Randall Moon - US grants

A protein skeleton lies beneath the plasma membrane of both erythroid and non-erythroid cells, including striated muscle. Little is known about how this skeleton assembles, and even less is known about its functions in nonerythroid cells. The principal aim of the proposed research is to answer the question of how cells regulate the assembly of spectrin, which is an abundant membrane skeleton protein composed of one alpha and one beta subunit. The approach will be to construct full-length cDNAs for each subunit, and to express functional spectrin mRNAs in vitro by transcription from SP6 vectors. To manipulate the process of spectrin assembly it would be useful to express spectrin from the SP6-generated mRNAs in a cell not actively synthesizing endogenous spectrin. Therefore, mRNA for the two subunits will be microinjected into Xenopus oocytes in the presence and absence of putative spectrin-binding sites, and in varying ratios of mRNA for each subunit. By following newly synthesized spectrin subunits by immunoprecipitation with monospecific antisera, we shall determine 1) whether spectrin which is synthesized in excess of the availability of its binding sites is preferentially degraded, as previously hypothesized (Moon and Lazarides, J,. Cell Biol. 98, 1984) and 2) whether assembly of the spectrin subunits in a 1:1 stoichiometry is independent of the ratio of synthesis of the subunits (op. cit.). Induceable constructs for each spectrin subunit will then be cotransfected into murine cells cultured in vitro, initially to further test the hypothesis that over-expression of spectrin will, in the absence of available binding sites, lead to degradation of excess subunits. Second, analysis of transfected cells will determine whether the levels of unassembled spectrin subunits feedback to regulate their synthesis, as proposed by several groups for regulation of tubulin synthesis. Finally, experiments will be initiated with these full-length cDNA constructs to determine the role of nonerythroid spectrin in the formation of specialized membrane-cytoskeletal domains in myogenic cells. The initial approach will be to use anti-sense RNA to induce phenotypic mutations in spectrin in myotubes. Collectively, these studies will determine some of the mechanisms by which cells regulate the synthesis of these membrane skeleton components, the mechanisms by which these polypeptides assemble with a specific stoichiometry, and after assembly has occurred, some of the functions of spectrin during myogenesis. Following the grant period mutagenesis of the full- length constructs will provide a powerful approach for identifying the functional domains of spectrin; defects in which may be causally linked to human hereditary hemolytic anemias (e.g., Lux, Semin. Hematol. 16, 1979).

The candidate's long-term goal is to elucidate the molecular mechanisms by which cytoskeletal proteins participate in the formation of functionally specialized membrane and cytoplasmic domains in developing embryos and differentiating cells. This will involve application of antisense RNA technology initially in Xenopus, but later in transgenic mice. Attached to the inner surface of the membrane of cells is a skeleton of proteins. The major protein in this membrane skeleton is spectrin, which is a very large, flexible, rod-like dimeric protein. It is clear that spectrin is important in human diseases since defects in the alpha-subunit of spectrin in red blood cells are linked to hereditary hemolytic anemia. A related spectrin, termed fodrin, is encoded by different genes and is present in muscle. It is likely that defects in fodrin also occur, and are linked to muscle diseases. The applicant's short term goal is to directly test the hypothesis that alpha-fodrin is important in the development muscle cells. Our approach is to use antisense RNA techniques to cause defects in alpha-fodrin during muscle development in the frog Xenopus laevis. This technique involves cloning an alpha-fodrin cDNA in an antisense orientation under control of one of several promoters. The recombinant plasmids are then microinjected into the cytoplasm of fertilized Xenopus eggs, which are cultured in vitro. The plasmids are transcribed into antisense RNA at the blastula stage of development. At various times during development into tadpoles, the embryos will be assayed at the level of RNA and protein to determine whether expression of antisense RNA from the injected plasmid leads to specific inhibition of alpha-fodrin mRNA and protein. We have already shown that this general approach works, and the present study will focus specifically on the muscle embryos defective in alpha-fodrin. If defects in muscle fodrin lead to defects in normal muscle structure and function, this will support our hypothesis. Once we understand how alpha-fodrin is involved in muscle defects in frogs, our long-term goal is to identify human muscle diseases which also involve defective alpha-fodrin. Related to this goal, we are presently developing full-length human cloned fodrin probes which may be useful in the diagnosis of human muscle diseases which involve fodrin. Dr. Moon is a tenure-track Assistant Professor of Pharmacology at the University of Washington School of Medicine. The Department of Pharmacology expects to provide the space and support necessary for development of the full potential of his research program. Award of an RCDA will enhance Dr. Moon's career development by increasing the portion of time that he can devote to development of his novel antisense RNA method for probing the roles of specific cytoskeletal proteins in morphogenesis and formation of specialized membrane and cytoplasmic domains. This work has important implications in cell biology, molecular pharmacology, and human disease will have broad impact.

