodopsin-2 and halorhodopsin. Third, I will record and analyze the neuronal responses in the PFC during specific periods of the task, thus enabling direct comparison of the effect of DA signals on neuronal activity and on animal behavior under specific cognitive demands. By integrating these strategies, I will elucidate the differential effects of dopaminergic modulation on prefrontal executive functions such as working memory and decision making.

The emergence of structure and function from the collective interactions of individual components such as biopolymers, cells and even whole organisms is an often seen but very poorly understood facet of the living world. We would like to use a bacterial system as a model to investigate collective behavior emerging in a predator-prey system. Such behavior gives rise to the rich socio-biology and evolution of co-operation in microbes. In particular, we want to study collective Myxobacterial behavior that manifests as a co-operative predation phenomenon of Myxococcus Xanthus, known as the predatory rippling motility, when they invade colonies of E. Coli. Using a combination of techniques such as microfluidics, genetic engineering and 3-D fluorescence microscopy, we would like to develop a picture of the events, at the level of individual cell interactions that lead to the large scale collective rippling motion of the Myxobacteria. The experiments would be performed with a view to isolate the purely physical mechanisms that lead to the collective behavior from those involving biochemical signaling. We will then use the knowledge of the individual interactions and apply physical and statistical mechanical theories on self-organization, to extract general principles that would be pertinent to a range of biological phenomena. We anticipate that such an approach will have the potential to generate a huge leap forward in our understanding, not just of M. xanthus predation, but also of how individual cells coordinate their activities to form large-scale structures in general, opening potentially new ways of thinking about biological organization.

Cancer progression often involves mutations in well-known oncogenes and tumor suppressors, but also depends on aberrations in other genes whose roles are only partially understood. These additional genes may play a major role in determining the unique features of specific cancer cells, and are thus of tremendous importance. In particular, chromatin factors have recently emerged as a specific class of recurrently-mutated genes in cancer, but their normal and aberrant functions are poorly understood. To decipher their role in cancer progression, I will study the effect of recurrent mutations in selected chromatin regulators on cancer gene expression and on genome-wide knock down effects. First, I will use available molecular and phenotypic profiles of a large set of cancer cell lines to develop computational models that predict which genes are essential for each cell-line. I will identify chromatin regulators with recurrent mutations and a predicted role in cancer and further explore their impact by deeply characterizing the epigenetic states of cell lines with and without such mutations. Finally, I will use computational modeling to generate specific hypotheses regarding the function of chromatin regulators, which will be further refined and tested by knocking down these regulators.

Among vertebrates, urodele salamanders show the highest potential for regenerative responses to injury-inflicted tissue loss. The neotenic salamander species Axolotl has proven particularly informative to study the process of limb regeneration after amputation, a multi-step process involving wound healing, blastema formation, re-growth and -patterning. Classical approaches such as grafting experiments have yielded key insights into these processes; yet the lack of a controllable genetic system has hampered their understanding at a more molecular level.
Here, I propose a novel combination of two different transgene delivery methods to circumvent these caveats. Their use will make the Axolotl limb regeneration system amenable to rapid and targeted interferences of gene function. Specifically, I will establish transgenic Axolotls expressing the avian retroviral receptor TVA in a tissue-specific manner, thereby allowing for viral-mediated mis-expression or knockdown of target genes. Delivery of TVA-expression constructs will be achieved through Tol2 transposon-mediated transgenesis. I plan to enhance the degree of transgene mosaicism in founder animals, by using recombinant transposase protein. This will by-pass the long generation time of the Axolotl system, (approx. 1 year to sexual maturity), and enable me to perform regeneration experiments straight in F0 animals.
As a proof of principle, I will apply the proposed transgenic toolkit to address the question of joint regeneration, as a model for the re-establishment of signaling centers and potential cellular re-specifications during vertebrate limb regeneration.

Metabolism-first theories on the origin of life state that there may have been prebiotic systems capable of propagation and Darwinian evolution that were not dependent on genetic, nucleotide-based replication. However, to date metabolism-first theories are not supported by experimental evidence. We propose to test this idea experimentally by observing the formose reaction compartmentalized in protocell analogues. Using high-throughput microfluidic techniques, we will react a simple formaldehyde feedstock in aqueous microdroplets, which can be split and fused to simulate protocell division and growth, respectively. We will identify the carbohydrates produced in the droplets, to determine if there is compositional heredity between generations of protocells. We will also investigate how the system responds to selection pressure: whether the sizes of the droplets or initial stochastic variations in their starting contents affect the nature and complexity of the carbohydrates produced. We hope to show that a network of chemical reactions is capable of heredity and evolvability, in the absence of canonical genetic macromolecules.

Over the last years much attention has been given to a new class of long non-coding transcripts that apparently does not code for proteins. While a few have been shown to be functional as RNAs, recent evidence from both genome-wide and targeted approaches suggests that a significant number of these transcripts harbor short open reading frames that are translated in vivo. Building on large-scale sequencing projects, the goal of my postdoctoral research is to discover and characterize novel secreted peptides that regulate embryonic development.

Aim 1 builds on preliminary research in the Schier lab that has identified a highly conserved, secreted short peptide expressed in a developmentally regulated manner in zebrafish embryos. Preliminary morpholino knockdown experiments suggest that this peptide is an important developmental regulator with potential roles in blood and/or mesoderm development. I will perform an in-depth characterization of the function of this particular peptide during embryogenesis.

