Robert E. Steele - US grants

The goal of the proposed research is to understand how pp60src functions to regulate structural and biochemical features of the Xenopus oocyte. The nature of cytoskeletal changes caused by expression in oocytes of pp60src with constitutive kinase activity will be examined. The pattern of tyrosine phosphorylation in oocytes producing kinase-constitutive pp60src is altered when such oocytes are made to reenter the cell cycle by progesterone treatment. Activation of the oocyte's endogenous maturation promoting factor (MPF) by microinjection of cyclin mRNA will be used to examine whether MPF is responsible for this change in substrate pattern. Immunochemical and biochemical methods will be used to determine whether changes in pp60src distribution or modification accompany the change in substrate pattern. Site directed mutagenesis will be used in an attempt to define the portion of pp60src which mediates the response to progesterone.

Partial support is requested to defray room and board costs for graduate students attending the 1992 West Coast Regional Developmental Biology Conference. The conference will be held on April 30-May 3 at the Stanford Sierra Camp, Fallen Leaf Lake, California. The program includes 25 speakers, reflecting as full a mix of gender, race and degree of professional advancement as is compatible with the primary criterion of scientific excellence. Topics to be addressed in the five formal sessions are as follows: 1) Regulatory Hierarchies, 2) Cell Polarity in Development, 3) Control of Cell Number, 4) Signal Transduction, and 5) Specification of Cell Fate. The conference will also include a keynote address to be given by Dr. Susan Bryant on the first evening. All attendeees will be encouraged to contribute to a formal poster session.

Protein-tyrosine kinase receptors are important mediators of developmental signals in multicellular animals. The goal of the project in this application is to understand how receptor protein-tyrosine kinases are used in developmental processes in the simple animal Hydra. The attraction of Hydra as a system for these studies lies in the extreme simplicity of its structural organization and development. The initial approach to be taken will involve completing the molecular characterization of a gene from Hydra which is a member of a developmentally important class of receptor protein-tyrosine kinases in vertebrates. The cloned gene for this receptor will provide reagents for determining what cells in the polyp produce the receptor. With methods which we have developed for introduction of cloned genes and synthetic oligonucleotides into Hydro, it will be possible to carry out gain of function and loss of function experiments to determine what developmental processes depend on the receptor whose gene we have isolated. By expression studies it may be found that the receptor is expressed in the stem cells or committed precursors of a particular developmental lineage. If so, we can use antibodies against the receptor to isolate the cells which express it. These isolated cells can be introduced into stem cell- depleted Hydra polyps to determine their developmental fates. Such information will help in understanding how input via this receptor directs the development of cells. Because receptor protein-tyrosine kinases have been so well conserved during animal evolution, determining their roles in Hydra development should provide insight into how these proteins function in development in more complex animals. The well- documented involvement of receptor protein-tyrosine kinases in cell growth and differentiation and their ability to be converted to oncogenic forms indicate that an understanding of the roles these proteins play will have implications for understanding, diagnosing, and treating human diseases ranging from cancer to birth defects.

A key innovation during the evolution of multicellular animals was the ability to form and maintain a body axis. Cnidarians are the earliest diverging animals in which a clearly defined axial pattern exists. This pattern could be established by molecular mechanisms evolutionarily related to those responsible for patterning of the anterior/posterior axis in the embryos of protostomes and deuterostomes. If this was the case, the investigator would conclude that axial patterning evolved only once during the evolution of extant metazoans. Alternatively, the molecular mechanisms of axial patterning in the Hydra polyp could be largely or even completely distinct from those used in more recently diverged animals. Such a finding would indicate that axial patterning evolved more than once during the evolution of metazoans. Either of these possible outcomes will have a major impact on understanding the evolution of multicellular animals.

A number of genes have been identified which are attractive candidates for involvement in axial patterning in the freshwater cnidarian Hydra. Functional tests of these roles of theses genes in axial patterning have, however, not been possible due to the lack of methods for introduction and expression of cloned genes in adult Hydra polyps. The goal of this project is the development of a method for obtaining expression of exogenous gene products in Hydra. Several avenues will be pursued. These include: (1) use of a pantropic retrovirus as a vector for stable introduction of cloned genes into Hydra embryos or into the spermatogonial cells of the adult polyp; (2) microinjection of DNA into embryos; (3) introduction of DNA into embryos and adult polyps via particle bombardment; and (4) introduction of proteins into the cells of adult polyps using the TAT-mediated protein uptake system.

A key innovation during the evolution of multicellular animals was the ability to distinguish self from non-self. The molecular mechanisms underlying such recognition are understood in considerable detail in vertebrates, particularly as mediated by the various classes of antigen receptors. Despite considerable effort, however, it has not been possible to trace the evolutionary history of vertebrate antigen receptors into any metazoan taxon which diverged earlier than the jawed fishes. Such efforts have been focused on the use of molecular methods in attempts to identify genes encoding orthologues of vertebrate antigen receptors. This project will makes use of a different approach to address the origins of antigen receptor signaling, based on the finding in the early-diverging invertebrate Hydra of a Syk family protein-tyrosine kinase (PTK). Syk PTKs interact with phosphorylated tyrosine residues on the cytoplasmic tails of antigen receptors in vertebrates through two src homology 2 domains. Thus the molecules with which Hydra Syk interacts are candidates for mediating recognition of foreign cells in this organism, and are potentially evolutionarily related to vertebrate antigen receptors. The objective of this project is to identify the molecules with which Hydra Syk interacts and thereby to trace the evolutionary history of the antigen receptor signaling pathway. Initial experiments will test the ability of Hydra Syk to bind to and function with vertebrate antigen receptors. A positive result in this case would provide support for conservation of the basic mechanics of Syk/receptor interaction over a wide range of metazoan evolution. Subsequent experiments will be directed towards identifying the molecules with which Syk interacts in Hydra. Comparisons of these molecules with vertebrate proteins should indicate whether there is any evolutionary continuity between them and vertebrate antigen receptors. If no such continuity is evident, the relationships of the interacting proteins in Hydra with the proteins of other metazoans will nonetheless reveal important, and currently unavailable, information regarding the evolutionary history of the signaling pathway which was modified for use with vertebrate antigen receptors.

