Understanding how neuronal circuits are formed during the development of the nervous system has been a major challenge for neuroscientists. The role of genetic programs in this process is increasingly well understood. However, our understanding of the way in which electrical activity affects the structure and function of developing circuits is quite limited.
This research will develop a coherent understanding of where electrical activity is important, when it is important, and what it is important for, using a simple three-neuron microcircuit that develops within a 10 hr period. The long-term goal of the project is to identify the role(s) of electrical activity in the assembly of microcircuits during the development of the nervous system. For this purpose we will use state-of-the-art techniques in genetics, molecular biology, imaging and neurophysiology: we will use a recently developed Gal4/UAS system in a vertebrate embryo to track the development of specific neurons and manipulate their activity selectively.

Cell division and migration are frequently recognized as isolated processes but their interdepence has been largely neglected in developmental biology. The roundworm C. elegans provides a unique opportunity to study the crosstalk of both processes during embryogenesis. The complete lineage and the divisions of all cells can be followed and the exact time of each cell division is known. Together with a recently established automated microscopy system for lineage tracing and the ease of genetic manipulation, a quantitative analysis of cell cycle timing at a level that exceeds the possibilities in any other model system can be achieved. This project will therefore exploit the invariant developmental timing of C. elegans to provide insight into how cell cycle regulation is linked to migration.

Brains evolution is the consequence of many years of selective pressure within specific environments, enriching genotypes and neural phenotypes that result in adaptive behaviors. The driving mechanisms of this critical process are largely unexplored. My long-term goal is to discover how environmental and biological mechanisms direct neural network changes, accomplishing behavioral adaptation. The objective of this proposal is to examine these adaptive changes in robotic and Drosophila olfactory networks during experimental evolution in controlled environments. It has been hypothesized that 1) the convergence of early olfactory networks across species towards discrete, glomerular topologies reflects an adaptive response to common features of olfactory environments and 2) that the divergence of odorant receptor repertoires and glomerular identity across species indicates pliability and natural diversity of olfactory networks within species. These hypotheses will be tested in three specific aims: 1) measure the impact of odorant spatial covariance on the topology of emergent robotic olfactory networks, 2) test the function of novel connections in the Drosophila olfactory system, and 3) identify natural biological strategies that modify the Drosophila olfactory system to enhance innate chemosensory behaviors. This approach innovates by leveraging the distinct advantages of robotics and Drosophila and exploring the dynamics of emergent and pre-existing olfactory networks, respectively. I expect to reveal the mechanistic principles underlying olfactory network evolution, an important step in understanding why brains are the way they are and how they can change.

Cells can respond strongly and specifically to mechanical cues in their environment, including the viscoelastic properties of the extracellular matrix. Remarkably, the lineage distributions of adult neural stem cells (NSCs) can be directed in vitro by tuning the elasticity (stiffness) of their environment.

We propose to develop a phase diagram of stem cell mechanoregulation in three steps: First, we will identify the viscoelastic parameters of the extracellular matrix substrate cells sense. Second, we will determine which parts of the signal transduction machinery must be present in an NSC for it to sense its mechanical environment. To this end, we will make use of lentiviral vectors to precisely modulate expression of targets in the mechanosening pathway. This will allow us to generate a “phase diagram” of cell response to stiffness of the material, in dependence of signal transduction machinery. Third, we will use lentiviral expression systems to address the question of whether the expression of cytoskeletal proteins characteristic for one lineage is simply correlated with differentiation, or whether it is a cause of differentiation. This is likely to have particularly high impact on the field, as the expression of these cytoskeletal proteins is frequently used as “gold standard” marker of cell fate.

