James David Jontes, PhD - US grants

DESCRIPTION (provided by applicant): The function of the central nervous system (CNS) is dependent upon the establishment of synaptic junctions during development and the proper patterning of neuronal connectivity to generate functional circuits. Failure to establish the appropriate synaptic connections can result in severe disorders of the nervous system, including mental retardation, autism, schizophrenia and depression. Thus, it is essential to understand the molecular mechanisms responsible both for assembling the synaptic junction, and for directing the selection of functionally appropriate synaptic partners. Our long-term goal is to understand the cellular and molecular mechanisms responsible for synaptic assembly and selection in the vertebrate CNS. To address these questions, we are using in vivo multiphoton imaging of synaptogenesis in the developing zebrafish CNS, focusing on the roles played by 3-protocadherins. The embryonic zebrafish is an ideal system in which to address these questions, as embryos are transparent, develop externally and rapidly, and have a relatively simple and highly stereotyped CNS. In addition, the recent development of site-specific modification of the genome through the use of engineered zinc-finger nucleases (ZFNs) will enable the efficient production of zebrafish lines that harbor mutations in genes of interest. In this exploratory R03 proposal, we will use the emerging ZFN technology to generate lines of zebrafish carrying lesions in each of the 3-protocadherin genes present in zebrafish, pcdh13 and pcdh23. Having generated and validated these lines, we will also perform an initial phenotypic analysis. As targeted deletion of pcdh3 in mice results in extensive neuronal death throughout the CNS, we will first assess the levels of programmed cell death occurring in the developing nervous system. We will also characterize any effects on the organization of the nervous system, using wholemount immunocytochemistry to label the early scaffold of axon tracts. Finally, we will use both immunocytochemistry and live imaging to identify any defects in the formation or maintenance of synaptic vesicle clusters. The pcdh3 mutant lines produced from this work will be invaluable resources for investigating both the cellular and developmental function of Pcdh3, allowing us to take full advantage of the zebrafish model system. PUBLIC HEALTH RELEVANCE: The formation of synaptic junctions and their patterning into circuits of appropriately connected neuronal populations is essential for the normal development of the central nervous system. Defects in these events can have a profound influence on human behavior, and may underlie numerous disorders of the nervous system, including autism, mental retardation and schizophrenia. The results of our studies will greatly enhance our knowledge of 3-protocadherin function in neural circuit assembly, and will provide a strong foundation for developing novel therapeutic strategies for addressing these disorders. These investigations will also contribute to a firm understanding of the developmental mechanisms responsible for synaptogenesis, which could be essential for creating effective approaches to promote repair after brain or spinal cord injuries.

DESCRIPTION (provided by applicant): Selective cell-cell interactions are required to establish the precise three-dimensional architecture of the vertebrate nervous system through coordinated cell movements, regional partitioning, axon targeting and synapse formation. The cadherin superfamily comprises a large, diverse array of cell surface receptors that includes both the classical cadherins and the protocadherins. Both classical cadherins and protocadherins have been proposed to play roles in establishing neural circuitry in the developing brain. While the classical cadherins are known to be homophilic cell adhesion molecules, the involvement of protocadherins in cell adhesion remains uncertain. In our previous work, we showed that Protocadherin-19 (Pcdh19) associates with N-cadherin (Ncad) to form a heteromeric cis-complex. In preliminary data, we show that Pcdh19 mediates homophilic adhesion as part of the Pcdh19-Ncad complex, although it is not adhesive itself. The main hypothesis of this proposal is that protocadherins associate with classical cadherins to form cis-complexes with distinct adhesive properties. To investigate this hypothesis in more detail we propose the following Specific Aims. In Aim 1, we will test the hypothesis that members of the d-protocadherin family form cis-complexes with classical cadherins and determine whether these complexes mediate homophilic adhesion, as we have shown for Pcdh19-Ncad. In Aim 2, we will first determine the residues in Pcdh19 that constitute the adhesive interface, and then determine if this mechanism is general to Pcdh-Cdh complexes. Insights gained from these experiments will elucidate the molecular underpinnings of a novel mechanism of cell adhesion and will advance our knowledge of protocadherin and cadherin function, providing an essential foundation for understanding the roles of these molecules in neural development and elucidating the mechanisms that establish patterns of neural connectivity. PUBLIC HEALTH RELEVANCE: Protocadherins and cadherins have been implicated in a number of neurodevelopment disorders, including autism, schizophrenia, epilepsy and mental retardation. Despite the importance of protocadherins to neural development, very little is known about their molecular and cellular functions or developmental roles. Our studies will dramatically advance our knowledge of the mechanism of cell adhesion by the protocadherins and provide a foundation for understanding their roles in the development of the vertebrate nervous system.