0120591
Bode

It is becoming increasingly clear that understanding how an organism interacts with its environment will require a complete accounting of the genes which make up the organism and the expression patterns of the genes under various conditions. Members of the phylum Cnidaria (jellyfish, corals, sea anemones, and hydras) are key members of their environments, yet very little is known about their genetic makeup. To address this problem, a collection of cloned DNAs representing the messenger RNA populations (cDNAs) of two well-studied cnidarians will be generated. The two organisms that will be used are the freshwater cnidarian Hydra and the colonial marine cnidarian Podocoryne. Bacteria containing the cloned cDNAs will be robotically arrayed to generate archives of the clones. Sequence data will be obtained from approximately 50,000 of the cDNA clones from each organism. The resulting sequence data will be analyzed in various ways using bioinformatic computing tools. The analyses will provide information on what genes are present in cnidarians and how those genes are evolutionarily related to those in animals which diverged more recently than cnidarians, such as vertebrates and insects. Such information will be very valuable for defining the processes by which multicellular animals have evolved. The availability of cloned cDNA sets from two model cnidarians will also make it possible to examine the expression of large numbers of genes in these organisms using the technique of DNA array analysis. In particular it will be possible to identify genes whose expression changes when the organisms are placed under conditions which reflect those present during periods of environmental stress (e.g. elevated temperature). Genes whose expression levels change under stress conditions in the laboratory may be useful tools for monitoring the health of cnidarians (e.g. corals) in their natural setting.

0208335
Steele

A complete description of the evolutionary history of multicellular animals will require detailed information on the content, organization, and regulatory mechanisms of the genomes of animals from phyla throughout the animal phylogenetic tree. Of particular importance for such studies are species that diverged early from the rest of the animals and unicellular organisms which are close relatives of animals. Features of the genomes of such organisms which are shared with more recently diverged animals would then be ones which were present in the ancestor of all modern animals. The goal of this project is to prepare and archive bacterial artificial chromosome libraries from Nematostella vectensis (a sea anemome which is a member of the early diverging animal phylum Cnidaria) and from the choanoflagellate Monosiga brevicollis, a unicellular organism closely related to multicellular animals. These libraries will provide important resources for addressing a variety of questions related to the origins, evolution, and development of multicellular animals. Such questions include: How did the Hox family of developmental regulatory genes evolve? When did various other developmental regulatory gene families appear during animal evolution? How closely related are choanoflagellates and multicellular animals, and what genes were necessary to make the transition from unicellularity to multicellularity? What happened at the genome level when nerves were added to multicellular animals? What cell adhesion and intercellular signaling molecules were present in the common ancestor of modern animals? Are cnidarian genes organized into operons, as suggested by the presence of spliced leader addition in this phylum? In addition to providing material for analysis of animal evolution, the libraries will facilitate the isolation of gene promoters, which will be essential for the development of functional assays in these organisms and for dissecting gene regulatory networks. The libraries generated by this project will be freely available to the research community. Information on the libraries and their availability will be provided through various means, including appropriate scientific newsgroups and web sites.

DESCRIPTION (provided by applicant): Because of its simple composition and construction, ease of experimental manipulation, regenerative capacity, and stem cell systems, Hydra is a potentially important model system for investigation of a wide range of biological and health-related problems at the tissue, cellular, and molecular levels. The recent completion of the Hydra genome sequence and the publication of a method for making stably transgenic Hydra provide important new tools for exploiting Hydra as a model system. The goal of the project described in this proposal is to generate resources that will allow and motivate the research community to exploit Hydra as an experimental system. To achieve this goal it will be necessary to have a set of vectors with various properties for use in constructing transgenic lines. Thus one of the aims of this project is the development and validation of vectors that will allow: 1. Conditional expression of transgenes;2. Expression of epitope- tagged proteins;3. Loss of function phenotypes by RNA interference and expression of a toxin gene. The second major aim of the project is to carry out high-throughput in situ hybridization to determine the expression patterns of a wide range of genes identified from the Hydra genome project. These will include genes encoding transcription factors, signaling proteins, extracellular matrix proteins, and cell junction proteins. Particular attention will be paid to Hydra genes homologous to human disease genes. The resulting catalog of expression patterns will provide a starting point for functional studies of genes using the transgenic tools developed by the project. Public Health Relevance: Given the currently available tools and resources for Hydra research and the additional resources that we propose to generate, we envision using these resources to exploit Hydra as a model system for basic biological and biomedical research.