During neuronal differentiation, spherical progenitor cells develop into asymmetric neurons with one axon to send signals to, and multiple dendrites to receive signals from, other neurons. The actin cytoskeleton, in concert with the microtubule network, plays a crucial part in this process. However, our understanding of cytoskeletal regulation remains fragmentary.

The actin-related protein-2/3 (Arp2/3) complex is of key importance as the only known regulator of actin filament branching, enabling the formation of complex, highly connected actin networks. Previous studies provide conflicting evidence regarding the role of Arp2/3 in the nervous system and Arp2/3 function has yet to be investigated at all in a physiological context. The aim of this project is to dissect the precise role of Arp2/3 in neuronal differentiation. With a conditional Arp3 knockout mouse available for the model system, immunohistochemistry techniques will be used to investigate the effect of Arp2/3 complex ablation upon neuronal organisation and morphology. After characterising the neuronal phenotype of Arp3 knockout mice, the mechanisms of Arp2/3 action will be explored using primary neuronal cultures in combination with live-cell imaging techniques and electron microscopy. A clearer understanding of the role of Arp2/3 in neuronal differentiation will offer valuable new insights into the regulation of the actin cytoskeleton in general during vertebrate nervous system development.

A challenging issue for bacterial pathogens is the need to modulate host cells behavior to allow their own successful propagation. Intracellular pathogens secrete a large number of proteins (“effectors”) into their host cells to either trigger entry, escape cellular defense, modulate cellular responses or interact with intracellular components that are critical for infection. We hypothesize that pathogens also secrete small RNAs that interact with host cellular mRNAs and non-coding RNAs, thus modulating cellular activity for their own benefit. Discovery of such RNAs among the vast amount of cellular RNAs is highly challenging due to lack of proper techniques; however, the emergence of ultra-highthroughput sequencing and computational inference (Sorek’s expertise), in combination with single-cell fluorescence Imaging techniques (Palmer) and extensive expertise in pathogenesis and cell biology (Cossart) will allow us to address this challenge. Our aim is to identify secreted RNAs using the model intracellular pathogen Listeria monocytogenes, and understand their roles in pathogenicity. Although scientifically 'risky', if successful these studies could lead to a major breakthrough in the fields of bacterial pathogenicity and RNA biology.

Phosphorylation serves as a nearly universal regulation of protein function. However, it is paradoxical that the phosphorylation of a number of extracellular, secreted proteins has been reported while most of the known kinases are localized in the cytoplasm or nucleus. In an attempt to elucidate how extracellular phosphorylation occurs, our laboratory recently identified a novel family of atypical kinases that are mainly localized in the ER and Golgi lumen and are secreted. We found that one family member, Fam20C, is the original Golgi casein kinase that phosphorylates a number of secreted substrates and regulates biomineralization. However, little is known about the cellular function and substrates of other family members. In this proposal, I plan to study the function of another family member, Fam198B, which is highly conserved in vertebrates and the deficiency of which likely results in impaired glucose metabolism in mice. I will characterize the kinase activity, cell line- and tissue-distribution and membrane topology of Fam198B. I will also attempt to identify substrates of Fam198B using multiple approaches, including peptide array library screening, quantitative phosphoproteomics and bioinformatics. Finally, I will study the in vivo function of Fam198B using mouse models, focusing on the potential metabolic dysregulation and phenotypes related to potential Fam198B substrates. I believe my study on Fam198B will greatly advance the understanding of this secreted kinase family and extracellular phosphorylation in general.

Neuronal differentiation is a fundamental process for the formation of neuronal circuits within the developing brain. In neurons the microtubule cytoskeleton plays an essential role during the different phases of neuronal differentiation. In most animal cells, the centrosome is the major microtubule organizing center (MTOC) coordinating cell division, motility and polarity. In neurons the role of the centrosome as a MTOC is highly debated, particularly during later stages of differentiation when the centrosome is thought to become inactivated. Thus, it remains unclear what the contribution is of centrosomal vs acentrosomal microtubule nucleation during neuronal development. The proposed project is aimed to investigate the role of centrosome inactivation in neuronal polarization and differentiation. Using a combination of different biochemical, molecular and cellular biology techniques and cutting-edge live imaging microscopy in hippocampal neurons we will address the role of the centrosomal and acentrosomal nucleation during neuronal development. In this application we propose the following objectives: 1) Determine how and when the centrosome becomes inactivated in hippocampal neurons, 2) Address the consequences of increasing centrosome number and changing their localization in neuron polarization and 3) Investigate whether non-centrosomal microtubule nucleation occurs at the Golgi and how it may be related to neuron polarization.