Inappropriate expression of proto-oncogenes may be causally related to a variety of cancers and to birth defects. This has led to considerable interest in elucidating the normal pattern of expression and functions of proto-oncogenes which are expressed during development of vertebrate embryos. The proto-oncogene Int-1 has a particularly interesting pattern of expression-it is expressed in a transient manner in the dorsal midline of the neural tube of vertebrate embryos, and may be involved in signalling between cells. The hypothesis that vertebrate embryos express additional genes related to int-1 has not been widely studied. In the proposed project, we shall test this hypothesis by completion of the following specific aims: 1. cDNAs related to int-1 will be cloned by PCR approaches from Xenopus embryo mRNA, using oligonucleotide primers complementary to conserved regions of int-1 and IRP from several species. That these techniques are appropriate is evident from our recent cloning of at least four unique Xenopus cDNAs homologous to int-1 by this approach. 2. To determine whether transcripts complementary to the int-1-related cDNAs are regulated in a temporal manner during development. To determine whether the expression of the transcripts related to the int-1-related cDNAs is restricted to specific regions of embryos, Xenopus embryos will be microdissected, RNA extracted, and RNA blot analyses undertaken. 3. The least conserved region of the int-1-related proteins will be expressed in E. coli following ligation of the cDNAs in frame in the expression vector pRIT2T. Polyclonal antisera will be prepared in rabbits. Whole mount immunocytochemistry of Xenopus embryos will be used to determine the spatial distribution of the int-1-related proteins. 5. mRNA transcribed from nearly full-length int-1-related cDNAs will be microinjected into fertilized Xenopus eggs to deregulate normal expression. Analysis of resulting embryonic phenotypes will provide insight into the normal functions of the int-1-related proteins. 6. Subtractive cDNA probes will be used to clone mRNAs changing in response to expression of int-1-related proteins. 7. Several lines of investigation will pursue cellular receptors for int-1 and int-1-related proteins, as well as second messenger systems involved in signal transduction. Collectively these specific aims will provide new information on the expression and functions of potential proto-oncogenes important cell signalling and communication in vertebrate embryos, as well as human cancers and birth defects.

Wnts are secreted ligands which activate receptor-mediated signaling pathways. Vertebrate Wnts and their signaling pathways have been implicated in playing key roles in axis specification, in patterning mesoderm and the nervous system, in providing polarity to limbs, and in organogenesis. When inappropriately activated, components of the Wnt pathways may be linked to various human cancers notably colorectal cancer and melanoma. The current proposal represents a competing continuation application, to maintain funding for our work on Wnt genes and their signaling pathways in Xenopus. As the previous specific aims have largely been answered, our present proposal introduces new specific aims to address recent questions arising from this prior support. Whereas many of the previous specific aims asked embryological questions, the current focus is on Wnt signal transduction. Aim 1 uses yeast-two hybrid screens, and mass spectroscopy analysis, to identify new components of the Wnt-1/Frizzled-1 pathway. Aim 2 investigates the biochemistry of the distinct Wnt-5a/Frizzled-2 pathway which we discovered in the prior two project periods. Aim 3 investigates the target genes of Wnt signaling and their functions in development. Given the high degree of conservation of the Wnt-1 signaling pathway in evolution, it is likely the results of this work in Xenopus will continue to directly apply to Wnt signaling in other vertebrates.