The proposed project requires expertise in materials science, mechanics, cell biophysics, and stem cell biology. We propose to use the support of the HFSP fellowship to combine my expertise in the former two areas with new training in the latter two to tackle the problem of neural stem cell mechanoregulation.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder next to Alzheimer’s disease. A major pathological hallmark of PD is the presence of cytoplasmic inclusions called Lewy bodies in the substantia nigra. Recently, several mutations in the leucine-rich repeat kinase, LRRK2, were found to be associated with autosomal dominantly inherited, late-onset PD. Mutations in LRRK2 is thought to cause disease by a toxic gain-of-function. LRRK2 is a multi-domain protein with dual enzymatic activity. LRRK2 has been shown to accumulate along with a-synuclein, the major constituent of Lewy bodies. The exact function of wild-type LRRK2 is however unclear. C. elegans is being increasingly used as a model organism to study human neurodegeneration. I will aim to develop and characterise a transgenic C. elegans model expressing either wild-type human LRRK2 or the most common PD mutation G2019S LRRK2. I will also attempt to study the role of C. elegans LRRK2 homolog, LRK1 on autophagy and whether autophagy contributes to LRRK2 accumulation and disease.

This HFSP short-term fellowship will be an excellent opportunity to establish in a new but related area of research and get some pilot data to apply for career development fellowships in the UK. Working at the forefront in the areas of autophagy and protein conformation diseases together with excellent support from my current supervisor, I strongly believe that this short-term fellowship would immensely help in this crucial stage of my career. This will be a stepping-stone towards establishing an independent and successful group in the coming years.

Hypochlorous acid (HOCl), the active ingredient of household bleach, is used as powerful antimicrobial strategy by the mammalian host defense. HOCl apparently kills bacteria by causing oxidative unfolding and aggregation of essential bacterial proteins. As defense mechanism, bacteria employ the redox-regulated chaperone Hsp33. Hsp33 uses the oxidative unfolding activity of HOCl to convert from a fully folded yet inactive protein to a partially unfolded, highly active chaperone, which protects cellular proteins against stress-induced unfolding and bacteria against HOCl-stress. In my postdoctoral research, I will H/D exchange in combination with mass spectrometry to visualize the structural dynamics of Hsp33 activation and inactivation process. These studies will provide me with a detailed mechanistic picture about how protein conformation regulates substrate binding and release in ATP-independent chaperones. Based on these results and the analysis of Hsp33’s substrate binding specificity using high affinity peptides, I will conduct computational modeling of Hsp33-substrate interactions. These studies will guide mutational studies, which will provide insights into the general mechanism of chaperone-substrate interactions and the central role Hsp33 plays in protecting proteins against HOCl-stress. Finally, I will exploit the modular character of Hsp33, which makes this protein uniquely suited for a directed evolution approach to generate oxidant-specific redox modules. These experiments have the clear potential to generate sensor proteins that detect specific oxidants in living cells, and to elucidate how distinct oxidative stress sensing mechanisms evolved in proteins.

Circadian clocks enable organisms to anticipate daily recurring physiological and environmental changes by modulating a plethora of body functions according to the time of day. In peripheral organs of mammals, metabolism and the circadian clock are strongly coupled, and a severe disruption of circadian rhythmicity can evoke diseases such as metabolic syndrome. It is therefore of high interest to unveil the mechanisms that connect metabolic and circadian regulation on the molecular level. Here I propose to study a novel regulator I identified in the host lab of my HFSP fellowship as a paradigm for metabolic sensor proteins that regulate the circadian clock. A number of projects will be initiated in order to analyze the function of this regulator, heat shock factor 1 (HSF1), in the circadian network. Specifically, I want to address the question of how HSF1 is mechanistically linked to the circadian clock by employing various hypothesis-driven and unbiased approaches. Furthermore, I want to identify the cues that regulate the activity of HSF1 under physiological conditions in order to better understand the metabolic parameters the body has to adjust to under normal and disease conditions. Finally, in a collaborative effort an attempt will be made to identify novel metabolic regulators of the circadian clock in a large scale loss-of-function screen. The CDA would help me to make a successful transition to an independent group leader by giving me the financial means to set up a competitive chronobiology lab. This would greatly help me to pursue my long term goal of creating a lab with a research focus at the interface between metabolic and circadian regulatory networks.

