Prof. Dr. Alexander Schier Department Biozentrum Profiles & Affiliations OverviewResearch Publications Projects & Collaborations Projects & Collaborations OverviewResearch Publications Projects & Collaborations Profiles & Affiliations Projects & Collaborations 15 foundShow per page10 10 20 50 Morphogen signaling and mechanotransduction in early killifish embryogenesis Research Project | 2 Project MembersImported from Grants Tool 4709749 The cis-regulatory code of Nodal morphogen interpretation Research Project | 1 Project MembersImported from Grants Tool 4701080 DevBoundaries - Formation of tissue boundaries during zebrafish embryogenesis Research Project | 2 Project MembersDuring animal development, boundaries need to be established between cell types to guarantee the physical and functional integrity of tissues. However, the underlying mechanisms are poorly understood because it has been challenging to analyze the coordination of gene expression, cell proliferation and cell movement needed for boundary formation. I will probe the in vivo mechanisms of boundary formation using the zebrafish embryonic shield region as a model system. The shield contains overlapping progenitor cells that give rise to various midline structures whose boundaries form and sharpen during gastrulation. I will use imaging and genetic approaches to determine how shield cells differentiate and generate tissue boundaries. Aim 1: To characterize lineage, movement and differentiation of shield progenitors and their descendants, I will use in toto light-sheet imaging. This approach will generate a dynamic atlas detailing the cellular basis of boundary formation. Aim 2: To characterize the gene expression changes during the separation of shield progenitors and their descendants, I will use spatial transcriptomics. This aim will create a dynamic atlas detailing the transcriptomic basis of boundary formation. Aim 3: To define the molecular basis of boundary formation, I will disrupt candidate genes involved in boundary formation. This approach will determine if differentiation and morphogenesis are coupled during boundary formation and define molecular pathways that ensure robust boundary formation. Together, the proposed approaches will identify cellular and genetic mechanisms controlling boundary formation during embryogenesis. Allen Discovery Center for Cell Lineage Renewal Research Project | 2 Project MembersMulticellular organisms develop by way of a lineage tree, a series of cell divisions and molecular changes that give rise to cell types, tissues and organs. Despite their fundamental relevance to development, our knowledge of cell lineages and their determinants remains fragmentary, and fundamental questions remain unanswered: What are the molecular and cellular programs that drive cells to acquire specific fates? How do these vary within an individual, between individuals, and across species? What are the cell lineage motifs that underlie consistencies and differences in form and function? In its first phase, our Center has established novel paradigms for recording cell lineage and cell states. We can introduce heritable and cumulative changes into the genome to record the lineage relationship between cells (scGESTALT; MEMOIR). We can measure the transcriptional and epigenetic states of cells and reconstruct the molecular trajectories underlying cell type differentiation (sci-seq). We can measure transcriptomes and lineage markers in tissues and reconstruct the spatiotemporal unfolding of development (seqFISH; sci-Space). In C. elegans , we have used such information to define developmental principles such as multilineage priming. In C. elegans , Drosophila , zebrafish and mouse, we have used our datasets to define cellular diversity and regulators of cell type differentiation. We now have the methodological foundation to address fundamental questions in developmental biology at the scale of the entire vertebrate embryo. In order to reveal developmental rules that are shared and divergent across individuals and species, we propose to map (Aim 1), model (Aim 2) and manipulate (Aim 3) embryogenesis. We will focus on zebrafish and mouse embryogenesis, because these are well-established and accessible model systems in which we have laid the requisite groundwork. In Aim 1 , we will use genomic and imaging approaches to generate high-resolution maps of natural zebrafish and mouse embryogenesis and of stem cell-derived synthetic mouse embryogenesis. Maps will include cell lineage, gene expression, chromatin accessibility data and signaling activity and will be complemented by live imaging of cell movements. In Aim 2 , we will integrate these datasets to generate a consensus scaffold of the molecular trajectories and lineage structures of embryogenesis, together with statistical models that seek to explain developmental processes in terms of rates of cell division and probabilistic differentiation. In a complementary approach, we will develop lineage motif analysis, a framework for extracting the key recurrent programs of cell lineage histories in relation to time, space and molecular state. We will then apply both the consensus model and lineage motif analysis to investigate different kinds of developmental variation: interclonal, interindividual and interspecies. We will make our tools, datasets, models and insights available to the community through a navigable 'virtual embryo'. In Aim 3 , we will functionally test model predictions and address how genetic, embryological or environmental manipulations affect cell fate decisions, lineage variation and developmental robustness. We will manipulate key parameters of development such as the activity of lineage regulators and intercellular interactions, cell number and proliferation rates in different lineages, and environmental and metabolic conditions. The observed phenotypic consequences will shed light on the mechanisms underlying developmental robustness and variation. DevUTRs - Uncovering the roles of 5′UTRs in translational control during early zebrafish development (Marie Curie Fellowship Madalena Pinto) Research Project | 2 Project MembersDuring early developmental stages, metazoan embryos are transcriptionally silent, and embryogenesis is controlled by maternally deposited factors. Developmental progression requires the synthesis of new mRNAs and proteins in a coordinated fashion. Many posttranscriptional mechanisms regulate the fate of maternal mRNAs, but it is less understood how translational