While chromosomes fill the nuclear space as decondensed chromosome territories during interphase, they are dramatically re-structured into compact rod-shaped rigid bodies during mitosis. The aim of my work is to elucidate the molecular mechanisms underlying this cell cycle-dependent chromosome reorganization. Unlike previous studies, I plan to focus on a yet poorly characterized chromosomal domain defined as ‘chromosome periphery’. I first plan to establish a detailed map of the proteins that target to the chromosome periphery in human cells, applying both high-resolution three-dimensional microscopy and superresolution fluorescence imaging techniques as well as molecular biology techniques, including chromatin immunoprecipitation combined with next generation sequencing (ChIP-seq) analysis. The combination of these approaches will provide me with information on different scales, first at the cell biological level and second at the DNA sequence level, to obtain a complete picture of the localization of the proteins that may organize the chromosome periphery. I will then investigate the functional relevance of chromosome periphery components in mitotic chromosome morphogenesis by RNAi screening in tissue culture cells. I expect that this work will provide insight into how a protein network sets up a chromosomal domain and how such a domain can contribute to the formation of mitotic chromosomes.

Clathrin-mediated endocytosis (CME) leads to the internalization of receptor-ligand complexes and nutrients. In a multistep pathway, modules of proteins are recruited to the plasma membrane and begin to form clathrin-coated vesicles (CCV). This process is initiated by proteins that bind to phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). The role of PI(4,5)P2 during CME has been studied extensively. However the roles of other lipids such as phosphatidylethanolamine (PE) or sphingolipids (SL) have not been investigated at similar depth. However, indirect evidence supports an endocytic role for these lipids. Impairing SL synthesis slows transferrin internalization. Furthermore, PE enhances endocytosis in living cells and clathrin coated pit assembly on liposomes. Here, I propose to dissect the role of these lipids in CME. I will first identify the lipid requirements for CME by comparing the lipid composition of the plasma membrane to that of CCVs by mass spectrometry. I hypothesize that PE will be enriched in the CCV fraction whereas SL will be absent. This approach will be useful to decipher the role of the lipids in CCV formation. Based on the information provided by these experiments, I propose to develop an in vitro assay in collaboration with Pr. Fletcher at Berkeley to elucidate lipid requirements for CCV formation. This assay will also be used to investigate the consequence of lipid localization in the inner vs. outer leaflet on CCP formation. I propose also to test the biological relevance of these results in vivo in genome-edited cells lines developed in the host lab. The proposed research promises to make important contributions to understanding CME.

Believe it or not, the white shark on “Shark Week” is at a disadvantage: whereas your bones house a microscopic universe of cells, working in concert to fix microscopic wear and tear we cause every day, the cartilage of shark skeletons can be added to, but not repaired. Think of it this way: with bone, you upgrade components just as they go out of style or fail, but with cartilage you’re stuck working with the same old model you started with (explaining why my knees hurt). But sharks and other cartilaginous fishes use a curious work-around: although their entire skeleton is cartilage —not unlike what you’d find on spareribs at the dinner table— it’s reinforced with a thin, outer shell. The shell itself is striking under a microscope: thousands of geometric blocks called ‘tesserae’ made of the same mineral as in our bones and fitting together like floor tiles to wrap the entire skeleton. Although tesserae were first observed more than 100 years ago, we still have no idea what they are actually doing as these spectacular fish swim, bite, and glower on our TV screens. We’re curious about the mechanical role tesserae play in skeletal performance. To get a true feel for how tesserae manage and distribute forces, we plan to make our own shark bits: imitation skeletons scaled up for easier mechanical testing. Using high-resolution engineering and materials science tools, we will characterize geometries and tissue properties that define tesserae of a model species, and then use those features and a state-of-the-art 3D, multi-material printer —typically for industrial applications and capable of printing physical models with rigid and flexible parts— to manufacture tesseral mats that can be pushed, pulled, and fractured in ways that echo biological conditions. Like any design process, when the model doesn’t work, we’ll return to the source (the fish) for a deeper understanding of the template; once the parameters are clear, we can apply our models to the wide range of tesserae shapes and sizes in different species with different ecologies. This will shine a light on the functional role of this biological tiling, but also point to generalizable features useful for manmade tiled composites. Given that sharks live long lives and have existed for millions of years with irreparable, tiled skeletons, we have much to learn: why replace the old model if it never breaks?