The goal of this project is to develop a computational system for displaying gene regulatory network information in an evolutionary context. It is envisaged that the preliminary version of the new system, at the end of the short research stay funded by this grant, will provide the relational schema to hold and query gene regulatory information and putative orthologues of every gene in the network. This will be presented in a text-based form in the PlnTFDB (http://plntfdb.bio.uni-potsdam.de/), GabiPD (http://www.gabipd.org/) and GRASSIUS databases, and will provide the basis for graphical display using e.g., Cytoscape (YEUNG et al., 2008). The understanding of the the evolution of gene regulatory networks (GRN) and their conservation patterns is being hindered by the lack of a computational system that displays such networks in an evolutionary context. A system encompassing a broad phylogenetic range of plant species, as the proposed in this application, will provide insights into the evolution and control of GRNs in the plant lineage.

Regeneration of tissues and organs is an enigmatic field in biology, with fundamental unsolved questions. Humans may regenerate finger and toe tips in a process which involves the integrated regeneration of many tissues, including nail matrix, nail bed, finger pulp, sensory organs, dermis and epidermis, all of which reform to a normal cosmetic and functional state. Mouse (newborn & adult) exhibit similar inherent capacities and may regenerate digit tips of a forelimb and hind limb within 4-5 weeks providing an experimental animal model on which to study this phenomenon. Both cellular and molecular mechanisms of digit tip regeneration are poorly defined. The specific research aims are: 1. To trace the cell and germ layer origin of digit tip regeneration and explore their somatic fates 2. To elucidate the molecular mechanism of digit tip regeneration and to study the functions of key genes in early regeneration events. My graduate studies involved in-depth familiarization with principle mechanisms and processes of tissue and organ development and regeneration. My post doctorate training will focus on the questions of the molecular mechanisms and the ‘cell of origin’ during mammalian digit tip regeneration. Integrating a single-cell level of understanding of the biology of stem cells within this regeneration processes. I plan to conduct this post doctoral activity in one of the leading stem cell biology laboratories, under the supervision of Irving L. Weissman at Stanford.

The overall goal of this project is to analyze the kinetic profile of protein sequences as they are synthesized, one at a time, by detecting the pattern and timing of optical signals emitted from labeled single ribosomes. The ribosomes and tRNAs will be labeled by FRET pairs and will be extensively characterized in a cell-free system. Various coding mRNAs will be utilized in order to follow the translation process in real time using total internal reflectance fluorescent (TIRF) microscopy.
The ribosomal protein L11 will be tagged with a FRET acceptor whereas the cognate FRET donor will label a specific tRNA. The signal will be in the form of temporal FRET pulses, reporting the accommodation of specific tRNAs. Knowing the mRNA sequence will allow real-time analysis of rhythm and pauses that modulate the translation rate. Factors that govern the kinetic profile of protein synthesis will be inspected; these include rare codon usage, mRNA secondary structure, and nascent chain sequence.
Single-molecule recording of protein synthesis directly reports the kinetic trajectory of that individual ribosome. Tabulation of the pausing times will enable statistical inferences concerning the kinetic mechanism of translation. In contrast to single molecule investigation, ensemble techniques suffer from lack of synchronization. Although bulk measurements are very informative, the mentioned disadvantage became apparent during my PhD studies. Acquiring the methodologies in my host labs will enable me to combine single molecule spectroscopy and imaging with bulk measurements in my future career. This will lead to a deep mechanistic understanding of dynamical processes at the molecular level.