control shapes early embryogenesis. Protein synthesis is primarily regulated at the translation initiation step by elements in the 5′ untranslated region (5′ UTR) of the mRNA. However, we currently lack a systematic understanding of the regulatory information contained within 5′ UTRs and how they functionally impact mRNA translation throughout development. Using zebrafish as a model of vertebrate development, we are developing an in vivo massively parallel reporter assay (MPRA) to identify 5′ UTR motifs involved in translation regulation. By integrating the translational behaviour of 5′ UTR reporters throughout embryogenesis with sequence-based regression models, we anticipate to uncover novel cis -regulatory elements in 5′ UTRs with developmental roles. The MPRA will lay the foundation for future studies dissecting the molecular mechanisms underlying the function of newly identified 5′ UTR motifs. Formation of tissue boundaries during zebrafish embryogenesis (EMBO Fellowship Yinan Wan) Research Project | 2 Project MembersTitle: Formation of tissue boundaries during zebrafish embryogenesis Abstract: During animal development, boundaries need to be established between cell types to guarantee the physical and functional integrity of tissues. However, the underlying mechanisms are poorly understood because it has been challenging to analyze the coordination of gene expression, cell proliferation and cell movement needed for boundary formation. I will probe the in vivo mechanisms of boundary formation using the zebrafish embryonic shield region as a model system. The shield contains overlapping progenitor cells that give rise to various midline structures whose boundaries form and sharpen during gastrulation. I will use imaging and genetic approaches to determine how shield cells differentiate and generate tissue boundaries. Aim 1: To characterize lineage, movement and differentiation of shield progenitors and their descendants, I will use in toto light-sheet imaging. This approach will generate a dynamic atlas detailing the cellular basis of boundary formation. Aim 2: To characterize the gene expression changes during the separation of shield progenitors and their descendants, I will use spatial transcriptomics. This aim will create a dynamic atlas detailing the transcriptomic basis of boundary formation. Aim 3: To define the molecular basis of boundary formation, I will disrupt candidate genes involved in boundary formation. This approach will determine if differentiation and morphogenesis are coupled during boundary formation and define molecular pathways that ensure robust boundary formation. Together, the proposed approaches will identify cellular and genetic mechanisms controlling boundary formation during embryogenesis. Genetic and neural regulation of sleep and arousal Research Project | 1 Project MembersStimuli that elicit responses in awake animals fail to do so during sleep. This increased arousal threshold and locomotor quiescence are hallmarks of sleep behavior, but the genetic and neural mechanisms that control arousal remain poorly understood. Inappropriate regulation of arousal contributes to insomnia or nonrestorative sleep, which affect up to a third of the population. Revealing how the brain regulates arousal states is thus of significant biomedical importance. Genetic studies have provided entry points to define genes and neural circuits regulating arousal. For example, studies of specific G-protein coupled receptors (GPCRs) have revealed sleep regulatory mechanisms in Drosophila, zebrafish, mouse and humans, while family linkage and genome-wide association studies have identified candidate genes involved in human sleep behavior and sleep disorders. Our proposed research extends these studies by analyzing the cellular roles of genes involved in human insomnia and Restless Legs Syndrome (Aim 1), and by revealing novel functions of G-protein coupled receptors in sleep and arousal (Aim 2). We use zebrafish as a model system because powerful imaging, genetic, genomics, and behavioral approaches can be combined to investigate vertebrate sleep and arousal. In preliminary studies, we disrupted genes associated with human insomnia and Restless Leg Syndrome (RLS) and profiled their effects on behavior. We discovered that mutations in the transcriptional regulators meis1b and skor1a/b lead to overlapping arousal phenotypes and perturbed cerebellum function. These results suggest that meis1b and skor1a/b regulate cerebellar development and function, and implicate the cerebellum in the regulation of sleep and arousal (Aim 1). In a complementary approach to identify novel sleep and arousal regulators, we disrupted 93 brain-expressed G-protein coupled receptor genes. In our initial screening efforts, we discovered several mutants with abnormal brain activity or behavior. In particular, galr2a/b and gpr101 mutants exhibit daytime hyperactivity and hypoactivity, respectively (Aim 2). We will extend these studies as follows: Aim 1: Dissect how the transcriptional regulators meis1b and skor1a/b regulate cerebellum circuitry and arousal. We hypothesize that disruption of specific subsets of cerebellar neurons generates aberrant locomotor drive and heightened sensory responsiveness in meis1b and skor1a/b mutants. To test this idea, we will use imaging, genomic, and neuronal manipulation approaches to define, phenocopy, and rescue cerebellum defects in meis1b and skor1a/b mutants. These studies will reveal previously unknown functions for Restless Legs Syndrome genes, as well as novel roles for cerebellum circuitry in arousal regulation. Aim 2: Identify G-protein coupled receptors that modulate sleep and arousal. We will complete our genetic screen to identify GPCRs that regulate arousal and identify common signaling pathways by intersecting phenotypes with previous pharmacological and overexpression screens. To prioritize hits for follow-up studies, we will characterize candidates from the primary screen with a battery of secondary behavioral tests, characterize GPCR expression vis-à-vis whole brain activity signatures, and determine relevant ligand/receptor relationships. These studies will help identify novel GPCRs and signaling pathways regulating sleep and arousal. These studies will enrich our genetic and circuit-level understanding of how the brain controls sleep and arousal, and inform potential diagnostic or therapeutic approaches to human sleep disorders. Jane Coffin Childs Fellowship Award - Dr. Emily Bayer Research Project | 2 Project MembersThe neuronal regulation of internal organs has been historically considered to be 'self-contained' and autonomous. However, it is increasingly appreciated that brain-body communication modulates not just the target organs, but also the brain itself. I'm combining the incredible specificity of the C. elegans pharyngeal nervous system with a study of the less-described visceral innervation in zebrafish, a system still compact enough that it can be detailed comprehensively. In C. elegans , I am using inducible neuronal silencing of pharyngeal neurons in combination with behavioral assays for somatic neuron function to identify novel behavioral outputs modulated by internal sensation. In zebrafish, the genetic identities, modalities, and functions of visceral neurons (from both the vagus nerve and cranial sensory ganglia) are still largely undescribed. I have used scRNAseq approaches to collect transcriptomic data on these cell types in adult animals, and will complement this approach with spatial transcriptomics to resolve the anatomical organization of molecular cell types. This will allow me to examine what kinds of information are passed between organs and the CNS (based on the nature of sensory receptors expressed), and what the circuits responsible for this look like (based on the anatomical properties and signaling capabilities of the involved neuron types). The molecular and genetic basis of kin recognition in zebrafish (Oded Mayseless) Research Project | 1 Project MembersKin selection is a widespread phenomenon through which individuals gain indirect fitness by directing altruistic behaviors toward genetically related individuals. The mechanism that permits recognition between individuals, based on genetic relatedness, needs to be highly selective and refined. Despite its critical nature, the sensory mechanisms governing this phenomenon remain incomplete. Larval zebrafish prefer kin-specific vs nonkin odors. Larvae that have been isolated or exposed to non-kin cues lose their capacity to recognize kin. Specific olfactory sensory neurons (OSNs) and the highly polymorphic major histocompatibility complex (MHC) have been implicated, however the molecular mechanisms which mediate the specificity of kin odor recognition are unknown. By combining behavioral, functional, and molecular approaches, we will unravel the molecular basis of kin recognition. Dissecting the emergence of neuronal cell populations in the habenulae of the vertebrate brain (BIF Fellowship Jakob El Kholtei) Research Project | 2 Project MembersThe vertebrate brain displays an incredible number and diversity of neurons. Neural stem cells, or progenitor cells, give rise to this neuronal diversity through the process of neurogenesis. Despite many scientific efforts, we are yet to attain a comprehensive understanding of how these progenitors establish the myriad of neuronal cell types in the correct locations and quantities. By establishing and using genomic and optical lineage tracing tools in the zebrafish, I will study the dynamics of neural stem cells in the context of the vertebrate habenulae, a conserved forebrain region. Taking advantage of the comparably simple structure and limited size of the habenulae, I will reconstruct a comprehensive, spatially resolved lineage tree of a complete vertebrate brain region. By combining this approach with a molecular characterisation of habenular development using single-cell RNA-sequencing and sequential fluorescent in situ hybridisation, I aim to gain a comprehensive understanding of neurogenesis in this brain region. The method development involved in this project and the anticipated findings will significantly contribute toour understanding of vertebrate brain development. 12 12 OverviewResearch Publications Projects & Collaborations
Projects & Collaborations 15 foundShow per page10 10 20 50 Morphogen signaling and mechanotransduction in early killifish embryogenesis Research Project | 2 Project MembersImported from Grants Tool 4709749 The cis-regulatory code of Nodal morphogen interpretation Research Project | 1 Project MembersImported from Grants Tool 4701080 DevBoundaries - Formation of tissue boundaries during zebrafish embryogenesis Research Project | 2 Project MembersDuring animal development, boundaries need to be established between cell types to guarantee the physical and functional integrity of tissues. However, the underlying mechanisms are poorly understood because it has been challenging to analyze the coordination of gene expression, cell proliferation and cell movement needed for boundary formation. I will probe the in vivo mechanisms of boundary formation using the zebrafish embryonic shield region as a model system. The shield contains overlapping progenitor cells that give rise to various midline structures whose boundaries form and sharpen during gastrulation. I will use imaging and genetic approaches to determine how shield cells differentiate and generate tissue boundaries. Aim 1: To characterize lineage, movement and differentiation of shield progenitors and their descendants, I will use in toto light-sheet imaging. This approach will generate a dynamic atlas detailing the cellular basis of boundary formation. Aim 2: To characterize the gene expression changes during the separation of shield progenitors and their descendants, I will use spatial transcriptomics. This aim will create a dynamic atlas detailing the transcriptomic basis of boundary formation. Aim 3: To define the molecular basis of boundary formation, I will disrupt candidate genes involved in boundary formation. This approach will determine if differentiation and morphogenesis are coupled during boundary formation and define molecular pathways that ensure robust boundary formation. Together, the proposed approaches will identify cellular and genetic mechanisms controlling boundary formation during embryogenesis. Allen Discovery Center for Cell Lineage Renewal Research Project | 2 Project MembersMulticellular organisms develop by way of a lineage tree, a series of cell divisions and molecular changes that give rise to cell types, tissues and organs. Despite their fundamental relevance to development, our knowledge of cell lineages and their determinants remains fragmentary, and fundamental questions remain unanswered: What are the molecular and cellular programs that drive cells to acquire specific fates? How do these vary within an individual, between individuals, and across species? What are the cell