Gail Martin - US grants

The overall objective of this work is to study what controls the phenomenon of X-chromosome inactivation in early female mouse embryos. Our working hypothesis is that methylation of specific sites in X-linked genes is involved in this process. We have carried out a study of the methylation patterns of the mouse Hprt gene when they are carried on the active and inactive X-chromosome. This was accomplished by making use of clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. This characterization revealed two regions of differential methylation in the mouse Hprt gene. First, a 5' region of the gene is completely unmethylated when carried on the active X and is extensively methylated when carried on the inactive X. We have extended this negative correlation between methylation and gene expression by showing that these 5' sites are demethylated when the Hprt gene is reactivated either spontaneously or following 5-azacytidine treatment. Second, we have identified several sites in the 3' 20kb of the gene, extending from exon 3 to exon 9, which are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. Our findings, taken in conjunction with previous studies by others of the human Hprt gene, indicate that there is conservation of the regions of differential methylation between mammalian species and this suggests that these regions might play a role in the regulation of expression and X-chromosome inactivation of the Hprt gene. The goal of our present studies is to determine whether methylation of the sites in the 5' region of the gene occurs concomitant with X-inactivation or if these changes in methylation are perpipheral to the phenomenon, perhaps acting as a final "locking" process. (M)

The mouse t-complex is a region of chromosome 17 in which there maps a number "lethal t-mutations" that, when homozygous, cause disturbances of early development leading ultimately to the death of the embryo. The main obstacle to the study of lethal t-mutant gene expression during embryogenesis has been the difficulty of obtaining appropriate experimental material. Recently we have devised a method of cell culture that enables us to establish pluripotent embryonic stem (ES) cell lines directly from early mouse embryos. Since each of these cell lines constitutes a virtually unlimited supply of cells from a single early embryo of a particular genotype, they can be used to circumvent many of the difficulties inherent in studies of intact early mouse embryos. We have already been able to use this method to establish ES cell lines that are either homozygous (t/t) or heterozygous (+/t) for one of the lethal t-mutations, tw5. We now propose to establish t/t and +/t ES cell lines from embryos carrying two different lethal t-mutations, t0 and t9. All of these cell lines will then be used in studies aimed at identifying the products of lethal t-mutant gene expression in ES cells. One approach will be to use a sophisticated computer-analyzed system of 2D gel electrophoresis to compare the patterns of protein synthesis in t/t and +/t with those of +/+ cells. This should enable us to identify polypeptides that might result from lethal t-mutant gene expression. Further studies will be aimed at determining which of these are lethal t-mutation-specific. In addition, we will test the hypothesis that lethal t-mutations cause alterations in the expression of cell surface molecules of early embryonic cells. t/t ES cells will be used as antigens to raise monoclonal antibodies that detect molecules present on the surface of t/t ES cells as a consequence of lethal t-mutant gene expression. Our immediate goal is to learn more about the nature of the lethal t-mutant gene products, as a first step toward understanding their function. Ultimately such information should help to elucidate the mechanism of the control of normal embryonic development. The results could have implications for research on human development since there are three examples of possible t-complex analogues in man.

The aim of this proposal is to study genes that control early mammalian development. The approach to be taken is founded on the observation that a 180 bp sequence, known as the "homeobox," is found in several of the genes that are known to regulate development in Drosophila. This highly conserved sequence is also present in approximately 10 copies in the mammalian genome, and mouse genomic clones containing homeoboxes have been isolated. Preliminary studies suggest that at least one of the "homeobox-containing genes" that has been isolated is expressed differentially during the development of the mouse embryo. The working hypothesis upon which this proposal is based is that the homeobox identifies genes that have a role in the control of developmental processes in mammals. The first specific aim of these studies is to carry out Northern blot analyses to determine at what stages of mouse embryonic development transcripts from these genes are expressed. Information gained from this study will be useful in carrying out the second specific aim, which is to isolate from cDNA librares, clones that encode these transcripts. The availability of such clones will make possible a study of the structure of these homeobox-containing genes. The third specific aim is to determine which cells in the developing embryo actively express homeobox-containing mRNAs by in situ hybridization with the cDNA probes. The cDNA clones will also be used to carry out the fourth specific aim, which is to obtain quantities of the homeobox-containing gene products in bacterial and mammalian expression vector systems. The proteins thus obtained will be used to raise specific antibodies against the gene products and to study their function. The antibodies will be used to study the subcellular localization of the gene products in developing embryos. Finally, the fifth specific aim is to determine the position of the homeobox-containing genes in the mouse linkage map and attempt to identify mutant alleles of these genes. In addition, the effects of induced alterations in homeobox-containing gene expression on the development of the mouse embryo will be evaluated. Taken together, the results of these studies should help to determine the function of these genes in the developing mouse embryo and may lead to a better understanding of how development in mammals is controlled.

