Projects & Collaborations 22 foundShow per page10 10 20 50 Implementing 3Rs in Switzerland: an interdisciplinary in-depth exploration of barriers and facilitators [Implement-3R] Research Project | 10 Project MembersThe 3R principles and regulation of animal research The use of animals in biomedical and other research presents an ethical dilemma: we do not want to lose scientific benefits, nor do we want to cause laboratory animals to suffer". In Switzerland, the majority of animals (64%) is used in fundamental research and a minority (ca. 20%) are used for developing and testing pharmaceutical and chemical products. The 3R-strategies ("replace, reduce, refine") are "today widely accepted by scientists as a moral obligation to treat animals humanely and if possible to use alternative methods in experiments". The national and international regulatory framework concerning the use of animals for research stipulates adherence to the 3R principles[3-5]. In addition, Swiss law requires researchers to demonstrate a favourable harm-benefit ratio to justify animal experiments[6, 7]. There is an ongoing discussion in the fields of ethics, law and science concerning the interpretation of the 3Rs and harm-benefit analysis. Eminent ethicists have recently proposed a more elaborated, argued way how to balance social benefit and animal welfare in this context. Angiogenesis in zebrafish: how are vascular networks formed? Research Project | 1 Project MembersNo Description available Angiogenesis in zebrafish: how do vascular networks form? Research Project | 1 Project Membersto be added ... Drosophila Branching 2.0 Research Project | 4 Project MembersHow animals acquire their distinct and species-specific shapes and three-dimensional morphologies and how such morphologies are encoded in the genome has been a fascinating topic for numerous scientists over many decades. While the question of how animal form and diversity is achieved is fascinating, gaining a cellular and molecular understanding of the phenomenon of morphogenesis requires breaking down the problem into more precise or better defined questions, such as how individual parts of an animal, for example external extremities or internal organs reach their precise shape during development.My laboratory has tried to answer questions related to the overall topic of animal morphogenesis by using genetic approaches, mainly in Drosophila melanogaster, the fruit fly, and more recently also in Danio rerio, the zebrafish. In this grant application, we would like to capitalise on recent developments in our lab and answer two sets of questions that have preoccupied us over a long period. We and others have studied tracheal development in drosophila as a paradigm for a complex morphogenesis process. We have shown that cell rearrangements are key to generate the distinct morphologies of different tracheal branches in the drosophila tracheal system, and that these cell rearrangements are controlled by transcription factors such as Spalt and Knirps and by cellular activities such as cell migration, cell shape changes and adherens junction remodelling. However, we know very little about the targets of these transcriptional regulators and their cellular and molecular functions in regulating cell rearrangements. We propose to undertake transcriptional profiling to unravel the key role of the Spalt transcription factor. Profiting from the efficiency of Crispr-based methods in drosophila and the short generation time, functional studies of candidate genes can then rapidly be performed and the data integrated into the existing molecular networks. While we hope to isolate more genes involved in the control of cell rearrangement starting from transcriptional profiling, it is very likely that many genes or proteins involved in tracheal morphogenesis have not been identified using classical zygotic loss of function mutagenesis, due to maternal contribution. We have recently developed a nanobody-based method allowing for the depletion of maternally provided proteins in specific tissues and study their role in morphogenesis processes. Using this novel approach, we would like to undertake a pioneering pilot screen to search for proteins with hitherto uncharacterized roles in the tracheal system and in the amnioserosa, a central force-producer during the tissue morphogenesis process of dorsal closure. Furthermore, and inspired by the wealth of nanobody-based novel tools we have already designed and used in drosophila, we want to introduce and validate novel protein-directed approaches based on short peptide tags