lineage motifs that underlie consistencies and differences in form and function? In its first phase, our Center has established novel paradigms for recording cell lineage and cell states. We can introduce heritable and cumulative changes into the genome to record the lineage relationship between cells (scGESTALT; MEMOIR). We can measure the transcriptional and epigenetic states of cells and reconstruct the molecular trajectories underlying cell type differentiation (sci-seq). We can measure transcriptomes and lineage markers in tissues and reconstruct the spatiotemporal unfolding of development (seqFISH; sci-Space). In C. elegans , we have used such information to define developmental principles such as multilineage priming. In C. elegans , Drosophila , zebrafish and mouse, we have used our datasets to define cellular diversity and regulators of cell type differentiation. We now have the methodological foundation to address fundamental questions in developmental biology at the scale of the entire vertebrate embryo. In order to reveal developmental rules that are shared and divergent across individuals and species, we propose to map (Aim 1), model (Aim 2) and manipulate (Aim 3) embryogenesis. We will focus on zebrafish and mouse embryogenesis, because these are well-established and accessible model systems in which we have laid the requisite groundwork. In Aim 1 , we will use genomic and imaging approaches to generate high-resolution maps of natural zebrafish and mouse embryogenesis and of stem cell-derived synthetic mouse embryogenesis. Maps will include cell lineage, gene expression, chromatin accessibility data and signaling activity and will be complemented by live imaging of cell movements. In Aim 2 , we will integrate these datasets to generate a consensus scaffold of the molecular trajectories and lineage structures of embryogenesis, together with statistical models that seek to explain developmental processes in terms of rates of cell division and probabilistic differentiation. In a complementary approach, we will develop lineage motif analysis, a framework for extracting the key recurrent programs of cell lineage histories in relation to time, space and molecular state. We will then apply both the consensus model and lineage motif analysis to investigate different kinds of developmental variation: interclonal, interindividual and interspecies. We will make our tools, datasets, models and insights available to the community through a navigable 'virtual embryo'. In Aim 3 , we will functionally test model predictions and address how genetic, embryological or environmental manipulations affect cell fate decisions, lineage variation and developmental robustness. We will manipulate key parameters of development such as the activity of lineage regulators and intercellular interactions, cell number and proliferation rates in different lineages, and environmental and metabolic conditions. The observed phenotypic consequences will shed light on the mechanisms underlying developmental robustness and variation. DevUTRs - Uncovering the roles of 5′UTRs in translational control during early zebrafish development (Marie Curie Fellowship Madalena Pinto) Research Project | 2 Project MembersDuring early developmental stages, metazoan embryos are transcriptionally silent, and embryogenesis is controlled by maternally deposited factors. Developmental progression requires the synthesis of new mRNAs and proteins in a coordinated fashion. Many posttranscriptional mechanisms regulate the fate of maternal mRNAs, but it is less understood how translational control shapes early embryogenesis. Protein synthesis is primarily regulated at the translation initiation step by elements in the 5′ untranslated region (5′ UTR) of the mRNA. However, we currently lack a systematic understanding of the regulatory information contained within 5′ UTRs and how they functionally impact mRNA translation throughout development. Using zebrafish as a model of vertebrate development, we are developing an in vivo massively parallel reporter assay (MPRA) to identify 5′ UTR motifs involved in translation regulation. By integrating the translational behaviour of 5′ UTR reporters throughout embryogenesis with sequence-based regression models, we anticipate to uncover novel cis -regulatory elements in 5′ UTRs with developmental roles. The MPRA will lay the foundation for future studies dissecting the molecular mechanisms underlying the function of newly identified 5′ UTR motifs. Formation of tissue boundaries during zebrafish embryogenesis (EMBO Fellowship Yinan Wan) Research Project | 2 Project MembersTitle: Formation of tissue boundaries during zebrafish embryogenesis Abstract: During animal development, boundaries need to be established between cell types to guarantee the physical and functional integrity of tissues. However, the underlying mechanisms are poorly understood because it has been challenging to analyze the coordination of gene expression, cell proliferation and cell movement needed for boundary formation. I will probe the in vivo mechanisms of boundary formation using the zebrafish embryonic shield region as a model system. The shield contains overlapping progenitor cells that give rise to various midline structures whose boundaries form and sharpen during gastrulation. I will use imaging and genetic approaches to determine how shield cells differentiate and generate tissue boundaries. Aim 1: To characterize lineage, movement and differentiation of shield progenitors and their descendants, I will use in toto light-sheet imaging. This approach will generate a dynamic atlas detailing the cellular basis of boundary formation. Aim 2: To characterize the gene expression changes during the separation of shield progenitors and their descendants, I will use spatial transcriptomics. This aim will create a dynamic atlas detailing the transcriptomic basis of boundary formation. Aim 3: To define the molecular basis of boundary formation, I will disrupt candidate genes involved in boundary formation. This approach will determine if differentiation and morphogenesis are coupled during boundary formation and define molecular pathways that ensure robust boundary formation. Together, the proposed approaches will identify cellular and genetic mechanisms controlling boundary formation during embryogenesis. Genetic and neural regulation of sleep and arousal Research Project | 1 Project MembersStimuli that elicit responses in awake animals fail to do so during sleep. This increased