Imported from Grants Tool 4701329

Chronic or acute diseases affecting the neuromuscular system can cause debilitating symptoms in human patients, yet they may manifest through very different etiologies. For example, either muscle or nerve tissue can present the primary pathological phenotype. However, the intricate functional connection between the two tissues, through so-called neuromuscular circuits, will eventually result in deficiencies in both, if disease is allowed to progress unchecked. Correct embryonic assembly of these neuromuscular circuits is essential, in order to ensure faithful muscle-nerve communication. The required circuit specificity is determined - at least in part - by molecularly defined subgroups of motor neurons, so-called motor neuron «pools». Neurons of a given pool project their axons to a single muscle in the periphery. Whether or not distinct molecular subtypes also exist among the various muscle groups is currently less clear. We know, however, that both antero- and retrograde signals between muscle fibers and motor neurons play a crucial role in refining these circuits during development, and maintaining robustness and functionality during homeostasis. The goal of this proposal is thus to profile molecular signatures of circuit-specific muscle and motor neuron pairs, and to functionally test their relevance in neuromuscular circuit formation. For this, I propose to combine state-of-the-art single-cell RNA-sequencing of axon-backfilled motor neurons with bulk transcriptomic analyses of micro-dissected limb muscles connected to the corresponding motor neuron pools. Furthermore, we will exploit a unique experimental setting, in which one of the main motor nerve branches is re-routed and ectopically connects to a duplicated muscle, thereby shunting the original circuit logic. We will evaluate the role of emerging candidate genes using gain- and loss-of- function approaches in both muscle- and neuron-lineages. A better understanding of the molecular crosstalk between muscle and motor neurons will provide new insights into the mechanisms of neuromuscular circuit formation, refinement and maintenance, as well as support the development of regenerative therapies for treating human muscle and nerve diseases.

Die kontrollierte Bewegung der Gliedmassen von Wirbeltieren beruht auf der Kontraktion peripherer Muskeln, welche durch Entscheidungsprozesse im zentralen Nervensystem gesteuert wird. Im Rückenmark leiten spezifische Gruppen von Motoneuronen diese Steuerungseingaben vom Gehirn an ihre entsprechenden Muskelziele weiter, und bilden zusammen mit sensorischen Neuronen hochspezifische neuromuskuläre Schaltkreise. Der korrekte Aufbau dieser Schaltkreise während der embryonalen Entwicklung ist essentiell, um später die richtigen Kontraktionsmuster der Muskeln und damit Bewegungskontrolle zu gewährleisten. Die erforderliche Spezifität dieser neuromuskulären Schaltkreise wird - zumindest teilweise - durch molekular definierte Untergruppen von Motoneuronen bestimmt, sogenannten Motoneuronen-«Pools». Neuronen eines bestimmten Pools innervieren immer nur einen einzelnen, spezifischen Muskel in der Peripherie. Dies geschieht durch wegleitende Signale, sogenannte 'axon guidance cues', welche die wachsenden Nervenstränge der molekular unterschiedlichen Motoneuronen-Pools in die Nähe ihrer jeweiligen Muskelziele lotsen. Ob unter den verschiedenen Muskelgruppen in der Peripherie auch molekulare Subtypen existieren oder nicht, ist derzeit noch unbekannt. Wir wissen jedoch, dass sowohl antero- wie auch retrograde Signale zwischen Muskelfasern und Motoneuronen eine entscheidende Rolle bei der Verfeinerung dieser Schaltkreise, deren Robustheit und deren Homöostase spielen. Ein besseres V erständnis der molekularen Wechselwirkungen in diesen Schaltkreisen wird uns daher neue Einblicke in die Ätiologie angeborener Fehlbildungen des neuromuskulären Systems der Extremitäten gewähren, sowie die Entwicklung von regenerativen Therapien für dieses Systems bei menschlichen Patienten unterstützen. Ziel des hier vorgeschlagenen Projektes ist es daher die Existenz unterschiedlicher molekularer Subtypen in den verschiedenen Muskelgruppen der Wirbeltiergliedmassen zu untersuchen, und sie mit den transkriptionellen Signaturen einzelner verbundener Motoneuronen zu integrieren. Basierend auf unseren eigenen Erkenntnissen in einem Modell der experimentell induzierten Polydaktylie, oder Vielfingerigkeit, gehe ich davon aus, dass die Ausbildung der Architektur dieser Schaltkreise eine erhebliche Plastizität aufweist - dies im Gegensatz zu dem, was man von der oben beschriebenen Logik erwarten würde. Bei einer durch Polydaktylie induzierten Verdoppelung des Muskels Flexor digiti quarti beobachten wir nämlich eine fehlgeleitete Nerv-zu-Muskel-V erbindung, außerhalb des normalerweise dafür vorgesehenen Schaltkreises. Wir werden diesen einzigartigen experimentellen Ansatz dazu benutzen, um die molekularen Wechselwirkungen von Motoneuronen und Muskeln innerhalb ihres normalen Schaltkreises zu untersuchen, wie auch nach dem Auftreten fehlgeleiteter Muskelkontakte. Durch die Analyse und Integration von Einzelzell-RNA-Sequenzieranalysen markierter Motoneuronen mit den molekularen Profilen der mit ihnen verbundenen Muskelgruppen, wollen wir Schlüsselregulatoren in dieser komplexen Wechselwirkung definieren, und deren Relevanz funktionell in vivo testen. Die Identifizierung molekularer Faktoren zur korrekten Bildung von Muskel- Nerv-Verbindungen wird unser Verständnis neuromuskulärer Schaltkreis-Architekturen verbessern, sowie die Entwicklung von therapeutischen Ansätzen zur Aufrechterhaltung ihrer motorischen Funktionen vorantreiben.

