Kathryn V. Anderson - US grants

DESCRIPTION (provided by applicant): The genetic pathways and networks that organize the body plan of the mammalian embryo are not well understood. Between implantation and midgestation, the critical events of axis determination, gastrulation, germ layer specification, morphogenesis and early differentiation of organ systems take place, but the majority of genes that regulate these processes have not yet been identified. This is a proposal to continue to use ethylnitrosourea (ENU) mutagenesis to identify genes that control early development in the mouse embryo. Recessive mutations are identified because they cause clear morphological abnormalities in the midgestation embryo. Positional cloning is used to identify the genes responsible for the developmental phenotypes. This approach has identified mutations in 30 genes required for neural and mesodermal patterning, including at least two dozen previously uncharacterized genes. The molecular lesions responsible for 6 mutations have been identified, including mutations in 4 genes that had not been studied previously in the mouse. The work will now focus on the early events of axis specification, patterning and morphogenesis. Recently identified mutations that affect early patterning and morphogenesis will be mapped and analyzed. Several genes that affect these processes will be cloned and characterized molecularly. To increase the efficiency of identification of genes that control early post-implantation development, including axis specification and morphogenesis, transgenic lines will be generated that express fluorescent proteins in defined subsets of cells in the e7.5-e8.5 embryo. These reporter lines will be used to help characterize mutations that have already been identified and to screen for new mutations that affect early patterning and morphogenesis more efficiently. New mutations will be identified based on inappropriate expression of these reporters and define groups of genes that affect specific aspects of early development. Identification of the genes that control development of the mammalian embryo is crucial for the detection, prevention and treatment of human birth defects. The signaling pathways that act in early development also have critical roles in tumor development, so it is likely that these genes identified in this study will define new steps in tumor development and progression.

DESCRIPTION (provided by applicant): Sonic hedgehog (Shh) signaling is essential for the organization of the mammalian central nervous system and for determination and maintenance of adult neural stem cells. Nevertheless, it is not known how the Shh signal is transmitted from the membrane protein Smoothened to the Gli transcription factors that implement the pathway. Four components are known to act at this step of the pathway: Kif7, Protein kinase A (PKA), Suppressor of fused (Sufu) and the primary cilium. The first two aims of this proposal will define how Kif7 and PKA function at the primary cilium to transduce the Shh signal. Kif7 has dual roles as a core component of the Shh pathway and as a kinesin required for cilia structure. Immunolocalization and co-immunoprecipitation experiments in wild-type and mutant cells will define how Kif7 regulates the activity of the Gli transcription factors. High-resolution static and live imaging will define whether Kif7 has global roles in ciliary trafficking or has a specific rol in trafficking of Gli proteins. PKA is a strong negative regulator of Shh signaling that is localized o the base of cilia. Genetic and cell biological experiments will test whether PKA needs to be localized to the base of the cilium to function, whether PKA controls trafficking in the cilium and whether Shh controls PKA activity. Development of new therapies for Hedgehog-dependent diseases and tumors will depend on a deep understanding of these signaling mechanisms. Cilia are templated by centrosomes, and human genetic diseases that disrupt the centrosome cause microcephaly. Aim 3 of the proposal will define the developmental and cellular functions of centrosomes in the early embryo and in the developing brain. The Sas4 gene (also called Cenpj or Cpap) is essential for centriole duplication and Sas4 mutant embryos lack centrioles, cilia and centrosomes. Analysis of the Sas4 mutant phenotype will define whether centrosomes regulate signaling, cell division, cell migration or cell survival. Data indicate that the early lethality o Sas4 embryos is rescued by removal of p53, and experiments will define the p53-dependent pathways activated in Sas4 mutants. To determine the roles of cilia and centrosomes in patterning and cell behavior in the developing brain, the phenotypes caused by conditional genetic deletion of Sas4 or of Ift88, which is required for formation of cilia, will be compared. Patterning, proliferation, cortical organization and cell death will be analyzed in mutants that lak either cilia or centrosomes in the developing brain. In utero electroporation of GFP- tagged Cre will be used to conditionally delete Sas4 and Ift88 in the brain and then follow the fate of individual cells that lack cilia or centrosomes. These experiments will define the roles of cilia ad centrosomes in the control asymmetric cell division and migration in the cortex.