and the corresponding binding proteins reported to be functional in cultured cells. If these tools work well in multicellular organisms where they have not been tested yet, a wealth of novel applications in developmental biology will emerge, which will help us studying candidate genes from our screens.Question 1: How is the process of cell intercalation regulated at the molecular level?Aim 1: Identify Spalt-regulated genes using RNAseq and study their role in cell intercalationQuestion 2: Can we identify many more proteins involved in tracheal branching morphogenesis, whose contribution has not yet been identified? Aim 2: Identify novel genes (proteins) regulating tracheal morphogenesis using deGradFP.Question 3: Can short peptides be used for in vivo tagging and subsequent protein manipulation?Aim 3: Generate/validate novel tools to directly study proteins in morphogenesis processes Anti-angiogenic Effects of BAL078 in Zebra-Fish Research Project | 1 Project MembersNo Description available In vivo cell biology of organ morphogenesis Research Project | 1 Project Membersto be added Roche financing employment of Verena Küppers Research Project | 2 Project Membersto be added Sub-cellular targeting microscopy - Signaling in Development and Oncology Research Project | 6 Project MembersIn the past decade, research in development and oncology has uncovered an impressive number of relevant signaling pathways. While a qualitative understanding of signals, and their connection to cellular outputs has been established, plasticity and feedback of signaling networks remain obscured. A better understanding of dynamic and locally constrained signaling events driving organ development and disease progression requires access to refined subcellular probe detection. The availability of optogenetic and chemical biology tools provides novel opportunities, but requires dedicated microscopy equiment. For this reason, six projects at the Department of Biomedicine (DBM) and the Biocenter (BC) of the Univerisity of Basel i) localized lipid signaling in disease (M. Wymann, DBM); ii) dynamic subcellular Wnt/b-catenin signaling in epithelial mesenchymal transition (G. Christofori, DBM); iii) DNA dynamics and confined epigenetic plasticity (P. Schär, DBM); iv) real-time monitoring of Sonic Hedgehog and Bone Morphogenetic Protein gradients in limb buds (R. Zeller, DBM); v) ultrastructural analysis of neuronal stem cell control (V. Taylor, DBM); and vi) molecular mechanisms determining the development of vascular networks (M. Affolter, BC), illustrate the need of the requested "subcellular targeting microscopy" equipment. The core of the platform is a highly sensitive microlens-enhanced spinning disk microscope linked to FRAP, ablation, and multiple excitation laser lines, and an integrated TIRF module to monitor plasma membrane events. Through its integration into the BioOptics core facility at the DBM, the subcellular targeting platform will be accessible to >500 regional researchers at the University of Basel, DBM, FMI, D-BSSE, Fachhochschule, etc. An image storage and analysis pipeline with remote user access capabilitiy is in place, to allow seamless operation and output. The requested equipment will allow the use of genetically encoded opto-genetic proteins, proteins tagged for reactivity with chemical inducers of dimerisation (CIDs), and the possibility to perform FRAP/TIRF and FRAP/confocal microscopy, and will greatly enhance the possibilities to manipulate and track subcellular localization of target proteins. The insights gained by these experimental approaches will be critical for a better understanding of dynamic biological processes, and will spur the design of innovative therapeutic approaches to counteract resistance mechanisms in oncology. In vivo cell biology of organ morphogenesis Research Project | 1 Project MembersOrgans and tissues acquire particular three-dimensional shapes during development, which are intimately linked to the particular function(s) an organ has to fulfil. Organ shape is to a large extent determined by cell behaviour, and cell behaviour is to a large extent regulated by cell-cell signalling and cell mechanics. A major interest of my laboratory over the last few years has been to determine how branching morphogenesis restructures epithelial tissues to generate such fascinating structures as the trachea or the vasculature. While we have initially put much effort in understanding this process in vivo in an invertebrate system (tracheal development in Drosophila melanogaster) using state of the art genetics in combination