arousal threshold and locomotor quiescence are hallmarks of sleep behavior, but the genetic and neural mechanisms that control arousal remain poorly understood. Inappropriate regulation of arousal contributes to insomnia or nonrestorative sleep, which affect up to a third of the population. Revealing how the brain regulates arousal states is thus of significant biomedical importance. Genetic studies have provided entry points to define genes and neural circuits regulating arousal. For example, studies of specific G-protein coupled receptors (GPCRs) have revealed sleep regulatory mechanisms in Drosophila, zebrafish, mouse and humans, while family linkage and genome-wide association studies have identified candidate genes involved in human sleep behavior and sleep disorders. Our proposed research extends these studies by analyzing the cellular roles of genes involved in human insomnia and Restless Legs Syndrome (Aim 1), and by revealing novel functions of G-protein coupled receptors in sleep and arousal (Aim 2). We use zebrafish as a model system because powerful imaging, genetic, genomics, and behavioral approaches can be combined to investigate vertebrate sleep and arousal. In preliminary studies, we disrupted genes associated with human insomnia and Restless Leg Syndrome (RLS) and profiled their effects on behavior. We discovered that mutations in the transcriptional regulators meis1b and skor1a/b lead to overlapping arousal phenotypes and perturbed cerebellum function. These results suggest that meis1b and skor1a/b regulate cerebellar development and function, and implicate the cerebellum in the regulation of sleep and arousal (Aim 1). In a complementary approach to identify novel sleep and arousal regulators, we disrupted 93 brain-expressed G-protein coupled receptor genes. In our initial screening efforts, we discovered several mutants with abnormal brain activity or behavior. In particular, galr2a/b and gpr101 mutants exhibit daytime hyperactivity and hypoactivity, respectively (Aim 2). We will extend these studies as follows: Aim 1: Dissect how the transcriptional regulators meis1b and skor1a/b regulate cerebellum circuitry and arousal. We hypothesize that disruption of specific subsets of cerebellar neurons generates aberrant locomotor drive and heightened sensory responsiveness in meis1b and skor1a/b mutants. To test this idea, we will use imaging, genomic, and neuronal manipulation approaches to define, phenocopy, and rescue cerebellum defects in meis1b and skor1a/b mutants. These studies will reveal previously unknown functions for Restless Legs Syndrome genes, as well as novel roles for cerebellum circuitry in arousal regulation. Aim 2: Identify G-protein coupled receptors that modulate sleep and arousal. We will complete our genetic screen to identify GPCRs that regulate arousal and identify common signaling pathways by intersecting phenotypes with previous pharmacological and overexpression screens. To prioritize hits for follow-up studies, we will characterize candidates from the primary screen with a battery of secondary behavioral tests, characterize GPCR expression vis-à-vis whole brain activity signatures, and determine relevant ligand/receptor relationships. These studies will help identify novel GPCRs and signaling pathways regulating sleep and arousal. These studies will enrich our genetic and circuit-level understanding of how the brain controls sleep and arousal, and inform potential diagnostic or therapeutic approaches to human sleep disorders. Jane Coffin Childs Fellowship Award - Dr. Emily Bayer Research Project | 2 Project MembersThe neuronal regulation of internal organs has been historically considered to be 'self-contained' and autonomous. However, it is increasingly appreciated that brain-body communication modulates not just the target organs, but also the brain itself. I'm combining the incredible specificity of the C. elegans pharyngeal nervous system with a study of the less-described visceral innervation in zebrafish, a system still compact enough that it can be detailed comprehensively. In C. elegans , I am using inducible neuronal silencing of pharyngeal neurons in combination with behavioral assays for somatic neuron function to identify novel behavioral outputs modulated by internal sensation. In zebrafish, the genetic identities, modalities, and functions of visceral neurons (from both the vagus nerve and cranial sensory ganglia) are still largely undescribed. I have used scRNAseq approaches to collect transcriptomic data on these cell types in adult animals, and will complement this approach with spatial transcriptomics to resolve the anatomical organization of molecular cell types. This will allow me to examine what kinds of information are passed between organs and the CNS (based on the nature of sensory receptors expressed), and what the circuits responsible for this look like (based on the anatomical properties and signaling capabilities of the involved neuron types). The molecular and genetic basis of kin recognition in zebrafish (Oded Mayseless) Research Project | 1 Project MembersKin selection is a widespread phenomenon through which individuals gain indirect fitness by directing altruistic behaviors toward genetically related individuals. The mechanism that permits recognition between individuals, based on genetic relatedness, needs to be highly selective and refined. Despite its critical nature, the sensory mechanisms governing this phenomenon remain incomplete. Larval zebrafish prefer kin-specific vs nonkin odors. Larvae that have been isolated or exposed to non-kin cues lose their capacity to recognize kin. Specific olfactory sensory neurons (OSNs) and the highly polymorphic major histocompatibility complex (MHC) have been implicated, however the molecular mechanisms which mediate the specificity of kin odor recognition are unknown. By combining behavioral, functional, and molecular approaches, we will unravel the molecular basis of kin recognition. Dissecting the emergence of neuronal cell populations in the habenulae of the vertebrate brain (BIF Fellowship Jakob El Kholtei) Research Project | 2 Project MembersThe vertebrate brain displays an incredible number and diversity of neurons. Neural stem cells, or progenitor cells, give rise to this neuronal diversity through the process of neurogenesis. Despite many scientific efforts, we are yet to attain a comprehensive understanding of how these progenitors establish the myriad of neuronal cell types in the correct locations and quantities. By establishing and using genomic and optical lineage tracing tools in the zebrafish, I will study the dynamics of neural stem cells in the context of the vertebrate habenulae, a conserved forebrain region. Taking advantage of the comparably simple structure and limited size of the habenulae, I will reconstruct a comprehensive, spatially resolved lineage tree of a complete vertebrate brain region. By combining this approach with a molecular characterisation of habenular development using single-cell RNA-sequencing and sequential fluorescent in situ hybridisation, I aim to gain a comprehensive understanding of neurogenesis in this brain region. The method development involved in this project and the anticipated findings will significantly contribute toour understanding of vertebrate brain development. 12 12