DESCRIPTION (provided by applicant): Recent advances in genome technology have made it possible to apply phenotype-based screens to identify genes that perform roles of fundamental importance during mammalian development. In ENU mutagenesis screens already carried out at Sloan-Kettering Institute, 41 recessive lethal mutations that disrupt specific aspects of embryogenesis have been identified. Of 30 mutations mapped to specific regions of the genome, three were found to be alleles of previously defined genes. Most of the new mutations affect genes that were not previously known to play a role in mammalian development. The 41 existing mutations will be characterized to provide enough information that other investigators interested in specific aspects of development will be able to identify those mutations that will advance their research. Three kinds of information will be obtained for each mutation: the morphology at the time of developmental arrest; map position; and expression of informative molecular markers. A website will be developed to make the information about these mutations available to the community and frozen sperm from mutant lines will be made available to interested investigators. Five investigators will combine their expertise to perform new screens to identify recessive mutations that cause defects in specific developmental processes at three stages of gestation, e9.5, e12.5, and e18.5. The e9.5 screen will identify mutants based on abnormal external morphology and abnormalities in gene silencing (genomic imprinting, retroposon silencing, and Xist expression in males). The e12.5 screen will identify mutants based on abnormal external morphology and inappropriate expression of molecular markers of neural patterning. The e18.5 screen will identify mutants based on abnormal external morphology, abnormal morphology of the genitourinary tract, and abnormal histology of the kidney. Information about the new mutants will be entered on website, and frozen sperm made available to interested investigators. For a small number of genes of interest to the consortium, map-based approaches will be used to identify the genes responsible for the mutant phenotype. All new polymorphic mapping markers and new methods will be made available to the community as electronic resources.

DESCRIPTION (provided by applicant): Abnormal canonical Wnt signaling causes developmental abnormalities and increased Wnt signaling drives the formation of a variety of tumors, including the childhood medulloblastoma and the vast majority of human colorectal carcinomas. This has generated considerable interest in the identification of small molecule inhibitors of the Wnt pathway for therapy. Recently, it has been suggested that small molecules that inhibit the Wnt pathway through stabilization of Axin, such as IWR-1 and XAV939, would be useful in therapy. Axin is a key negative regulator of the Wnt pathway because of its role as a scaffold of the ?-catenin destruction complex, which keeps the pathway off in the absence of ligand. We recently discovered that stabilization of Axin through either mutation or by treatment with IWR-1 has the opposite effect of promoting Wnt signaling in certain stem/progenitor populations during development. These findings raise the possibility that the circuitry of the canonical Wnt pathway is different in stem cells. We will test the hypothesis that stabilized Axin can activate the Wnt pathway by favoring the formation of an Axin-LRP5/6 membrane complex that increases the level of free ?-catenin. We will define the mechanisms responsible for the cell type specificity of the effects of stabilized Axin protein. We will use a genetic approach to identify other cells in the mouse where stabilization of Axin2 protein activates the canonical Wnt pathway, focusing on the Wnt-dependent stem cells in the intestine, brain, breast and skin that could act as cancer stem cells. The vast majority of cases of human colorectal carcinoma are associated with mutation or silencing of APC, a negative regulator of the Wnt pathway. We will test whether the stabilized allele of Axin2 suppresses or enhances intestinal polyp formation in the mouse ApcMin model. The studies will provide a paradigm for how stem/progenitor cells differ from other cells in their sensitivity to Wnt pathway inhibitors, which will define appropriae targets for therapy.