with high resolution live imaging, we have recently moved much of our research efforts to vertebrates, in particular to zebrafish (Danio rerio). Using high resolution in vivo imaging, we have described the cellular activities during angiogenesis, in particular during sprouting, vessel fusion and vessel pruning. To our surprise, we find that the plasticity of developing vessels is accompanied by unexpected cell behaviour; endothelial cells can fission and self-fuse during anastomosis and pruning, respectively. With the advent of the recently introduced genome manipulation tools (TALEN, CRISPR/Cas9), a genetic dissection of the different steps in angiogenesis is now possible at unprecedented level, and proteins can be tagged at endogenous loci and used as marker for high resolution live imaging. We have recently applied intracellular nanobodies for the first time in developing drosophila embryos to directly manipulate protein function, in particular for protein degradation. We used an anti-GFP nanobody fused to an F-box in degrade GFP-fusion proteins (we called the method "deGradFP" for degrading GFP). In the meantime, we have functionalized the anti-GFP nanobody manifold, so that it can be used for in vivo protein trapping, protein localisation and for post-translation modification of proteins of interest. During the next granting period, we will introduce the use of intracellular nanobodies (and other protein binders) for studying the zebrafish vascular system, in particular to answer the following questions: Q1: How is the dynamic cell behaviour during sprouting controlled? Q2: How do endothelial cells recognize each other in order to connect? Q3: Which molecular processes are involved in luminal membrane expansion? Q4: Which molecular processes are involved in membrane fusion and membrane fission? In order to answer these questions, we propose to undertake the following experimental strategies : 1) Investigate the role of VE-cad in the dynamic rearrangements of endothelial cells. 2) Generate and use cutting edge in vivo live imaging tools to characterize molecular aspects of cell behaviour during angiogenesis processes. 3) Generate and analyse mutations in candidate genes affecting distinct cellular activities. 4) Generate and use novel tools to manipulate protein function in vivo - a step closer to a synthetic biology approach to organ formation. Lightsheet microscopy Research Project | 5 Project MembersResearch at the Biozentrum of the University of Basel focuses on basic cellular and molecular aspects of living systems. The recent technological developments in Lightsheet microscopy have opened up new possibilities for light microscopy-based live-cell imaging of whole organisms or cells. To install this novel technology at the Biozentrum, we plan to purchase an instrument for light sheet microscopy. This acquisition will be embedded within the new imaging core facility set up to provide state-of-the-art microscopy technology with the necessary support and training to the Biozentrum research community. 123 123
Implementing 3Rs in Switzerland: an interdisciplinary in-depth exploration of barriers and facilitators [Implement-3R] Research Project | 10 Project MembersThe 3R principles and regulation of animal research The use of animals in biomedical and other research presents an ethical dilemma: we do not want to lose scientific benefits, nor do we want to cause laboratory animals to suffer". In Switzerland, the majority of animals (64%) is used in fundamental research and a minority (ca. 20%) are used for developing and testing pharmaceutical and chemical products. The 3R-strategies ("replace, reduce, refine") are "today widely accepted by scientists as a moral obligation to treat animals humanely and if possible to use alternative methods in experiments". The national and international regulatory framework concerning the use of animals for research stipulates adherence to the 3R principles[3-5]. In addition, Swiss law requires researchers to demonstrate a favourable harm-benefit ratio to justify animal experiments[6, 7]. There is an ongoing discussion in the fields of ethics, law and science concerning the interpretation of the 3Rs and harm-benefit analysis. Eminent ethicists have recently proposed a more elaborated, argued way how to balance social benefit and animal welfare in this context.
Angiogenesis in zebrafish: how are vascular networks formed? Research Project | 1 Project MembersNo Description available
Angiogenesis in zebrafish: how do vascular networks form? Research Project | 1 Project Membersto be added ...