Morphogen signaling and mechanotransduction in early killifish embryogenesis Research Project | 2 Project MembersImported from Grants Tool 4709749
The cis-regulatory code of Nodal morphogen interpretation Research Project | 1 Project MembersImported from Grants Tool 4701080
DevBoundaries - Formation of tissue boundaries during zebrafish embryogenesis Research Project | 2 Project MembersDuring animal development, boundaries need to be established between cell types to guarantee the physical and functional integrity of tissues. However, the underlying mechanisms are poorly understood because it has been challenging to analyze the coordination of gene expression, cell proliferation and cell movement needed for boundary formation. I will probe the in vivo mechanisms of boundary formation using the zebrafish embryonic shield region as a model system. The shield contains overlapping progenitor cells that give rise to various midline structures whose boundaries form and sharpen during gastrulation. I will use imaging and genetic approaches to determine how shield cells differentiate and generate tissue boundaries. Aim 1: To characterize lineage, movement and differentiation of shield progenitors and their descendants, I will use in toto light-sheet imaging. This approach will generate a dynamic atlas detailing the cellular basis of boundary formation. Aim 2: To characterize the gene expression changes during the separation of shield progenitors and their descendants, I will use spatial transcriptomics. This aim will create a dynamic atlas detailing the transcriptomic basis of boundary formation. Aim 3: To define the molecular basis of boundary formation, I will disrupt candidate genes involved in boundary formation. This approach will determine if differentiation and morphogenesis are coupled during boundary formation and define molecular pathways that ensure robust boundary formation. Together, the proposed approaches will identify cellular and genetic mechanisms controlling boundary formation during embryogenesis.
Allen Discovery Center for Cell Lineage Renewal Research Project | 2 Project MembersMulticellular organisms develop by way of a lineage tree, a series of cell divisions and molecular changes that give rise to cell types, tissues and organs. Despite their fundamental relevance to development, our knowledge of cell lineages and their determinants remains fragmentary, and fundamental questions remain unanswered: What are the molecular and cellular programs that drive cells to acquire specific fates? How do these vary within an individual, between individuals, and across species? What are the cell lineage motifs that underlie consistencies and differences in form and function? In its first phase, our Center has established novel paradigms for recording cell lineage and cell states. We can introduce heritable and cumulative changes into the genome to record the lineage relationship between cells (scGESTALT; MEMOIR). We can measure the transcriptional and epigenetic states of cells and reconstruct the molecular trajectories underlying cell type differentiation (sci-seq). We can measure transcriptomes and lineage markers in tissues and reconstruct the spatiotemporal unfolding of development (seqFISH; sci-Space). In C. elegans , we have used such information to define developmental principles such as multilineage priming. In C. elegans , Drosophila , zebrafish and mouse, we have used our datasets to define cellular diversity and regulators of cell type differentiation. We now have the methodological foundation to address fundamental questions in developmental biology at the scale of the entire vertebrate embryo. In order to reveal developmental rules that are shared and divergent across individuals and species, we propose to map (Aim 1), model (Aim 2) and manipulate (Aim 3) embryogenesis. We will focus on zebrafish and mouse embryogenesis, because these are well-established and accessible model systems in which we have laid the requisite groundwork. In Aim 1 , we will use genomic and imaging approaches to generate high-resolution maps of natural zebrafish and mouse embryogenesis and of stem cell-derived synthetic mouse embryogenesis. Maps will include cell lineage, gene expression, chromatin accessibility data and signaling activity and will be complemented by live imaging of cell movements. In Aim 2 , we will integrate these datasets to generate a consensus scaffold of the molecular trajectories and lineage structures of embryogenesis, together with statistical models that seek to explain developmental processes in terms of rates of cell division and probabilistic differentiation. In a complementary approach, we will develop lineage motif analysis, a framework for extracting the key recurrent programs of cell lineage histories in relation to time, space and molecular state. We will then apply both the consensus model and lineage motif analysis to investigate different kinds of developmental variation: interclonal, interindividual and interspecies. We will make our tools, datasets, models and insights available to the community through a navigable 'virtual embryo'. In Aim 3 , we will functionally test model predictions and address how genetic, embryological or environmental manipulations affect cell fate decisions, lineage variation and developmental robustness. We will manipulate key parameters of development such as the activity of lineage regulators and intercellular interactions, cell number and proliferation rates in different lineages, and environmental and metabolic conditions. The observed phenotypic consequences will shed light on the mechanisms underlying developmental robustness and variation.