DESCRIPTION (provided by applicant): Funds are requested for partial support of the 2013 Gordon Research Conference on Developmental Biology. This five-day conference, which has been running since 1970, is recognized as the major and most prestigious mid-size meeting in Developmental Biology, bringing together ~150 outstanding senior and junior scientists for discussions of the recent advances in the field. The conference has several features that make it unique. It spans a wide variety of experimental systems and focuses on areas of exceptional activity or promise. This leads to fruitful comparative analyses and raises new questions about underlying mechanisms. 31 invited speakers, chosen based on their creative contributions to the field the field and their ability to promote fruitful discussions, have confirmed their attendance. None of the invited speakers spoke at the last conference in 2011. For the first time, the 2013 conference will take place at the Gordon Conference site in at Il Ciocco Tuscany Resort in Lucca (Barga) Italy, which will encourage an international exchange of ideas. The site is geographically isolated, which will keep participants in close proximity for five days of in-deph discussion, without distractions. The conference format will consist in the mornings and evenings of ~45 short talks followed by discussion and in the afternoon of informal interactions and presentation of ~90 posters. The nine sessions cover classic topics and emerging areas in the field: Organogenesis; Cellular Mechanisms of Early Development; Developmental Genetics; Regulatory Networks of Gene Expression during Development; Evolution of Morphological Diversity: Epithelial Patterning and Morphogenesis; Stem Cell Biology; Patterning and Cell Fate. Some of the session time has been kept uncommitted to choose speakers from abstracts submitted by the participants. By maximizing both formal discussion and informal interactions, the Gordon Conference on Developmental Biology will help to define both the present state and the future of the field.

Project Summary/Abstract Epithelial-mesenchymal transitions (EMTs) are highly regulated dynamic processes in which cells in stable epithelia acquire the ability to migrate and organize new tissues. EMTs are essential for establishment of tissues layers in developing embryos and for the development of many different organs, and disrupted EMTs can cause birth defects. In the adult, abnormal EMTs can cause fibrosis in the kidney, liver and lung, and they can also drive tumor progression and metastasis. Despite their importance, the dynamics and regulation of the cellular events of mammalian EMTs in vivo are poorly understood. The mouse gastrulation EMT provides an unparalleled context to combine the tools of genetics, imaging and cell biology to define the cellular, tissue and mechanical mechanisms that control cell behavior during mammalian epithelial-to- mesenchymal transitions in vivo. The mouse gastrulation EMT, which generates the three body layers of the animal, provides a uniquely advantageous context to dissect the molecular and cellular processes that regulate an EMT. The gastrulation EMT is relatively rapid: individual cells move from the epithelium to the mesenchymal layer in less than an hour. Signaling pathways and transcription factors that are required for this EMT have been identified, and the EMT can be visualized using fluorescent transgenic reporters in real time. This proposal uses a novel set of mouse mutations to define the cell biological events required for the EMT. Genetic and cell biological experiments will test the novel hypothesis that a self-organizing network of interactions among a set of apical epithelial proteins controls the stochastic ingression of cells during gastrulation. Experiments will test whether FGF signaling directly promotes the cell biological changes that drive cells to exit the epithelium, in addition to its established role in the regulation of gene expression. The final step of the EMT is the organized and directed migration of the newly formed mesenchymal cells to generate the organs of the animal. Experiments will test the hypothesis that regulated migration of mesoderm cells drives elongation of the anterior-posterior body axis and that directed mesoderm migration depends on the Striatin Interacting Phosphatases and Kinases (STRIPAK) protein complex. This work will provide the first dynamic analysis of a genetic network that controls the cellular events of a mammalian EMT in vivo and will provide a foundation for understanding other normal EMTs, as well as how aberrant EMTs cause human disease.