Understanding how histone post-translational modifications regulate chromatin structure and a range of chromatin-related processes is fundamental to our understanding of, epigenetics, differentiation, stem cell biology, and human pathologies. While the importance of histone acetylation and methylation is well established, the role of histone ubiquitylation is only beginning to unravel, and insights have been largely restricted to monoubiquitylation of H2A on lysine 119 and H2B on lysine 120. Given the dynamic and transient nature of protein ubiquitylation and chromatin modifications, it is very likely that additional histone ubiquitylation sites exist that play important roles in chromatin-related processes. I provide herein a comprehensive strategy for the analysis of histone ubiquitylation states and functions. By combining yeast genetics, mass spectrometry, modification-specific antibodies, “designer chromatin”, and in vitro transcription assays I intend to identify novel histone ubiquitylation sites, study their function, and explore the biological machineries that deposit and interpret them. The newly identified modifications and the underlying enzymatic machineries will be verified in higher organisms, and their function will be studied in relevant experimental systems such as mammalian stem cells. This work has the potential to shed important light on the biological significance of novel and previously known histone ubiquitylation, and improve our understanding of chromatin-based processes. Mechanistic insight into these fundamental issues lies in the heart of chromatin biology with far-reaching implications for human biology and human diseases.

Fission yeast mating-type switching occurs in one of two sister cells based on the inheritance of older ‘Watson’ vs. ‘Crick’ DNA chains at mat1 locus. Possibly this mechanism of asymmetric cell division also functions in diploid organisms. Here, epigenetically differentiated sister chromatids, based on the replication history of their DNA chains, are selectively segregated to daughter cells. Analysis of mouse chromosome 7 segregation patterns - which is biased in embryonic stem cells (ESCs), ESC-derived endoderm and ectoderm, but not in mesoderm cells - supports this theory. Biased segregation is dependent on a dynein motor (Dnahc11) previously implicated in embryonic left/right body axis specification.

I propose to study segregation patterns of mouse chromosomes 7, 11 and X in vivo, by generating mice harbouring chimeric transgenes, consisting of part of the coding sequences of GFP and RFP, separated by a loxp site. Upon Cre-recombination, chromatids acquire either functional GFP or RFP. Depending on segregation pattern, either red and green or yellow (red+green) and colorless cell clones develop. I will investigate if segregation pattern is affected in Dnahc11 mutant mice in vivo. In addition, I will test predictions of the strand segregation model regarding embryonic lethality rates. Moreover, biochemical and cell biological studies with FLAG and GFP tagged Dnahc11 protein in ESCs will address mechanistic aspects of selective chromatid segregation.

One central aim in neuroscience is to relate the cortical anatomical structure to its function with the expectation to find answers to fundamental questions about neural processing, learning, memory, and even cognition. A major gap in the understanding of cortical circuit function arises from the difficulty to integrate the knowledge about the functional properties of synapses and neurons, because on one hand experiments usually include only small cell/synapse numbers, and on the other hand the knowledge of the circuit anatomy lacks fine detail required for biologically realistic models. The goal of my proposal is to bridge this gap. I will use channelrhodopsin assisted stimulation of axons together with postsynaptic calcium imaging to explore the properties and dendritic locations of the synapses from a large number of inputs simultaneously. This will provide crucial information for understanding synaptic integration and how the cortex samples for appropriate synaptic connections. Furthermore, I will use 2-photon glutamate uncaging to stimulate single synapses in vivo, in order to analyze the impact of the activity of a single synapse on its parent neuron, which is crucial to understand how structural and functional plasticity at the synapse level relates to circuit performance. The Career Development Award will help me accomplishing these goals by providing the necessary funds to establish a strong, productive team applying state of the art techniques to fundamental scientific questions at the Max Planck Institute for Neurobiology.

All higher animals use a complex muscle - tendon network to execute their daily movements. For these to happen properly all muscles have to form at the correct positions, grow to the appropriate size and produce just enough force. Muscle development is a multi-step process; myoblasts proliferate, migrate and fuse with each other at particular locations within the body depending on their identity. The forming myotubes search for appropriate tendon cells for a stable connection. My aim is to understand how the complex muscle network of an adult fly is built. What are the molecular mechanisms creating diversity and instructing the different muscles to target to specific tendon cells? As an entry point we developed and applied genome-wide, inducible RNAi in Drosophila and systematically surveyed the entire genome for genes with a potential role in adult muscle morphogenesis. 1) From this screen I aim to focus on two types of molecules for a phenotypic characterisation: transcriptions factors to investigate muscle diversity and cell surface molecules to study muscle targeting. 2) For a detailed phenotypic and mechanistic characterisation of these genes I will apply in vivo imaging. This will enable me to directly follow the entire process from muscle fusion to targeting in the intact animal. 3) Complementary to the above efforts I wish to compare the myoblast proteome of wild type to mutants in identified transcriptions factors. This should result in a quantitative measure of muscle diversity. I envision to unravel developmental principles instructing muscle morphogenesis that will be conserved and directly applicable to vertebrates and hence be of wide general interest.