Cell fate specification - that is, how distinct cell types arise from a pool of precursor cells - is one of the hallmarks of embryonic development. During this process, a genome common to all cells of an organism is differentially interpreted at the gene regulatory level, to result in individualized cellular phenotypes. So far, most studies have focused on the specification of different cell types from a single progenitor pool. However, notable exceptions exist to this trajectory, such as in the vertebrate skeleton that, depending on anatomical location, develops from three distinct progenitor populations (neural crest, somitic and lateral plate mesoderm). Despite this diversity in embryonic origins and thesurrounding tissue environments, these three progenitor populations converge phenotypically to give rise to functionally equivalent skeletal cell types. With the current project, we aim to define the gene regulatory logic underlying such cell fate convergence. Specifically, we will decode how transcriptional networks assimilate differences in progenitor transcriptomes and tissue niches to produce analogous cell types from distinct embryonic sources. Based on preliminary data from our lab, I hypothesize that progenitor-specific transcription factor profiles, resulting from cell-intrinsic and -extrinsic differences in their embryonic origins, are integrated at the cis-regulatory level, via lineage-specific enhancer elements, to result in the transcriptional and phenotypic convergence of the three skeletal precursor pools. To functionally test these hypotheses, I propose to combine state-of-the-art functional genomics at single cell- and lineage-resolution with experimental embryology and targeted CRISPR/Cas9-genome modifications. Capitalizing on the superior temporal control in avian embryos, their ability to sustain transplantation experiments, and our expertise in developmental biology, molecular genetics and transcriptome analyses, we will: Define, at single cell- and lineage-resolution, the transcriptome and chromatin dynamics of the three progenitor pools, as they converge towards a common skeletal cell fate Elucidate the interplay of cell-intrinsic and cell-extrinsic parameters in this gene regulatory convergence using quail-chick grafts followed by single-cell RNA-sequencing and chromatin accessibility profiling Functionally validate candidate regulatory factors underlying this convergence in vivo, using the CRISPR/Cas9system in a lineage-specific manner in chicken embryos Collectively, our work will delineate the gene regulatory logic instructing the transcriptional and phenotypic convergence towards a common skeletal cell fate, across three distinct embryonic progenitor pools. As such, the project promises to define novel paradigms of cell fate specification control, for the skeletal system and beyond, and open new avenues applicable to regenerative medicine.

The adult human body consists of hundreds, if not thousands, of distinct cell types. Examples include different neurons in the brain, epithelial cells lining the gut, or the cells that produce the bone or cartilage in our skeletons. During embryogenesis, all of these diverse cell types develop from a single progenitor cell - the fertilized egg. Understanding the different molecular mechanisms that orchestrate this diversification can help us re-create a particular cell type in a cell culture dish. Having access to such 'in vitro'-generated cells would then enable their use in tissue repair and replacement strategies in human patients. What are the potential difficulties in achieving these goals? Many cell type specification processes require complex interactions with the surrounding tissue. Hence, only in the context of a developing embryo can these processes be fully understood, making the use of non-human 'model organisms' indispensable. This, however, raises additional questions: how can we minimize the number of experimental animals used in these studies, and how can we ensure that findings in such model organisms also translate to us humans? To address these challenges, here I propose to integrate comparative genomics data to define conserved 'core regulatory switches' that specify a given cell type across species. We will then functionally test the relevance of these candidate 'switches' using genetic perturbations in chicken embryos. Our experimental approach will prevent the euthanasia of any pregnant female animal while at the same time maximize its relevance for the subsequent in vitro specification of human cell types.

