Clifton Ragsdale - US grants

DESCRIPTION (provided by applicant): The broad goal of this research is to understand the cellular and molecular mechanisms by which brain nuclei of the appropriate size, shape and neuronal cell-type constituency are generated during development. We study this problem in the midbrain, a principal division of the vertebrate brain. During the period of neurogenesis, the chick ventral midbrain is transiently organized into regularly spaced, longitudinal zones distinguished by morphology and histochemistry. Our hypothesis is that these zones, called midbrain arcs, are a patterning mechanism by which neurons are allocated to different nuclear fates. The aims of our research are (1) to test a prediction of this hypothesis, that the midbrain arcs differ in their fiber-connections, and (2) to discover the molecular mechanisms by which arcs are generated during midbrain development. In the initial funding period, we established that the most medial arc gives rise to two nuclear structures, the oculomotor complex and the red nucleus. We also demonstrated that a complete set of midbrain arcs could be elicited by an ectopic source of the morphogen Sonic Hedgehog (SHH). In the proposed experiments we will extend our test of the arcs-as-pronuclei hypothesis to lateral midbrain, and we will address whether a SHH source patterns the arcs directly, as a long-range morphogen, or acts in part through bone morphogenetic protein (BMP) intermediaries. The first set of experiments studies the anatomy of the lateral midbrain arcs with anterograde and retrograde tract-tracing methods. A finding of that the lateral arcs have characteristic neuronal connections would not only support the pronuclear hypothesis, but could serve to identify particular arcs as specific pronuclei. The second set of experiments tests the competency of SHH to specify midbrain arc cell-types directly, as a long-range morphogen. These experiments will be carried out in wild-type and talpid(2) mutant chick embryos using in ovo electroporation and explant tissue culture methods. Our finding that ectopic SHH induces BMP gene expression raises, however, the possibility that BMP2 and BMP7 may participate in long-range signaling by a SHH source. The third set of experiments tests the contributions of BMP signaling to midbrain arc patterning. The ventral midbrain is a frequent site of pathology in stroke and other neurological disorders. Understanding its developmental biology should facilitate research into therapeutics, including those based on tissue transplants. More generally, ventral midbrain development provides a model for understanding how brain nuclei form. Insight into these mechanisms may suggest novel approaches to neurological disorders due to nuclear ablations or disconnections.

A key advantage in mouse molecular genetics is the ability to tailor transgene expression to particular tissues at particular times in development. Such ability allows for increasingly more sophisticated analysis of gene function and detailed understanding of developmental fates, and the molecular and cellular mechanisms underlying those fates. These methodologies rely on germ-line manipulations in mouse embryos and are not directly applicable to other models of development, such as chick embryogenesis. Dr. Ragsdale and his coworkers will explore a more broadly applicable method of heritable, conditional transgenesis, one that could be used in a range of animal embryos. Avian retroviruses have a restricted host range, and even among chickens there are strains of birds that are susceptible to infection by only certain viral subgroups. The relevant viral receptors have now been cloned and can confer viral infectivity to heterologous cells. In addition, in a growing number of experimental systems, transient transgenesis can be achieved by direct tissue electroporation of embryos. Dr. Ragsdale will develop a transgenesis system in which electroporation delivers avian retroviral receptors for transient misexpression. The location and timing of the exogenous receptor expression is controlled by the time and site of the electroporation and can be further restricted by tissue-specific promoters. Subsequent retroviral infection then provides for heritable transgene delivery only to those cells expressing the viral receptor at the time of the infection. Dr. Ragsdale will explore this system in chick neural development, but its applicability is potentially much broader and includes the range of embryos, from ascidians to mice, where in vivo electroporation can be used to introduce plasmid DNA into developing organs.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the molecular development of the nuclei and circuitry of the brainstem oculomotor control system. The focus of this project is on the regulatory mechanisms underlying oculomotor neuron production in the midbrain oculomotor complex (OMC) and the hindbrain trochlear nucleus (TrN). The proposed experiments will be carried out in chick embryos, which have the experimental advantage of site-directed transgenesis by electroporation and which share with other birds a highly organized OMC composed of discrete subnuclei with distinct extraocular muscle targets. The first set of experiments assesses the roles of homeodomain transcription factors in specifying oculomotor neuron identity and generating discrete OMC subnuclei. These experiments include a molecular dissection of the function of the CFEOM2 candidate gene PHOX2A. The second set of experiments investigates the molecular mechanisms that regulate the specification of midbrain progenitor cells to an oculomotor neuron fate, with a focus on the roles of NKX6 and basic helix-loop-helix transcription factors in these processes. The third set of experiments is based on our findings of pronounced transcription factor heterogeneity within the TrN and OMC subnuclei. We will investigate the anatomical correlate of this heterogeneity, focusing on the specific hypothesis that these molecular divisions identify motor neurons with distinct targets in the oculomotor plant. Study of the mechanisms that govern OMC and TrN development may provide insight into genetic diseases of the oculomotor system, most notably those such as CFEOM in which specific pools of oculomotor neurons are lost. More generally, understanding of the unique molecular specification of oculomotor cell types may suggest novel therapeutic strategies, including stem cell-based approaches, for the treatment of oculomotor disorders, and may give insight to the relative resistance of the oculomotor unit to some motor neuron diseases.