Fundamental phenomena such as cellular identity and genome integrity are specified by a basal network of gene regulation. Central players are small RNAs, which base-pair to target RNAs and mediate gene silencing at a transcriptional or post-transcriptional level. These effects are mediated by Argonaute proteins, through transcript cleavage, block of translation, or methylation of homologous DNA. Novel technologies allowed the extensive sequencing of small RNAs and uncovered a great diversity in the model plant Arabidopsis. However, despite the large general impact of small RNAs on gene regulation, the individual roles of their majority remain mysterious.
This work aims to dissect the molecular functions of Arabidopsis small RNAs and thereby answer three fundamental questions: what are the roles of the numerous small RNAs that have not been implicated in silencing so far? What are the activities of the Argonaute proteins that bind those small RNAs? And how many small RNAs are needed to silence a single locus?
I have designed a set of molecular and biochemical tools to directly monitor the silencing activities of individual Argonautes as well as small RNAs in different contexts. In order to map the minimal set of small RNAs required for silencing, I will establish a method to quickly and specifically determine the sequences of all small RNAs produced from a trigger locus of interest.
The obtained results will thus enable me to unify novel and classic small RNAs to similar pathways or define an entirely novel role for small RNAs. Furthermore I will be able to assess the redundancy among small RNAs and design novel tools to screen for additional silencing effectors.

Cellular aging is associated with gradual accumulation of damage to cellular components. However, some types of cells, for example, adult stem cells (SCs) of epithelia, are especially long-lived and therefore appear to be better protected from aging than the others. SCs divide highly asymmetrically, giving rise to cells of different fate and often of different life expectancy. The goal of my project is to establish whether SCs and their offspring are unequal in terms of damaged components and activity of the damage repair machineries, and if so to unveil the molecular mechanisms that drive such asymmetry. I hypothesize that the asymmetry of SC division contributes to asymmetric segregation of cellular damage and/or cellular repair components, thus helping rejuvenation of the long-lived daughter. To test this hypothesis, I will use Drosophila intestinal SCs and neuroblasts as model systems. I believe that these studies would help us to understand better stem cell functioning and reasons of stem cell deterioration in normal aging and pathology.

About two years ago an experimental system was developed in which the reprogramming of somatic cells to a new pluripotent embryonic stem cell-like population, using only a handful of stemness factors, was achieved (1-4). However, until now this technique is very inefficient and potentially causing cancer and for these reasons, inapplicable in human therapy. To improve this process it is indispensable to learn to understand the complex network of molecular pathways that occur during reprogramming and to asses the functions and mechanism of each inducing factor. Since all the four reprogramming factors are related to signalling pathways on the one hand, and on the other hand Hh-Gli signalling has an important role in regulating patterning, proliferation, survival and growth in the embryo and the adult (5), this signalling pathway seems to play an important role in the process of reprogramming. Interestingly, it was shown that Hh-Gli signalling regulates a key stemness signature in cancer stem cells (6). With this background it becomes essential to study the role of Hh-Gli signalling in pluripotency. Moreover, a form of reprogramming could take place in advanced cancers characterized by the appearance of aggressive cancer stem cells. Comprehending the origin of cancer stem cells would provide multitudinous possibilities for new therapies with the final hope to be able to cure, until now incurable, forms of cancer. To this end I want to study the role of Gli function in reprogramming. Furthermore, I propose to focus on the investigation of the role of the reprogramming factors Nanog, Sox2, Oct4, and Klf4 in stem cells of the brain and in brain tumors in mice and in human.