DESCRIPTION (provided by applicant): The development of the vertebrate brain requires complex genetic networks to coordinate myriad processes across time and space, which include cell proliferation, differentiation and migration, along with axon guidance, synapse formation and elimination. Orchestration of these processes results in the assembly of microcircuitry and distributed systems that constitute a functional nervous system. Disruptions in neural development can have a dramatic impact, resulting in complex brain disorders, such as autism spectrum disorders, schizophrenia, mental retardation and epilepsy. Increasingly, genetic data implicate genes involved in axon guidance and synaptogenesis in these complex brain disorders. In addition, evidence suggests a common etiology for these disorders, as mutations associated with one disorder are also associated with increased incidence of others. Among the molecules that have been implicated in these complex brain disorders are the protocadherins. In particular, mutations in pcdh19 result in a female-limited form of infant-onset epilepsy. Despite the clear role for these molecules in neural development and the etiology of neurodevelopmental dysfunction, relatively little is known about their function in vivo. Here, we propose to generate lines of zebrafish in which pcdh7a, pcdh9, pcdh17 and pcdh19 have been inactivated using zinc finger nucleases (ZFNs). In addition, we propose to generate BAC transgenic lines expressing GFP under the control of the pcdh7a, pcdh9, pcdh17 and pcdh19 regulatory elements. To study the effects of the protocadherin mutations on neural development, we will cross the GFP lines into the mutant backgrounds and use in vivo 2-photon microscopy to determine the impact of the mutations on nervous system development. This work will significantly advance our understanding of protocadherin function, providing a foundation for more mechanistic studies and facilitating a better understanding of how disruption of protocadherins can lead to complex brain disorders. PUBLIC HEALTH RELEVANCE: This project is relevant to human health, as mutations in pcdh19 cause a female-limited form of epilepsy and are associated with an increased incidence of other brain disorders. In addition, pcdh7, pcdh9 and pcdh17 have been implicated in autism or schizophrenia.

Animals and their constituent tissues are assembled from large numbers of cells, which are held together by specialized adhesion molecules. As the three-dimensional structure of embryos emerges in development, cell-cell adhesion regulates the coordinated changes in cell shape, and movement, as well as cell number. How cell-cell adhesion coordinates these cell properties during development is not well understood. The long-term goal of this research program is to understand how cell adhesion controls development using the vertebrate brain as a model. The Principle Investigator has previously found that two adhesion molecules (Protocadherin-19 and N-cadherin) appear to cooperate in early stages of brain development. Investigators will use time-lapse imaging of living zebrafish embryos to visualize the functions of Protocadherin-19 and N-cadherin during the formation of the embryonic brain. As both Protocadherin-19 and N-cadherin are members of larger families of molecules, the proposed studies will be broadly relevant to the mechanisms by which vertebrate animals are formed during development. The proposed research will provide a rich training environment at the graduate, undergraduate and high school levels, with exposure to state-of-the-art genetic tools, sophisticated imaging approaches and quantitative image analysis.