Project Summary/Abstract Primary cilia are microtubule-based organelles that extend from the surface of mammalian cells and are specialized to respond to Hedgehog ligands and other signals. Abnormalities in primary cilia cause obesity, cystic kidney disease, and birth defects that affect development of the brain, skeleton and heart. Although primary cilia are widely distributed in embryonic and adult tissues, recent studies showed that the formation of primary cilia is regulated by lineage- and stage- dependent processes. The mechanisms that control cell-type specific primary cilia are not known. The goal of this project is to define the mechanisms that regulate primary cilia formation in specific tissues of the mouse embryo, with the long-term goal of developing therapies to restore or ablate cilia to treat human disease. Studies carried out in cells derived from mouse embryos will define the roles of specific proteins in the regulatory network that controls cilia initiation. The functions of specific proteins in that network, including RSG1, other RGK proteins and KIF24, will be validated in mutant mouse embryos. Studies will be carried out to determine why four specific cell types in the mouse embryo lack primary cilia: the extraembryonic endoderm (which contributes to the yolk sac of the fetus), the trophectoderm (an essential component of the placenta), the mature intestinal epithelium, and primordial germ cells. Developmental signals that regulate the dynamic gain or loss of primary cilia in the intestine and primordial germ cells will be identified. As it is clear that many regulators of tissue-specific formation of primary cilia have yet to be identified, screens will be carried out to identify genes that can promote formation of primary cilia in mouse extraembryonic endoderm stem cells, which never bear primary cilia.

? DESCRIPTION (provided by applicant): Advances in genomic technologies over the past decade have yielded an unprecedented level of information about animal genomes. However, much of the information underlying these 2D models of cell and tissue function stills awaits experimental verification and further investigation in intact living organisms. Another factor limiting the application of genome wide approaches is the absence of functional characterization for ? 1/2 of all annotated genes. The targeted mutations generated by the International Knockout Mouse Consortium (IKMC), including KOMP, provide a revolutionary resource for functionally annotating the mammalian genome. We propose to characterize the phenotypes of mutations in the KOMP2-generated KO lines that result in develop- mental arrest or abnormal morphology at or prior to E9.5. We will functional define ~200 previously uncharacterized genes whose functions are essential during pre- and early post-implantation development. The investigators of this group have previously studied the phenotypes of ~200 embryonic lethal mouse mutants, leading to many surprising findings, including the novel cellular mechanisms that create the mammalian endoderm, the requirement for primary cilia in the Hedgehog signaling pathway and many more. Our specific goals are to (1) Characterize the functions of new genes essential for early embryonic development, by second tier phenotyping of ?200 lines lethal at or before e9.5. We will define the stage of arrest, analyze morphological abnormalities, assess proliferation and cell death. Only genes in which knock- outs have not been characterized will be studied, and those encoding uncharacterized proteins will have highest priority, making this an unbiased screen for novel essential genes, and providing the first evidence on bio- logical function of 200 essential genes. (2) Use our expertise to characterize at cellular resolution the roles of previously uncharacterized genes in preimplantation development, early embryonic morphogenesis and placental development. We will characterize novel regulators of pluripotency of early embryonic lineages, the gastrulation epithelial-mesenchymal transition, early mesoderm migration and ventral folding, which are reiteratively used morphogenetic programs essential for many aspects of human development. Characterization of cellular and developmental functions of regulators of placentation is crucial for fetal and child health. Our specific Aims are: Aim 1. - Establishing a Phenotyping Pipeline, which will initiate at three trans NIH KOMP2 production and phenotyping centers. Aim 2 - Tier two phenotyping of ~200 mutations that cause lethality before E9.5. Aim 3 - Tier 3 phenotyping of lethal mutations that affect development of the blastocyst, early post-implantation morphogenesis and placentation. The embryonic lethal KO mutations generated by KOMP and IKMC provide an unprecedented opportunity to expand and enrich the functional annotation of the mammalian genome. The embryonic lethal genes thus identified will not only be indispensable for early mouse development, but also critical for multiple aspects of human development and tissue homeostasis.