The ability of cells to detect and transduce mechanical stimuli is a fundamental biological process that underlies touch, pain and control of muscle tension. In vertebrates, a subpopulation of somatosensory neurons is specialized to detect these different mechanical stimuli but how they accomplish this task is at present not understood. Similarly, the developmental program that allows these neurons to differentiate into a diversity of cell types with very specific mechanosensitive traits is largely unknown.
We will employ a multidisciplinary approach geared towards the identification and characterization of molecules that are necessary for the development and function of mechanosensory neurons. Using the mouse as a model system, we will conduct a genetic screen that will allow us to isolate molecules such as transcription factors and signaling molecules involved in the differentiation of mechanosensory neurons. Subsequently, we will test their potential to drive differentiation of neuronal precursors into specific mechanosensory neurons using stem cell biology. The resulting cell populations will be functionally analyzed for their mechanosensitive properties using live-cell imaging and electrophysiology and subsequently used to identify the underlying mechanosensory proteins.
This line of experiments will not only illuminate the genes necessary and sufficient for the generation of mechanosensitive neurons but will also allow the production of pure populations of sensory neurons, an invaluable tool for subsequent functional characterization of mechanosensory transduction that is currently impossible due to the heterogeneity of these cells in sensory ganglia in vivo.

Fast orientation in complex, unknown environments is one of the most challenging sensory tasks for mammals. While most mammals meet this challenge with stereoscopic vision, one large group has pursued an alternative and highly successful strategy: Bats probe their surroundings via a biological sonar system, by analyzing the echoes of their ultrasonic vocalizations. In visual animals, including humans, the perceptual distortions caused by mirrors allow studying fundamental questions in brain processing of 3D objects in 3D space. We propose to bring these concepts into the auditory domain by conducting a set of novel studies on the effects of acoustic mirrors in bats. Water surfaces act as acoustic mirrors and represent a dramatic distortion of physical space, a common problem facing bats in the wild. We will study the question of how animals perceive 3D objects and 3D space, and how 3D environments are represented in the brain, by using acoustic mirrors as a novel experimental tool – and we will approach this problem by combining the expertise of three labs, located in Israel and Germany. Methods span from behavioral experiments in the field and in large flight rooms with naïve bats, via formal psychophysical experiments with trained bats looking at the effects of acoustic mirrors on the sonar perception of 3D objects and their cortical neural representation, and ending with the effect of mirrors on the neural representation of 3D space itself at the level of the bat hippocampus.

EukaryoticDNA is densely packaged in the form of chromatin. Despite the dense packaging,a eukaryotic cell responds to signals of growth, development and adaptationwithin minutes by specifically turning on a few defined genes. This raises thefundamental question of how the transcription machinery finds these few genesamongst the large mass of packaged DNA. The discovery of “pioneer” factors thatrecognize specific DNA sequences in the context of chromatin helped solve alarge part of this question. Sequence-specific binding of these factorsdirectly de-condenses chromatin making it accessible for downstream effectors.Yet the mechanism of action of these factors is not known. I propose to addressthis question by applying recent advances in nuclear magnetic resonance (NMR)to study pioneer factor-nucleosome interactions. I will use FoxA as a modelsystem because it the best-studied pioneer factor and plays key roles in liverdevelopment. I will first obtain a high-resolution map of nucleosome dynamics usingNMR and then determine how FoxA binding perturbs nucleosome structure. Theresults will distinguish whether FoxA binds to transiently unpeeled nucleosomalDNA or whether, and if so how, it recognizes a composite binding site made upof histones and DNA. The results will also provide insight into whether FoxAcauses chromatin de-condensation by displacing linker histones, which mediatecompaction or whether FoxA disrupts inter-nucleosomal interactions throughconformational changes in the nucleosome. The NMR-derived map of the nucleosomedynamics will also be very powerful tool for studying the mechanisms of otherfactors that modulate chromatin structure.