The hypothesis on which this proposal is based is that the presumed proto-oncogene int-2 plays an important role in the control of early mammalian embryogenesis. Data from our previous study of int-2 expression in mouse embryos and teratocarcinoma cells demonstrated that there are four embryonic transcripts and that their expression is differentially regulated during normal embryogenesis and during the course of differentiation of teratocarcinoma stem cells in vitro. We propose to clone and characterize these four embryonic int-2 RNAs. Their protein products will be obtained by the use of a bacterial expression vector system, and anti-int-2 antibodies will be raised. The nucleic acid and immunological probes obtained in these studies will then be used to determine the stage- and cell type- specificity of int-2 expression in the embryo. In particular, we will determine which cells in the embryo express the gene and the subcellular location of its product(s). If it is a secreted protein, we will determine where in the embryo it becomes localized. Finally, we propose a series of experiments aimed at determining the effect of altering or abolishing the expression of int-2 in teratocarcinoma cells and/or embryos. The results of this study should provide us with information that will enable us to determine the function of int-2 in the embryo, which in turn should lead to a better understanding of the processes that control normal embryonic development. Furthermore, we anticipate that knowledge of the normal function of the gene will provide some insight into why abnormal expression of this proto-oncogene in the mammary gland results in carcinogenesis.

The ultimate goal of this proposal is to obtain mice heterozygous for mutations in specific genes thought to play an important role in the control of normal embryonic development. We propose two alternative plans for obtaining such mice. Both involve the creation of insertion mutations in the particular gene of interest in mouse embryonic stem (ES) cells in vitro. One plan employs a method that is termed "gene replacement," whereby we will attempt to target the insertion into the gene of interest by injecting into ES cells a mutated segment of that gene, and then isolate cells in which the incoming, mutated DNA has replaced one of the endogenous copies of that gene segment. The other plan involves the establishment of a library of frozen male ES cells in which "saturation mutagenesis" has occurred following retroviral integration. This library will be screened by the "polymerase chain reaction" (PCR) method to identify and isolate individual ES cell lines heterozygous for a proviral insertion in the gene of interest, once cells with mutations in the gene of interest are obtained, they will be injected into mouse blastocysts, which will be placed in foster mothers and allowed to develop to term. Any chimeric mice that are born will be mated to determine if the mutated ES cells have colonized the germ line. The successful conversion of mutated ES cells into functional germ cells would allow the establishment of new strains of mice heterozygous for an insertion mutation in the gene of interest. Once such mice are available, we would use them to obtain, by breeding, mutant homozygous embryos, which could then be analyzed to determine the effects on development of a lack of the wild-type gene product. The information gained from such studies could lead, ultimately, to an understanding of the functions of these genes in development.

The objective of this proposal is to train pre-doctoral students in Developmental Biology. Twenty-four faculty propose continuation of a broad, interdisciplinary training program. These faculty use molecular, cell biological and genetic approaches to address fundamental problems in developmental biology. Participating faculty have appointments in a total of 12 basic science and clinical departments within the university. All have extremely active research programs. Productive interactions between laboratories and trainees are promoted by an annual retreat, symposium/journal club series, joint research meetings, shared supervision, and a variety of collaborations. The program is conducted within the broader context of the Program in Biological Sciences, a consortium of seven UCSF graduate programs with more than 100 participating faculty. It attracts students of exceptionally high caliber. The features of our training program that are especially attractive to prospective students are the following: (1) a wide choice of laboratories for thesis research, (b) a laboratory rotation system, (c) an excellent st of courses, (d) a tutorial in how to present a seminar, (3) a highly cooperative spirit, and(f) an awareness that graduate training is important to the faculty at UCSF. We strive to facilitate the intellectual growth and scientific skills of each trainee so that each graduates as an independent scientist who will be able to contribute to the field for many decades into the 21st century.