DESCRIPTION (provided by applicant): The ability to regenerate structures and biological function would be of considerable value to a large number of patients. For many years experiments have focused on identifying what types of structures, in what organisms, undergo regeneration. More recently, studies have begun to identify signal transduction pathways that might be involved in regeneration. Among these pathways are those stimulated by the 19 members of the Wnt family of secreted ligands. Wnt signaling components are expressed during regeneration in multiple species but whether Wnt signaling is occurring and whether it is important for regeneration is unknown. Our specific aims seek to resolve the major outstanding questions regarding Wnts and regeneration. Aim 1: Is Wnt signaling activated during regeneration and if so, in which cells? Aim 2: Is Wnt signaling necessary for regeneration to occur, as determined by loss of function experiments? Aim 3: Can gain of function of Wnt signaling enhance regeneration? Finally, Aim 4 will seek to identify Wnt-responsive genes during regeneration and to study their roles in regeneration. By studying regeneration in Xenopus tadpoles, adult zebrafish, and the injured mouse spinal cord we have preliminary data supporting the first three aims, thereby establishing that Wnt signaling is indeed involved and functional in regeneration. The benefit of simultaneously employing frogs, fish, and mice in our project is that we can determine whether the roles of Wnt signaling in regeneration are conserved between species. Thus the general significance of the proposal is that we have a focused set of experiments that will reveal whether or not Wnt signaling is necessary and sufficient for stimulating regeneration in vertebrates. The immediate relevance to human medicine is that we directly test whether Wnt signaling improves the ability of neural stem cells to promote regeneration in a mouse model of spinal cord injury. This research therefore builds upon experiments in various model organisms to contribute to the development of therapies.

Wnt signaling pathways regulate cell proliferation, differentiation, polarity, and behavior in embryonic development, in adult stem cells, and in cancers and other disease states. Wnts are therefore reasonable candidates for playing essential roles in the normal biology of human embryonic stem cells (hESCs) and for altering the proliferation and differentiation of hESCs cultured in vitro. At least two Wnt signaling pathways exist in vertebrates, the Wnt/li-catenin pathway that controls gene expression through a well-defined pathway, and one or more "non-canonical" pathways that can antagonize the Wnt/B-catenin pathway. In various stem cells the Wnt/li-catenin pathway has been implicated in controlling self-renewal, proliferation, and differentiation. In contrast, the existence or functions of the non-canonical pathways in hESCs have not been reported. The overall goal of this project is to test the hypothesis that distinct Wnt signaling pathways are functionally involved in the in vitro proliferation and differentiation of hESCs, to elucidate the different mechanisms of signaling, and to leverage these insights to direct hESC differentiation in vitro. The specific aimsare: Aim 1. Interrogating distinct Wnt signaling pathways in hESC Aim 2. Regulation of Wnt pathways in hESCs by miRNAs Aim 3. Responses of hESCs to gradients of Wnt-3a and Wnt-5a ^ Collectively, the proposed studies will lend further insight into the mechanisms underlying the regulation of hESCs by Wnt signaling, particularly with regards to the role of Wnts in hESC proliferation, differentiation and motility. These findings can be exploited to manipulate hESCs in vitro with the eventual goal of generating protocols for hESC differentiation that can have therapeutic impact on human disease.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aberrant Wnt/[unreadable]-catenin signal transduction is involved in many diseases. In colorectal cancer and melanoma, mutational disruption of proteins involved in the degradation of [unreadable]-catenin, the key effector of the Wnt signaling pathway, results in stabilization of [unreadable]-catenin and, in turn, activation of transcription. We are employing tandem-affinity protein purification and mass spectrometry to define the protein interaction network of the [unreadable]-catenin destruction complex.

Work in model organisms has revealed that a small number of signal transduction pathways, including the Wnt/b-catenin pathway, play key roles throughout development, as well as in tissue repair and stem cell homeostasis in adults. Interestingly, the composition of the Wnt/b-catenin pathway differs in distinct cellular contexts, depending on expression of unique modifiers of the signaling pathway, or expression of different isoforms of conserved pathway components. We and others have evidence (see Preliminary studies) that Wnt/b-catenin signaling is repressed in pluripotent human embryonic stem cells (hESCs) undergoing selfrenewal, and that signaling is active during differentiation into both early and later mesodermal cell lineages. We hypothesize that context-dependent modifiers of Wnt/b-catenin signaling play key roles in the self-renewal and differentiation of hESCs. The initial goal of this proposal is to test the hypothesis that context-dependent modulators of Wnt/b-catenin signaling play key roles in regulating self-renewal and differentiation in hESCs. We believe that pursuit of this goal will reveal detailed mechanisms by which Wnt/beta-catenin signaling regulates self-renewal and specification of cell fate in hESCs. Our second goal is to expand our investigation of the roles of signal transduction pathways in stem cells to include the Hedgehog, Notch, and TGFb pathways, using novel multiplexed fluorescent reporters to enable simultaneous monitoring of multiple pathways in live cells. We believe that the development of the technology to simultaneously visualize the state of activity of multiple signaling pathways in live cells will have numerous uses in studies of signaling pathways in normal and diseased tissues.