The concept of 'cell fate', central to both developmental biology and regenerative medicine, has seen dramatic shifts in recent years. Thanks to emerging single-cell technologies and cellular (re-)programming, we are now able to address questions resulting therefrom at unprecedented detail. While many studies have focused on the emergence of cellular diversity from a single precursor type, there are instances where distinct embryonic progenitor pools converge to give rise to functionally analogous cell types. What is the gene regulatory logic underlying such cell fate convergence? And how amenable are these transcriptional networks to evolutionary change, to result in distinct, species-specific morphologies? Here, I propose an integrative approach to elucidate this question using vertebrate skeletogenesis as a model system, over developmental and evolutionary timescales, at lineage- and single-cell resolution, during cell fate convergence and embryonic pattern formation. Building on my expertise in comparative transcriptomics and developmental biology, we will (1) study the gene regulatory logic of analogous cell fate specification from three distinct embryonic progenitor pools; (2) delineate the essential core regulatory nodes through a deep sampling of the vertebrate phylogeny and functionally test them via targeted in vitro specifications; and, ultimately, (3) integrate these insights to investigate species- specific and patterning-relevant in vivo cell fate decisions using single-cell RNA-sequencing and mathematical modeling. Collectively, our work will define core regulatory networks that specify a given skeletal cell fate across developmental and evolutionary levels, during cell fate convergence and embryonic patterning. As such, it will help to define novel paradigms of developmental cell fate decision control, for the skeletal system and beyond, and open new avenues for regenerative medicine.

During development, the musculoskeletal apparatus of the vertebrate limb integrates and patterns diverse tissue types with distinct embryonic origins. Namely, the bones of the appendicular skeleton originate from the lateral plate mesoderm, while the musculature and its innervating motor neurons derive from the somatic mesoderm and the neural tube, respectively. How is the patterning of such distinct embryonic progenitor populations coordinated, in order to give rise to a fully functional, moveable limb? Moreover, what are the potential developmental constraints originating from such patterning interdependency between different tissue types? We are studying the patterning of these three tissue types in vertebrate autopods, hands and feet, where the appendicular skeleton shows the highest degree of morphological diversity and functional specialization. We are using chicken experimental embryology as well as genetic mouse models (in collaboration with Rolf Zeller's group, Department of Biomedicine, Uni Basel) to address these questions.

The highest degree of morphological diversification, as well as functional specialization, of the vertebrate limb skeleton has occurred in its most distal part, the so-called autopod. Most of the diversity relates to the number of digits present, as well as the skeletal patterns of each individual digit. Each digit pattern is determined by the number and size of its bony elements, the phalanges, and how they are connected to each other via synovial joints. These configurations are specified by an embryonic sequence of inducing phalanx versus joint cell fates, as the individual digits are growing out during autopod development. Understanding how these phalanx versus joint cell fate decisions are made would thus allow us to decipher the underlying developmental mechanism of autopod morphological diversification. We are using experimental embryology, single-cell RNA-sequencing and bioinformatics to unravel the molecular aspects of these cell fate decisions. In collaboration with Dagmar Iber's group (D-BSSE ETH Zürich) we aim to develop in silico models of this patterning process, to better understand its evolutionary flexibility.

Embryonic skeletogenesis occurs through the initial condensation of mesenchymal progenitors, followed by differentiation into the various skeletal cell types. These include chondrocytes, the bone-forming osteoblasts and joint progenitors that build the connections between mature skeletal elements. Depending on anatomical location, three distinct mesenchymal progenitor pools contribute to the different parts of the vertebrate skeleton: the somitic mesoderm forms the axial skeleton, whereas the lateral plate mesoderm and the neural crest give rise to the appendicular skeleton and parts of the cranial skeleton, respectively. We are studying the gene regulatory mechanisms underlying the generation of these cell types, originating from distinct embryonic sources and in different species. We use next-generation sequencing techniques to interrogate the transcriptional output, chromatin state and transcription factor binding profiles of these cells during maturation, both in vivo and in vitro.