My research has three complementary aims:

Aim 1: Develop a high-resolution temporal map of transcription factor binding in pluripotent precursor population (the mesoderm) and three of its derived tissues.

I will use isolate 2-5 million mesodermal nuclei at 5 equally spaced time points starting prior to mesodermal invagination but before mesodermal tissue differentiation. I will isolate tissue specific nuclei from three mesoderm-derived tissues (visceral muscle, heart muscle, and somatic muscle) after these cells are specified, at 8 time points after these tissues are specified. Three replicates representing different F1 crosses between inbred lines isolated from a natural population will represent biological replicates in this aim and form the basis for Aim 3. I will measure genome-wide DNase sensitivity in each chromatin sample using DNaseI-seq and infer transcription factor binding using DNase footprinting analysis.

Aim 2: Identify natural variation that impacts transcription factor binding at 2 timepoints with association mapping.

I will isolate mesodermal chromatin samples at T1 and T13 from 50 independent fly lines from a natural population and perform DNase-seq. Analysis of these data will allow us to identify cis-associated variants that modify DNase sensitivity and TF binding using previously developed QTL mapping approaches.

Aim 3: Track the effects of functional DNA variants identified in Aim 2 over the entire developmental time course with allele-specific analysis.

I will characterize the effect of individual SNPs that change TF binding over the full developmental time course using allele-specific analysis of the DNaseI data collected in Aim I. For each dsQTL identified in Aim 2, I will measure allele specific DNase sensitivity in the cis-region surrounding the dsQTL at each of the 13 time points. I will then characterize the effects of cis-regulatory variants on regional TF binding over time.

While inflammation has generally been seen as a consequence of microbial presence in wounds, it can also occur in sterile conditions, in which case it is usually triggered by ligands released by dying cells. While these two conditions call for different responses – microbial clearance or tissue repair – it remains unclear if and how organisms discriminate between these two processes. Neutrophils are usually among the first immune cells recruited during inflammation, either microbial or sterile. Given their rapidity of action and short lifespan, few studies have focused on the regulation of neutrophil function. Yet, while neutrophils are critical for microbe elimination and produce pro-inflammatory TNFa, they also participate in inflammation resolution and produce anti-inflammatory IL-10, so that regulation of these roles is likely to affect the course of inflammation. We here report that neutrophils elicited by microbial products rapidly upregulate TNFa when restimulated. Strikingly, endogenous ligands released during sterile inflammation, like necrotic cells or uric acid, can efficiently recruit neutrophils, but do not enable TNFa production. We thus hypothesize that neutrophil activation can be altered by recruitment into distinct microenvironments. In this proposal, we will solidify the nature of this priming phenomenon and test how neutrophils interact with the microenvironment of the inflammatory site. The regulation of neutrophil functions may enable the early distinction of sterile and microbial inflammation and allow the immune system to efficiently eliminate microbes while limiting unnecessary inflammation in the absence of pathogens.