Drosophila Branching 2.0 Research Project | 4 Project MembersHow animals acquire their distinct and species-specific shapes and three-dimensional morphologies and how such morphologies are encoded in the genome has been a fascinating topic for numerous scientists over many decades. While the question of how animal form and diversity is achieved is fascinating, gaining a cellular and molecular understanding of the phenomenon of morphogenesis requires breaking down the problem into more precise or better defined questions, such as how individual parts of an animal, for example external extremities or internal organs reach their precise shape during development.My laboratory has tried to answer questions related to the overall topic of animal morphogenesis by using genetic approaches, mainly in Drosophila melanogaster, the fruit fly, and more recently also in Danio rerio, the zebrafish. In this grant application, we would like to capitalise on recent developments in our lab and answer two sets of questions that have preoccupied us over a long period. We and others have studied tracheal development in drosophila as a paradigm for a complex morphogenesis process. We have shown that cell rearrangements are key to generate the distinct morphologies of different tracheal branches in the drosophila tracheal system, and that these cell rearrangements are controlled by transcription factors such as Spalt and Knirps and by cellular activities such as cell migration, cell shape changes and adherens junction remodelling. However, we know very little about the targets of these transcriptional regulators and their cellular and molecular functions in regulating cell rearrangements. We propose to undertake transcriptional profiling to unravel the key role of the Spalt transcription factor. Profiting from the efficiency of Crispr-based methods in drosophila and the short generation time, functional studies of candidate genes can then rapidly be performed and the data integrated into the existing molecular networks. While we hope to isolate more genes involved in the control of cell rearrangement starting from transcriptional profiling, it is very likely that many genes or proteins involved in tracheal morphogenesis have not been identified using classical zygotic loss of function mutagenesis, due to maternal contribution. We have recently developed a nanobody-based method allowing for the depletion of maternally provided proteins in specific tissues and study their role in morphogenesis processes. Using this novel approach, we would like to undertake a pioneering pilot screen to search for proteins with hitherto uncharacterized roles in the tracheal system and in the amnioserosa, a central force-producer during the tissue morphogenesis process of dorsal closure. Furthermore, and inspired by the wealth of nanobody-based novel tools we have already designed and used in drosophila, we want to introduce and validate novel protein-directed approaches based on short peptide tags and the corresponding binding proteins reported to be functional in cultured cells. If these tools work well in multicellular organisms where they have not been tested yet, a wealth of novel applications in developmental biology will emerge, which will help us studying candidate genes from our screens.Question 1: How is the process of cell intercalation regulated at the molecular level?Aim 1: Identify Spalt-regulated genes using RNAseq and study their role in cell intercalationQuestion 2: Can we identify many more proteins involved in tracheal branching morphogenesis, whose contribution has not yet been identified? Aim 2: Identify novel genes (proteins) regulating tracheal morphogenesis using deGradFP.Question 3: Can short peptides be used for in vivo tagging and subsequent protein manipulation?Aim 3: Generate/validate novel tools to directly study proteins in morphogenesis processes
Anti-angiogenic Effects of BAL078 in Zebra-Fish Research Project | 1 Project MembersNo Description available
Sub-cellular targeting microscopy - Signaling in Development and Oncology Research Project | 6 Project MembersIn the past decade, research in development and oncology has uncovered an impressive number of relevant signaling pathways. While a qualitative understanding of signals, and their connection to cellular outputs has been established, plasticity and feedback of signaling networks remain obscured. A better understanding of dynamic and locally constrained signaling events driving organ development and disease progression requires access to refined subcellular probe detection. The availability of optogenetic and chemical biology tools provides novel opportunities, but requires dedicated microscopy equiment. For this reason, six projects at the Department of Biomedicine (DBM) and the Biocenter (BC) of the Univerisity of Basel i) localized lipid signaling in disease (M. Wymann, DBM); ii) dynamic subcellular Wnt/b-catenin signaling in epithelial mesenchymal transition (G. Christofori, DBM); iii) DNA dynamics and confined epigenetic plasticity (P. Schär, DBM); iv) real-time monitoring of Sonic Hedgehog and Bone Morphogenetic Protein gradients in limb buds (R. Zeller, DBM); v) ultrastructural analysis of neuronal stem cell control (V. Taylor, DBM); and vi) molecular mechanisms determining the development of vascular networks (M. Affolter, BC), illustrate the need of the requested "subcellular targeting microscopy" equipment. The core of the platform is a highly sensitive microlens-enhanced spinning disk microscope linked to FRAP, ablation, and multiple excitation laser lines, and an integrated TIRF module to monitor plasma membrane events. Through its integration into the BioOptics core facility at the DBM, the subcellular targeting platform will be accessible to >500 regional researchers at the University of Basel, DBM, FMI, D-BSSE, Fachhochschule, etc. An image storage and analysis pipeline with remote user access capabilitiy is in place, to allow seamless operation and output. The requested equipment will allow the use of genetically encoded opto-genetic proteins, proteins tagged for reactivity with chemical inducers of dimerisation (CIDs), and the possibility to perform FRAP/TIRF and FRAP/confocal microscopy, and will greatly enhance the possibilities to manipulate and track subcellular localization of target proteins. The insights gained by these experimental approaches will be critical for a better understanding of dynamic biological processes, and will spur the design of innovative therapeutic approaches to counteract resistance mechanisms in oncology.