In Aim 2 I will use polyribosome profiling, a new experimental technique for the genome-wide identification of translated regions, to discover novel short peptides expressed during embryogenesis. In addition to analyzing translated regions at different stages of embryogenesis in wild-type zebrafish embryos, I will identify peptides that are regulated by Nodal signaling, a pathway essential for mesendoderm induction. Combined with expression data from existing RNA-seq data and conservation analysis, I will phenotypically characterize promising candidates and identify novel signaling pathways that regulate development.

We have previously discovered that there is a characteristic chromatin profile for embryonic pluripotency and that this profile is established during the maternal-zygotic transition. While our studies have begun to clarify the in vivo relationship between chromatin state, the onset of zygotic transcription, and pluripotency, they have raised several important questions that are the focus of this proposal. First, what is the functional relationship between the appearance of specific histone modifications, the activation of the genome and embryonic pluripotency? To address this question I will interfere with histone modification marks in vivo, both genome-wide and gene-specifically, and analyze the effect on transcription and pluripotency. Second, how is the chromatin signature characteristic for pluripotency set up during development? Here, I will use a combined experimental and computational approach to predict whether sequence motifs and/or pre-existing histone modifications direct the recruitment of histone methyltransferases to their targets and test these predictions in vivo. Finally, it is unclear how developmental signaling pathways interact with chromatin to establish and maintain lineage-specific gene expression programs. I will analyze the chromatin profile of Nodal target genes during the transition from pluripotency to fate specification in the presence and absence of Nodal signaling in order to address this question. Together, my in vivo studies will clarify the role of chromatin modifications in the transitions between genome activation, pluripotency, and cell fate specification.

The immune system and the nervous system on the other have been considered autonomous systems operating independent of each other. However, a recent discovery described that neurons are able to attenuate the release of pro-inflammatory mediators from cells of the immune system, via the secretion of acetylcholine. This challenged the long maintained paradigm and established an important interaction between the immune and nervous system. While it has been shown that expression of the nicotinic acetylcholine receptor (nAChR) on macrophages is essential for reduced cytokine production, the contribution of this specific modulator on function and cytokine release of immune cells other than macrophages is unclear. Additionally, the contribution of this particular neuro-immune loop during the initiation, chronic or resolution phase of infections remains elusive. To reveal the relative contribution of acetylcholine as a specific immune modulator during infections I am going to investigate the effect of acetylcholine signaling on immune cells during different phases of skin infection. To address the relative contribution of T cells, innate lymphocytes and dendritic cells I will make use of mouse strains were deficiency of the nAChR is limited to those immune cells. Additionally, I am aiming to identify new factors of neuro-immune regulation by performing gene expression analysis on neurons from the dorsal root ganglia following skin infection. This approach aims to establish previously unrecognized neuro-immune interactions during various models of skin infection as a general mechanism of the fine-tuning of an ongoing immune response by the nervous system.

In the quest to understand biophysical principles that govern protein folding (i.e. the process through which polypeptide chains adopt their functional three dimensional structure), single molecule spectroscopy offers new insights into stochastic nature of structural or kinetic factors that drive this process, not detectable through conventional spectroscopic techniques that allow ensemble averaged measurements. To this end, I propose to use single molecule experiments to investigate the early stages of folding; where the nascent chains are just emerging from the ribosome, the machinery that produces proteins inside the cell through a process called translation. The goal of this proposal is to resolve the underlying heterogeneity in nascent protein conformations previously unaddressed in the myriad of ensemble approaches used to investigate co-translational folding. In particular, I will investigate the role of non-native competing protein-ribosome interactions. The methodologies developed and showcased here can easily be extended to studying other systems such as natively disordered proteins where stochastic properties of conformational heterogeneity has been postulate to contribute to promiscuity in function allowing natively disordered proteins to participate in various functions in side the cell. This proposal provides fundamental insights into the nature of forces that drive folding; especially at early stages where nascent chains face cellular milieu for the first time. I hope to be able to resolve this heterogeneity in vivo at single molecule level inside the cell to bridge the gap between what in vitro and in vivo studies of protein folding and function.

Cell membranes composed of thousands of lipid species, differing in their alkyl chains, headgroups and degree of saturation. Changes in lipid composition or even the absence of a single lipid have shown to lead to severe pathologies and death. The leading hypothesis which explains the role of lipids in membrane functionality is that the lipids segregate into distinct domains. These lipid domains can with high specificity incorporate or exclude proteins, hence inhibit or accelerate biological processes at the membrane surface. Knowledge of protein-domain interactions is essential to understand cell functionality, and yet today we cannot distinguish between the thousands of possible lipid domains, nor is there a method to determine with which domains a protein associates with.
This research would provide a new methodology, which will determine the lipid composition a protein has a high affinity to. The method utilizes a liposome library composed of ~1500 liposomes, each made of a different lipid composition. The library, which will be created by advanced microfluidics, has the largest and most diverse composition of lipid liposomes ever made. Protein-domain interactions will be measured on all liposomes in library. These results will be strengthened by in vivo assays, where the effect of manipulating the synthetic pathway of the lipids on protein localization will be monitored.
The results in this study may explain the diversity of lipid composition among different cells and cell organelles, and could have big implications on fields such as membrane targeted drugs, virus-membrane interactions, drug delivery, etc. They may also change our basic concepts of membrane function.