tissue /cell culture

DESCRIPTION (provided by applicant): Understanding normal human development depends on animal model systems for experimental insight into underlying biological mechanisms. The long-term goal of this project is to use cephalopod biology to understand human organogenesis, including brain development. Among animals, human beings are large organisms with disproportionately large brains. Cephalopods, which include squids, octopuses, and cuttlefish, are the largest non-mammalian marine organisms and have the largest brains among all invertebrates. To study cephalopod development at a mechanistic level requires the identification of a cephalopod with ready availability, easy husbandry, and an embryology permitting experimental manipulation. The investigators have identified such a preparation, the Californian mud-flat octopus, O. bimaculoides. To date, they have developed methods for ex ovo culturing and for whole mount in situ hybridization for embryonic stages from the blastoderm to hatching. The investigators have also isolated a large panel of cDNAs for key developmental control genes. To complete this model system, they need to develop reliable methods of gene manipulation in these cephalopod embryos. Specific Aim 1 is to generate cephalopod-specific expression vectors based on octopus EF1-a and ubiquitin C genes. Specific Aim 2 is to develop methods of plasmid delivery to octopus embryos, for which the investigators will test as methods electroporation and chemical transgenesis using cationic polymers. Development of gene manipulation resources and methods will permit hypothesis-driven research of the molecular and cellular mechanisms of cephalopod development.

The purpose of the Neural Cell Culture Core is to provide MRDDRC funded researchers with primary cultures of embryonic and postnatal dissociated rat and mouse neurons, organotypic brain slices, wellcharacterized chick neural cell cultures, established neural cell lines, and customized gene transfection using the AMAXA system. A key objective of this core is to complement the Model Organism Core by providing high quality, standardized primary cell cultures of neural cells of genetically modified organisms. The University of Chicago has had a long history of primary neural cell culture dating back to the original aggregate studies of Beatrice Garber and Aaron Moscona and we have the expertise to isolate pure cultures of neurons, astrocytes, oligodendrocytes, microglia and neural stem cells as well as slices and organotypic cultures. Since many important questions such as ligand-receptor interactions and cell signaling pathway regulation can best be addressed in pure cell cultures, the investigators identified as users of the Model Organisms Core have identified a primary cell culture core facility as key to their future research progress.We envision that investigators supported by the Center will be able to use the core facilities to test specific ideas in purified cells or organotypic cultures. At present such studies are not possible for the individual investigator because of the time it takes to obtain the necessary expertise in cell isolation and quality control. The Core will also save users the many capital expenses involved in obtaining equipment necessary for preparation of primary cultures and transfection with the AMAXA system. In addition, the Core will be able to provide the reagents, consumables, media, and work hours required to prepare such cultures at a fraction of the cost that would be required if the MRDDRC researcher were to prepare the cultures themselves.