DevUTRs - Uncovering the roles of 5′UTRs in translational control during early zebrafish development (Marie Curie Fellowship Madalena Pinto) Research Project | 2 Project MembersDuring early developmental stages, metazoan embryos are transcriptionally silent, and embryogenesis is controlled by maternally deposited factors. Developmental progression requires the synthesis of new mRNAs and proteins in a coordinated fashion. Many posttranscriptional mechanisms regulate the fate of maternal mRNAs, but it is less understood how translational control shapes early embryogenesis. Protein synthesis is primarily regulated at the translation initiation step by elements in the 5′ untranslated region (5′ UTR) of the mRNA. However, we currently lack a systematic understanding of the regulatory information contained within 5′ UTRs and how they functionally impact mRNA translation throughout development. Using zebrafish as a model of vertebrate development, we are developing an in vivo massively parallel reporter assay (MPRA) to identify 5′ UTR motifs involved in translation regulation. By integrating the translational behaviour of 5′ UTR reporters throughout embryogenesis with sequence-based regression models, we anticipate to uncover novel cis -regulatory elements in 5′ UTRs with developmental roles. The MPRA will lay the foundation for future studies dissecting the molecular mechanisms underlying the function of newly identified 5′ UTR motifs.
Formation of tissue boundaries during zebrafish embryogenesis (EMBO Fellowship Yinan Wan) Research Project | 2 Project MembersTitle: Formation of tissue boundaries during zebrafish embryogenesis Abstract: During animal development, boundaries need to be established between cell types to guarantee the physical and functional integrity of tissues. However, the underlying mechanisms are poorly understood because it has been challenging to analyze the coordination of gene expression, cell proliferation and cell movement needed for boundary formation. I will probe the in vivo mechanisms of boundary formation using the zebrafish embryonic shield region as a model system. The shield contains overlapping progenitor cells that give rise to various midline structures whose boundaries form and sharpen during gastrulation. I will use imaging and genetic approaches to determine how shield cells differentiate and generate tissue boundaries. Aim 1: To characterize lineage, movement and differentiation of shield progenitors and their descendants, I will use in toto light-sheet imaging. This approach will generate a dynamic atlas detailing the cellular basis of boundary formation. Aim 2: To characterize the gene expression changes during the separation of shield progenitors and their descendants, I will use spatial transcriptomics. This aim will create a dynamic atlas detailing the transcriptomic basis of boundary formation. Aim 3: To define the molecular basis of boundary formation, I will disrupt candidate genes involved in boundary formation. This approach will determine if differentiation and morphogenesis are coupled during boundary formation and define molecular pathways that ensure robust boundary formation. Together, the proposed approaches will identify cellular and genetic mechanisms controlling boundary formation during embryogenesis.