It is by now well accepted that gene expression is not only controlled through the interplay of information stored in the DNA sequence, chromatin modifications and the binding of regulatory proteins such as transcription factors, but also through the spatial organization of the genome within the cell nucleus. Indeed, not only cell-type specific transcriptional programs but also cell-to-cell variability in gene expression (i.e. transcriptional noise) seems to be mediated by differential genome topology, probably because it either permits or prevents regulatory sequences (such as enhancers) to find their target regions (such as promoters). In this project, I will investigate how such chromatin contacts are established, which molecular players regulate these interactions, whether some or all of the chromatin contacts can be remembered through cell division and how the subtle differences in genome organization cause variegated gene expression among otherwise identical cells. This work will therefore further elucidate the impact of genome topology on gene expression and will provide mechanistic insight into a novel concept of biological variability and versatility at the single-cell level.

Our knowledge of the world is organized in concepts. Bert and Ernie, for example, are two person-concepts, well known to “Sesame Street” viewers. The ease with which we recognize Ernie and distinguish him from Bert belies the daunting complexity of the computational tasks at hand. This is, first, because the retinal activity patterns generated by an object as complex as Ernie is ever changing. Thus representations need to be computed that lump all the different instantiations of Ernie patterns in the retina, into an activity pattern in the brain that is stable across these different instantiations, yet selective enough to tell one person from another. Second, repeated encounters of the same object need to be grouped together over time and with other information -e.g. the name and the voice- to form a modality-free representation: the concept of “Ernie”. Understanding how the brain achieves these remarkable feats is the goal of this project. Our starting point is the finding of ‘concept cells’ by team member Rodrigo Quian Quiroga, which respond almost like a switch to any representation of one individual (e.g. different pictures, the name), but not to other stimuli. Our central hypothesis is that conceptual knowledge rests on explicit concept cell representations: to understand the neurobiology of knowledge, we need to understand concept cells. Conceptual representation is in fact crucial for perception and memory. We bring in expertise on concept cells in humans (through team member Rodrigo Quian Quiroga), the processing of sensory information in rats (team members Davide Zoccolan and Mathew E. Diamond), face recognition in macaque monkeys (team member Winrich Freiwald), and on theoretical neuroscience (team member Haim Sompolinsky) to tackle the problem of concept representations from different angles. Our goals are to find, localize and understand the functional properties of concept cells in three different species with very different brain sizes; to understand, mechanistically, how concept representations are built from sensory inputs; and to determine how conceptual knowledge might be acquired over time. Exploiting unique advantages of our interdisciplinary approach and the complementary use of three biological systems, our project will be the first concise effort to develop a neuroscience of knowledge.

Many animals can detect and use the Earth’s magnetic field for orientation and navigation functions (Lohmann, 2010; Mouritsen and Ritz, 2005). However, the receptor for magnetoreception in vertebrates remains unknown. Members of our team (Dickman Team) have recently discovered cells in the vestibular brainstem of pigeons that encode the direction, intensity, and polarity of the magnetic field (Wu and Dickman, 2012), as well as a magnetic sense neural pathway that includes regions in the brain known to be involved with spatial orientation and navigation tasks (Wu and Dickman, 2011). In addition, our team has obtained preliminary evidence for biogenic magnetic particles in specialized cellular regions of an inner ear receptor (the lagena) that could function as a magnetoreceptor transduction mechanism. Our preliminary data has suggested that these magnetic particles are made of hematite and maghemite, ferromagnetic compounds with permanent magnet properties. Thus, our primary hypothesis is that the lagena contains cells that perform as a magnetoreceptor. Afferent fibers that innervate those receptor cells are hypothesized to signal magnetic information to the brain. Other team members (Winklhofer Team) have recently developed a magnetic microscope that can identify and isolate cells that contain magnetic particles, then extract these cells for subsequent cellular/molecular and biophysical analyses. In fact, they have identified magnetic cells in the nasal epithelium of fish and performed elemental analyses on the magnetic particles contained within those cells. The primary goal of the proposed project is to determine the nature of the lagena receptor and the neural response properties of primary afferent fibers innervating the receptor cells. The project represents a new collaboration between J. David Dickman (Baylor College of Medicine, USA), a systems neurobiologist and Michael Winklhofer (Munich University, Germany) a biophysicist specializing in biomagnetics. Together, the two research teams will focus on two objectives. The first is to delineate the magnetic receptor mechanism in the avian lagena using state-of-the-art methodologies to isolate and characterize the magnetic particles and their cellular location within the lagena. The second is to characterize the response properties of neural afferents innervating the receptor by using both magnetic and motion stimulation. The findings from these objectives will then be used to develop a novel working model for a new magnetic sense.