Octopuses, with their endlessly flexible arms, their vertebrate-like eyes, and their sophisticated behaviors are a source of ongoing fascination to scientist and public alike. Octopus brains are large, with many lobes, and from a distance look much more like the brain of an insect or crab than that of a fish or mammal. How the connections of the octopus brain are organized is unknown, so researchers can only begin to guess how octopuses control their arms or learn how to see. The research aim of this project is to study octopus brain circuitry with the tools of modern molecular cell biology in order to understand how the largest invertebrate brains are organized. The broader impacts of this research include the participation of high school and College students in octopus research each summer and the training of graduate students in comparative neuroscience. Research findings will be incorporated into an evolutionary neurobiology teaching module that will be offered in two introductory courses on the fundamentals of neuroscience at the University of Chicago, Marine Biological Laboratory. The Ragsdale laboratory engages directly in public outreach on octopus brain biology, behaviors and evolution. The forums for this outreach include laboratory tours for local high school students, internet video presentations, and feature stories on Science blog sites.

A modern investigation of the neural bases of cephalopod behavior depends on neuroanatomical and genomic databases, ideally ones for a model organism. The Ragsdale laboratory has identified a candidate model cephalopod, Octopus bimaculoides. This project will describe the systems neuroanatomy of O. bimaculoides, including its molecular organization with transcriptome-­based gene expression analysis. The proposed experiments focus on two systems best understood from lesion and stimulation studies: the axial nerve cords of the arms (Aim 1) and the learning and memory lobes (LMLs) of the supraesophageal brain (Aim 2). Aim 1 will ask how the sensory-­motor circuits of the arms are organized. Aim 2 will study the systems architecture of the LMLs. In many taxa, learning and memory organs feature orthogonal arrays of neuronal processes that form longitudinal zones. Such organization has been found in insect mushroom bodies and the vertebrate cerebellum. Findings with the Golgi method suggest that octopus LMLs also have orthogonally organized connections, but to date, no zonal organization has been reported. Aim 2 of this project will use techniques of modern neuroscience to determine how the octopus LML is organized.

DESCRIPTION (provided by applicant): The long-term goal of this project is a cephalopod model system for studies into mechanisms of embryogenesis, neural development and regeneration. Among animals, human beings are large organisms with disproportionately large brains. Cephalopods, which include squids and octopuses, are the largest non-mammalian organisms and have the largest brains among all invertebrates. They also have a cellular pattern of early embryogenesis that is strikingly similar to that of vertebrates. To study cephalopod development at a mechanistic level requires the identification of a cephalopod species with ready availability, easy husbandry and an embryology permitting experimental manipulation. We have identified such a preparation, the California Two-spot Octopus (O. bimaculoides). An important additional advantage in choosing an octopus for a cephalopod model system is that octopus arms, each of which contains a massive central nervous system, regenerate rapidly after amputation and could serve as a new model in regenerative medicine. To date, we have developed methods for octopus in situ hybridization and immunohistochemistry, engineered an octopus plasmid expression vector and demonstrated gene delivery into octopus embryos and arm tissue. We have also isolated cDNAs for a small panel of octopus developmental regulatory genes by degenerate primer PCR and through plasmid cDNA library sequencing. For O. bimaculoides to be a credible model system will, however, require a much greater depth of information about octopus gene expression and regulation. The first aim of the proposed work is to develop RNAseq databases for O. bimaculoides using state-of-the-art sequencing technologies and assembly programs. The second aim is to apply this transcriptome information about message structure to a concurrent O. bimaculoides genome sequencing project, and to build out a cephalopod community web site (www.cephseq.org) for sharing sequence data. Cephalopod genomes are large and repeat-rich so transcriptome data will be essential for genome assembly and annotation. The generation of these transcriptomic and genomic resources will drive hypothesis-driven research on the molecular and cellular mechanisms of cephalopod development and regeneration.