During animal development, changes in cell number, cell shape and cell movement are coordinated to bring about the morphogenesis of complex three-dimensional tissues. Cell-cell adhesion is particularly important for morphogenesis, as it facilitates cell motility, maintains cell polarity and coordinates collective cell migration. Despite this central importance, relatively little is known about the mechanisms that dynamically regulate cell-cell adhesion during development. The classical cadherins (including N-cadherin) comprise a family of hemophilic cell adhesion molecules that are essential for vertebrate morphogenesis, and participate in a range of developmental processes. Protocadherins are a large family of molecules that are related to classical cadherins, although much less is known about their cellular and developmental functions. Previous work from this investigator's laboratory showed that Protocadherin-19 (Pcdh19) is involved in anterior neurulation in zebrafish, that it functions synergistically with N-cadherin (Ncad), and that Pcdh19 and Ncad interact in cis to form an adhesive complex. This interaction could provide a mechanism for dynamically switching between functional adhesive states within the cell, thus coordinating distinct adhesive systems during complex sequences of morphogenetic movements. The core objectives of this proposal are to: 1) understand how adhesion by Ncad and Pcdh19-Ncad coordinate collective cell behaviors during anterior neurulation, and 2) understand how these distinct adhesion mechanisms regulate actin dynamics during cell movements. To accomplish these objectives, the Principle Investigator and colleagues have established a zebrafish line mutant for pcdh19, as well as transgenic lines generated with ncad and pcdh19 promoters. These transgenic and mutant zebrafish lines will be used in conjunction with in vivo 2-photon time-lapse microscopy to dissect the functional significance of the Pcdh19-Ncad complex during development of the nervous system, and to determine how they influence actin dynamics during coordinated cell movements.

ABSTRACT The neural circuits underlying perception and behavior are assembled during development through intricate and coordinated processes that include neurogenesis and migration, axon and dendrite extension and arborization and synapse formation. In turn, conserved genetic programs orchestrate these dynamic developmental processes. While much progress has been made in identifying genes that are important for neural function, the mechanisms linking the action of gene products to the assembly of neural architecture and to function are poorly understood. To address this important question, we have generated zebrafish mutants and transgenic lines for several ?-pcdhs, evolutionarily conserved homophilic cell adhesion molecules that are strongly expressed in the developing nervous system. We show that ?-pcdhs are expressed within radial columns of neurons in the developing zebrafish optic tectum, and that the neurons within these columns are siblings derived from one or a small number of progenitors. Loss of pcdh19 degrades the columnar organization of pcdh19-expressing neurons, indicating that protocadherin function is required for column maintenance. Moreover, pcdh19 mutants exhibit defects in visually guided behaviors. This proposal tests the hypothesis that the shared inheritance of a ?-pcdh confers an identity to a column of neurons, and that differential expression ?-pcdhs defines a code for organizing tectal circuitry and is essential for neural function and behavior. Specifically, we will map the 3D distribution of neurons expressing individual ?-pcdhs, use in vivo timelapse to determine the cellular roles of ?-pcdhs during column formation, and use in vivo calcium imaging to determine the effects of ?-pcdh loss on the development of neural activity patterns. This study will shed light on a fundamental aspect of neural organization and generate essential new insights into the relationships between genes, the development of neural architecture and the origins of a range of neurodevelopmental disorders attributed to members of the protocadherin subfamily of cell adhesion molecules.

ABSTRACT During development, the division of progenitor cells is tightly regulated in time and place to produce the appropriate number and types of cells; misregulation of proliferation or differentiation can lead to morphogenetic defects and a variety of developmental disorders. The ?-pcdhs are a family of homophilic cell adhesion molecules that have been linked to neurodevelopmental disorders, including autism and epilepsy. They have additionally been identified as tumor suppressor genes in an array of cancers. Our preliminary data reveal that mutant zebrafish lacking individual ?1-pcdhs (pcdh1a, pcdh7a or pcdh9) or ?2-pcdhs (pcdh17, pcdh18b or pcdh19) all display increased cell proliferation in the early neural tube, resulting in excess neurons later in development. In a preliminary study using in vivo timelapse, we show by direct observation that the spatiotemporal dynamics of cell divisions is altered in the mutants. We further provide evidence that the ?- pcdhs are regulators of the canonical Wnt signaling pathway and that both canonical Wnt signaling and the Wnt receptor Ryk are required for the increased proliferation in ?-pcdh mutants. This proposal will test the hypotheses that: 1) cell-cell interactions, mediated by ?-pcdhs, coordinate cell proliferation and neural progenitor cell dynamics in the neuroepithelium; and 2) ?-pcdhs influence cell proliferation by regulating canonical Wnt/?-catenin signaling through Ryk. These experiments will elucidate the mechanics of fundamental events in nervous system development and provide important insights both into the underlying causes of neurodevelopmental disease and to the role of ?-pcdhs in cancer.