A wide variety of post-transcriptional modifications, with over a hundred known so far, decorate RNA molecules from all domains of life. N6-methyladenosine (m6A) is the most prevalent internal mRNA modification. It is essential to cell survival and organismal development, strongly impacting gene expression through unknown mechanisms. Recent mapping of the m6A landscape in human and mouse revealed over 12,000 m6A sites characterized by a typical consensus in transcripts of over 7,000 genes. Remarkably, sites preferentially cluster in three distinct landmarks of telling biological significance: at transcription start sites, around stop codons and within long internal exons - are dynamic, and are highly conserved between human and mouse. The primary aim of this proposal is to elucidate the modes of action of m6A in regulation of gene expression and shed light on this novel post-transcriptional regulatory layer. I will address the major challenges facing this evolving field, employing three chemical biology oriented approaches: 1) Profiling, by generating quantitative, single-nucleotide resolution methylation maps of systems under varying physiological conditions, aimed at correlating the dynamic methylome with observed phenotypes; 2) hypothesis-driven, aimed at probing one of most intriguing, innovative and well substantiated questions that arise from the current state of knowledge, namely the influence on protein translation; 3) open-ended, aimed at identifying interacting cellular machineries, acknowledging that what we know is only a tip of the iceberg of cross-talk between methylation and other regulatory layers.

Communication between cells is vital to the development and homeostasis of multicellular organisms. In vivo, signaling pathways function not in isolation but in combinations and are influenced by the specific cell type’s basal gene expression. A major gap in the field of cell signaling is to elucidate how pathways are integrated in real time. In particular, in most cases when two pathways have been implicated in a process, it is not clear whether they function sequentially, (eg. one pathway activating expression of another’s ligand), or synchronously (eg. regulating a common target). I propose to use Drosophila intestinal stem cells (ISCs), whose behaviors are regulated by multiple signaling pathways including Notch, Wingless, EGFR and Insulin, as a model system to dissect signaling pathway interactions. While some interactions between pathways have been identified in ISCs the full extent and nature of crosstalk is unclear. I will first compare the changes in gene expression following single or combinatorial pathway activation in vitro to characterize concomitant and sequential signaling crosstalk. Then, using target genes that respond to multiple pathways concomitantly, I will examine whether signal integration occurs by independent transcriptional responses or by convergence of upstream signals to regulate a transcription factor’s activity. Finally, I will use this information to develop a screen for regulators of crosstalk in cultured cells and validate the findings in vivo in ISCs. Together, these studies will clarify how multiple pathways are coordinated in the maintenance of stem cell homeostasis and provide mechanistic insights into crosstalk between pathways.

Signal transduction pathways enable cells to process information essential to their survival and proliferation, yet the genesis of these pathways remains poorly understood. These pathways are typically assembled using components from a small number of protein families that are organized in different ways to process diverse extra- and intracellular stimuli. We aim to investigate the evolutionary mechanisms that mold the interactions between pathway components following gene duplication or lateral gene transfer, leading to changes in the pathway structure and the emergence of novel signaling functions.
We specifically focus on the evolution of the two-component signaling pathway that drives sporulation initiation in Firmicutes. Sporulation is an ideal model for investigating signaling evolution: the pathways in Bacilli and Clostridiales arose from a common ancestral pathway, have similar components and engender analogous phenotypes, yet are characterized by different network architectures: a two-component system in Clostridiales and a complex phosphorelay in Bacilli.
We propose to reconstruct the evolutionary steps leading to the divergence of these pathways at the level of the protein, domain, interacting interface and active site. We will employ a multidisciplinary approach, combining computational phylogenetics and evolutionary systems biology with genetic manipulations and experimental assays.
A network evolution model will be developed to connect phylogenetic data on sequence evolution directly with the probabilities of gain and loss of interaction. This will allow us to reconstruct the most likely states of the sporulation pathway in key ancestral species and hence to predict network rewiring events: changes in protein dimerization and phosphotransfer specificities. The evolutionary histories produced will allow us to understand how the rewiring of modular proteins supports functional innovation at the network level, for example through the insulation of different signals by the elimination of crosstalk between related signal kinases.
The inferred interaction specificities will be tested in vitro using FRET and phosphotransfer assays. Using ancestral sequence reconstruction, we will probe the viability of the inferred ancestral pathways and the likely sequence of evolutionary transitions leading from one pathway structure to another.
We anticipate that our integrated approach will serve as a blueprint for probing the evolution of other signaling systems.