In vivo cell biology of organ morphogenesis Research Project | 1 Project MembersOrgans and tissues acquire particular three-dimensional shapes during development, which are intimately linked to the particular function(s) an organ has to fulfil. Organ shape is to a large extent determined by cell behaviour, and cell behaviour is to a large extent regulated by cell-cell signalling and cell mechanics. A major interest of my laboratory over the last few years has been to determine how branching morphogenesis restructures epithelial tissues to generate such fascinating structures as the trachea or the vasculature. While we have initially put much effort in understanding this process in vivo in an invertebrate system (tracheal development in Drosophila melanogaster) using state of the art genetics in combination with high resolution live imaging, we have recently moved much of our research efforts to vertebrates, in particular to zebrafish (Danio rerio). Using high resolution in vivo imaging, we have described the cellular activities during angiogenesis, in particular during sprouting, vessel fusion and vessel pruning. To our surprise, we find that the plasticity of developing vessels is accompanied by unexpected cell behaviour; endothelial cells can fission and self-fuse during anastomosis and pruning, respectively. With the advent of the recently introduced genome manipulation tools (TALEN, CRISPR/Cas9), a genetic dissection of the different steps in angiogenesis is now possible at unprecedented level, and proteins can be tagged at endogenous loci and used as marker for high resolution live imaging. We have recently applied intracellular nanobodies for the first time in developing drosophila embryos to directly manipulate protein function, in particular for protein degradation. We used an anti-GFP nanobody fused to an F-box in degrade GFP-fusion proteins (we called the method "deGradFP" for degrading GFP). In the meantime, we have functionalized the anti-GFP nanobody manifold, so that it can be used for in vivo protein trapping, protein localisation and for post-translation modification of proteins of interest. During the next granting period, we will introduce the use of intracellular nanobodies (and other protein binders) for studying the zebrafish vascular system, in particular to answer the following questions: Q1: How is the dynamic cell behaviour during sprouting controlled? Q2: How do endothelial cells recognize each other in order to connect? Q3: Which molecular processes are involved in luminal membrane expansion? Q4: Which molecular processes are involved in membrane fusion and membrane fission? In order to answer these questions, we propose to undertake the following experimental strategies : 1) Investigate the role of VE-cad in the dynamic rearrangements of endothelial cells. 2) Generate and use cutting edge in vivo live imaging tools to characterize molecular aspects of cell behaviour during angiogenesis processes. 3) Generate and analyse mutations in candidate genes affecting distinct cellular activities. 4) Generate and use novel tools to manipulate protein function in vivo - a step closer to a synthetic biology approach to organ formation.
Lightsheet microscopy Research Project | 5 Project MembersResearch at the Biozentrum of the University of Basel focuses on basic cellular and molecular aspects of living systems. The recent technological developments in Lightsheet microscopy have opened up new possibilities for light microscopy-based live-cell imaging of whole organisms or cells. To install this novel technology at the Biozentrum, we plan to purchase an instrument for light sheet microscopy. This acquisition will be embedded within the new imaging core facility set up to provide state-of-the-art microscopy technology with the necessary support and training to the Biozentrum research community.