Genetic and neural regulation of sleep and arousal Research Project | 1 Project MembersStimuli that elicit responses in awake animals fail to do so during sleep. This increased arousal threshold and locomotor quiescence are hallmarks of sleep behavior, but the genetic and neural mechanisms that control arousal remain poorly understood. Inappropriate regulation of arousal contributes to insomnia or nonrestorative sleep, which affect up to a third of the population. Revealing how the brain regulates arousal states is thus of significant biomedical importance. Genetic studies have provided entry points to define genes and neural circuits regulating arousal. For example, studies of specific G-protein coupled receptors (GPCRs) have revealed sleep regulatory mechanisms in Drosophila, zebrafish, mouse and humans, while family linkage and genome-wide association studies have identified candidate genes involved in human sleep behavior and sleep disorders. Our proposed research extends these studies by analyzing the cellular roles of genes involved in human insomnia and Restless Legs Syndrome (Aim 1), and by revealing novel functions of G-protein coupled receptors in sleep and arousal (Aim 2). We use zebrafish as a model system because powerful imaging, genetic, genomics, and behavioral approaches can be combined to investigate vertebrate sleep and arousal. In preliminary studies, we disrupted genes associated with human insomnia and Restless Leg Syndrome (RLS) and profiled their effects on behavior. We discovered that mutations in the transcriptional regulators meis1b and skor1a/b lead to overlapping arousal phenotypes and perturbed cerebellum function. These results suggest that meis1b and skor1a/b regulate cerebellar development and function, and implicate the cerebellum in the regulation of sleep and arousal (Aim 1). In a complementary approach to identify novel sleep and arousal regulators, we disrupted 93 brain-expressed G-protein coupled receptor genes. In our initial screening efforts, we discovered several mutants with abnormal brain activity or behavior. In particular, galr2a/b and gpr101 mutants exhibit daytime hyperactivity and hypoactivity, respectively (Aim 2). We will extend these studies as follows: Aim 1: Dissect how the transcriptional regulators meis1b and skor1a/b regulate cerebellum circuitry and arousal. We hypothesize that disruption of specific subsets of cerebellar neurons generates aberrant locomotor drive and heightened sensory responsiveness in meis1b and skor1a/b mutants. To test this idea, we will use imaging, genomic, and neuronal manipulation approaches to define, phenocopy, and rescue cerebellum defects in meis1b and skor1a/b mutants. These studies will reveal previously unknown functions for Restless Legs Syndrome genes, as well as novel roles for cerebellum circuitry in arousal regulation. Aim 2: Identify G-protein coupled receptors that modulate sleep and arousal. We will complete our genetic screen to identify GPCRs that regulate arousal and identify common signaling pathways by intersecting phenotypes with previous pharmacological and overexpression screens. To prioritize hits for follow-up studies, we will characterize candidates from the primary screen with a battery of secondary behavioral tests, characterize GPCR expression vis-à-vis whole brain activity signatures, and determine relevant ligand/receptor relationships. These studies will help identify novel GPCRs and signaling pathways regulating sleep and arousal. These studies will enrich our genetic and circuit-level understanding of how the brain controls sleep and arousal, and inform potential diagnostic or therapeutic approaches to human sleep disorders.
Jane Coffin Childs Fellowship Award - Dr. Emily Bayer Research Project | 2 Project MembersThe neuronal regulation of internal organs has been historically considered to be 'self-contained' and autonomous. However, it is increasingly appreciated that brain-body communication modulates not just the target organs, but also the brain itself. I'm combining the incredible specificity of the C. elegans pharyngeal nervous system with a study of the less-described visceral innervation in zebrafish, a system still compact enough that it can be detailed comprehensively. In C. elegans , I am using inducible neuronal silencing of pharyngeal neurons in combination with behavioral assays for somatic neuron function to identify novel behavioral outputs modulated by internal sensation. In zebrafish, the genetic identities, modalities, and functions of visceral neurons (from both the vagus nerve and cranial sensory ganglia) are still largely undescribed. I have used scRNAseq approaches to collect transcriptomic data on these cell types in adult animals, and will complement this approach with spatial transcriptomics to resolve the anatomical organization of molecular cell types. This will allow me to examine what kinds of information are passed between organs and the CNS (based on the nature of sensory receptors expressed), and what the circuits responsible for this look like (based on the anatomical properties and signaling capabilities of the involved neuron types).
The molecular and genetic basis of kin recognition in zebrafish (Oded Mayseless) Research Project | 1 Project MembersKin selection is a widespread phenomenon through which individuals gain indirect fitness by directing altruistic behaviors toward genetically related individuals. The mechanism that permits recognition between individuals, based on genetic relatedness, needs to be highly selective and refined. Despite its critical nature, the sensory mechanisms governing this phenomenon remain incomplete. Larval zebrafish prefer kin-specific vs nonkin odors. Larvae that have been isolated or exposed to non-kin cues lose their capacity to recognize kin. Specific olfactory sensory neurons (OSNs) and the highly polymorphic major histocompatibility complex (MHC) have been implicated, however the molecular mechanisms which mediate the specificity of kin odor recognition are unknown. By combining behavioral, functional, and molecular approaches, we will unravel the molecular basis of kin recognition.
Dissecting the emergence of neuronal cell populations in the habenulae of the vertebrate brain (BIF Fellowship Jakob El Kholtei) Research Project | 2 Project MembersThe vertebrate brain displays an incredible number and diversity of neurons. Neural stem cells, or progenitor cells, give rise to this neuronal diversity through the process of neurogenesis. Despite many scientific efforts, we are yet to attain a comprehensive understanding of how these progenitors establish the myriad of neuronal cell types in the correct locations and quantities. By establishing and using genomic and optical lineage tracing tools in the zebrafish, I will study the dynamics of neural stem cells in the context of the vertebrate habenulae, a conserved forebrain region. Taking advantage of the comparably simple structure and limited size of the habenulae, I will reconstruct a comprehensive, spatially resolved lineage tree of a complete vertebrate brain region. By combining this approach with a molecular characterisation of habenular development using single-cell RNA-sequencing and sequential fluorescent in situ hybridisation, I aim to gain a comprehensive understanding of neurogenesis in this brain region. The method development involved in this project and the anticipated findings will significantly contribute toour understanding of vertebrate brain development.