The vast majority (>95%) of human proteins is thought to be affected by alternative splicing. In fact, alternative splicing is one of the mechanisms thought to be underlying the increased organismal complexity of higher mammals. Without a doubt, the combinatorial complexity enabled by alternative splicing is mediated by changes on the protein level. However, only few studies thus far have addressed the consequences of alternative splicing on protein function, e.g. in the context of interaction networks. Particularly, large-scale interactome network mapping so far ignores the increased complexity and dynamic modulation of network connectivity by differential splicing. This project aims to fill this gap and identify protein interactions that are regulated by tissue-specific alternative splicing. We will use a bioinformatic modeling approach and analysis of deep-sequencing transcriptional profiling data to identify physiologically relevant splice isoforms that are likely to affect physical protein-protein interactions. Subsequently, we will experimentally identify differential interaction partners of the identified proteins and their isoforms using high-quality high-throughput interactome mapping by screening the different isoforms for interactions against a genome-wide set of ORFs and subsequent systematic verification. In the third part of the project we aim to genetically demonstrate the biological role of experimentally validated differentially-interacting-splice-isoforms using phenotypic screens and biological follow-up studies for selected high-confidence candidates. All information will be integrated with existing network maps and other biological information.

Light is not only important as an energy source for photosynthesis, but also a critical environmental signal for regulating plant growth and development. Plants have sensory photoreceptors that monitor different parameters of the light environment, including wavelength, intensity, direction and duration, and help plants to adapt their growth pattern to diverse and ever-changing environmental conditions. The photoreceptors and downstream signaling components in lower and higher plants, e.g. in the moss Physcomitrella patens and the seed plant Arabidopsis thaliana, are very similar. However, they respond to their surrounding light environment differently to meet their needs. The long-term goal of our project is to understand how species-specific response patterns to light evolved. In Physcomitrella and Arabidopsis, the phytochrome (PHY) family of photoreceptors, the PHY INTERACTING FACTORS (PIFs) and the nuclear transport protein FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) are critical components of light perception networks that promote photomorphogenesis in response to red/far-red light. Although the topologies of light perception networks, i.e. components (e.g. PHYs and PIFs) and wiring (e.g. PHY-PIF interactions), in higher and lower plants appear to be conserved, their response profiles to light are different. One possibility is that the evolution of species-specific light perception networks may have relied in part on changes in the network topology, which in many cases have been shown to be important for network evolution. Alternatively or in addition, the evolution of species-specific light response modes may have relied on modifications of network parameters, such as RNA/protein synthesis/degradation rates and complex association/dissociation constants. By systematic examination of the light perception networks in Arabidopsis and Physcomitrella using biochemical, genetic and systems biology approaches, we will build mathematical models that will help to distinguish between these possibilities. Using a reiterative method of model based guidance of experimental design and mathematical modeling, these models will identify key steps in the evolution of species-specific light perception networks in terrestrial plants.

Synaptic communication relies on neurotransmitter release from a specialized compartment, the synaptic vesicle (SV), followed by formation of new vesicles from the plasma membrane, in what is termed SV recycling. This highly regulated process needs to be homeostatically optimized throughout the organism’s life and implies the continual biogenesis of new proteins and organelles, coupled with the degradation of non-functional components.

Biochemical experiments were able to demonstrate that in culture several abundant vesicle components were degraded rapidly, with a half-life of less than 24 hours (Daly and Ziff, 1997). Surprisingly, although protein aging and degradation are necessary components of the life cycle of any cellular specialization, including the synapse, this finding has received little attention.

How fast are different proteins/vesicles replaced? Are SVs replaced as whole units, or are proteins first segregated and degraded separately? Is the degradation of vesicles related to their inclusion in different functional pools (readily releasable, reserve)? How are soluble (non-vesicular) proteins degraded? Is there a role for endosomes in degradation? Does the accumulation of non-functional synaptic vesicles correlate with aging and/or neurodegenerative diseases? Answering these questions will be essential to increase our basic understanding of synaptic function and will help to test novel hypothesis about neurological disease mechanisms.

I will employ here a host of advanced biochemical and imaging methods, including super-resolution microscopy and novel fluorescent protein-based assays, to address the spatio-temporal regulation of pre-synaptic boutons. I will study the turnover of SVs and important non-vesicular presynaptic proteins, in relation to their function. Both temporal and spatial aspects will be analyzed, in an attempt to understand the dynamic nanoscale organization of SV turnover.

The development of the central nervous system (CNS) is one of the most spectacular processes in biology. Key aspects include the formation of neuronal axons, their subsequent growth and guidance through thick layers of nerve tissue, and the folding of the brain. All these processes involve motion and must thus be driven by forces. However, while our understanding of the biochemical and molecular control of these processes is increasing rapidly, the contribution of the dynamic interplay between cellular forces and tissue elasticity remains poorly understood – mostly due to a lack of suitable measurement techniques. In this highly interdisciplinary project, we will integrate international expertise in optics, biophysics and neurobiology to investigate the role of mechanics in CNS development. We will develop a novel photonic toolbox for in situ, label-free and non-contact tracking of mechanical force and elasticity at cellular and intra-cellular levels with microscopic spatial and real-time temporal resolution. Force sensing will be based on a radically new approach that will enable the continued measurement of cellular forces down to the pN range without the need for detaching cells from their substrate as required in traction force microscopy. To measure the elasticity of cells and extra-cellular matrix material with 3D microscopic resolution, we will develop Brillouin-scattering based ElAsticity Mapping (BEAM). Unlike standard methods for cellular level elasticity measurements (eg atomic force microscopy), which are usually either invasive or limited to surfaces, BEAM will provide a non-contact, non-perturbative probe. These innovative experimental techniques will be applied to illuminate how forces exerted by neurons contribute to axon formation and neuronal guidance. Ultimately, we will investigate the involvement of mechanics in brain morphogenesis. Our approach will allow to look at these crucial biological processes from a new angle and shed new light on CNS development. Beyond this, our novel platform will be broadly applicable to areas far beyond the boundaries of neurobiology, as it provides unique cell-microenvironment metrics to relate mechanical interactions to cell behavior.

It has long been assumed that because early mammalian development relies on almost immediate zygotic transcription, the requirement for translational control during embryogenesis and pattern formation may not be very significant. Our findings have, however, uncovered a surprising role for ribosome-mediated specificity in tissue patterning and gene expression. In particular, our studies show that expression of Homeobox (Hox) mRNAs is tightly regulated at the translation level to promote restricted patterns of protein expression within the developing vertebrate embryo. This strongly suggests that tight regulation in translational control of transcripts, largely at the level of 5’UTR activity, may provide a critical additional layer of regulation to the mammalian genome. At present, there is a strong bias in examining patterns of gene expression within the developing vertebrate embryo at the transcript level and we lack an even basic understanding of which transcripts are being translated into functional proteins. In this proposal, I will apply a new powerful technology that I have optimized for small embryos known as ribosome profiling to delineate the translational landscape of gene expression in mesoderm, one of the most important cell lineages specified during vertebrate development. This will provide unprecedented insight into the classes of mRNAs and the topography of regulatory elements positioned in their 5’UTRs that are regulated at the translation level. I will extend these studies into chick embryos where I will be able to subsequently functionally characterize regulatory elements that impart transcript-specific translation control.