The ultimate goal of this proposal is to identify, characterize, and study the function of genes involved in critical steps in early mammalian development. Initially, we will focus our attention on the Evx-1 gene, a mouse cognate of the Drosophila homeobox-containing even- skipped gene. Based on the observation that Evx-1 is expressed in a gradient in the primitive streak, we have hypothesized that Evx-1 functions in a dose-dependent fashion to provide dorso-ventral patterning of the newly formed mesoderm. To test this hypothesis we will determine the consequences of expressing Evx-1 RNA at high levels throughout the primitive streak, thereby ablating the gradient of Evx-1 RNA that is normally observed. We will also determine the consequences of ablating Evx-1 gene function, by creating embryos homozygous for a null allele of Evx-I. In addition, we will identify the genomic sequences that determine the tissue-specificity of Evx-1 expression in the developing embryo. A second major goal is to identify and isolate the gene encoding a molecule, VE-1, that is expressed in the visceral endoderm overlying the future anterior region of the embryo during the early post-implantation stages of development. Our long-term goal is to determine the function of this earliest known marker of A-P polarity in mouse embryogenesis. In the course of pursuing these experiments, we propose to test the efficacy of a new method (the cre/lox "binary" transgenic mouse system) based on DNA recombination for manipulating gene expression in vivo. In these experiments the desired effect, e.g. ectopic gene expression, is achieved by mating mice of two different transgenic strains. One (the "target" mouse) carries a target transgene that is innocuous but has the potential to cause the desired effect, and the other (the "effector" mouse) carries a DNA recombinase capable of altering the target transgene so that it can produce the desired effect. Because the target transgene has no effect prior to recombination, and the effector (DNA recombinase) alone has no phenotype, both target and effector mice are normal. However, in offspring that inherit both transgenes the target transgene is altered by the effector, producing the desired phenotype. Specifically, the system we propose to test employs the cre gene of bacteriophage P1 as the effector. If this approach is successful, we propose further experiments aimed at increasing its versatility, by generating a standard target mouse strain that can be used with a variable effector strain to produce a new lineage map of the mouse, based on gene expression rather than anatomical position. We hope that in the long-term these experiments will not only lead to greater insights into the basic mechanisms controlling embryonic development, but will make a contribution towards the development of a new genre of transgenic mouse technology.

laboratory mouse

There is increasing evidence that the FGF family of signaling molecules plays a central role in the regulation of many aspects of vertebrate embryogenesis, and may also play a role in tumorigenesis. Recently, a gene called sprouty (spry), which apparently encodes an inhibitor of FGF function, has been identified in Drosophila melanogaster. We have isolated and begun to characterize two mouse genes, Spry1 and Spry2, that are closely related to Drosophila spry (D-spry). The experiments described here take advantage of recent improvements in transgenic mouse technology to study in detail the functions of these two genes. Advanced gene targeting methods employing both Cre and Flp DNA recombinases will be used to produce an allelic series of mutations at the Spry1 and Spry2 loci, and to obtain tissue-specific knock-outs of these genes. Studies of the mutant animals will be performed to elucidate the specific functions of Spry1 and Spry2 during development, and to investigate their possible function as tumor suppressor genes. In addition, we describe experiments using a gene replacement strategy to explore the uniqueness or equivalent of Spry1 and Spry2 gene functions.

The ultimate goal of this study is to identify, characterize, and determine the function of genes involved in critical steps in early vertebrate development. This proposal describes experiments that exploit recent advances in transgenic mouse technology as well as new methods for perturbing gene expression in chick embryos to study in detail the functions of two genes, Evx1 and Gbx2, which are known to play essential roles at early stages of vertebrate development. These methods will be used to perform both loss- and gain-of-function experiments to test hypotheses about Evx1 and Gbx2 function in the vertebrate embryo, including the idea that Evx1 functions to pattern the primitive streak and the idea that Gbx2 functions to pattern the embryonic brain via a repressive effect on Otx2 gene expression. In the course of these studies mouse lines will be produced that express the gene that encodes the site-specific DNA recombinase Cre in specific cell types. These lines, Hnf4-cre, T-cre, Sox1-cre, and Ap2alpha-cre, should be useful for specifically inactivating genes in the visceral endoderm, the nascent mesoderm and endoderm, the prospective neuroectoderm, and the distal limb mesenchyme, respectively. In addition to their utility in the experiments described here, these mouse lines should provide a valuable resource for studies aimed at analyzing the function of many other genes in the early embryo. By using them to elucidate the specific functions of Evx1 and Gbx2 insights should be gained into the fundamental mechanisms by which the embryonic ectoderm is prepared for gastrulation, the primitive streak is patterned to establish the basic body plan during gastrulation, and the neuroectoderm is patterned at the earliest stages in its development.

DESCRIPTION (Investigator's abstract): The FGF family of signaling molecules plays a central role in the regulation of many aspects of vertebrate embryogenesis, and is also important in the control of physiological and pathological processes in the adult. There is substantial evidence from previous genetic studies that one member of the FGF family, FGF8, is essential at multiple stages of embryogenesis. This proposal is focused on three key developmental processes that are dependent on FGF8 signaling: left-right axis- determination, limb development, and kidney development. The experiments proposed are aimed at using a genetic approach to elucidate the function of FgJ8 and other FGF family members in these processes. They take advantage of recent advances in methods for analyzing gene function in mice, including conditional loss- and gain-of-function in specific tissues, and reporter alleles for gene expression and protein distribution. Each Specific Aim includes the development of new lines of transgenic mice that will not only make it possible to answer specific questions and test hypotheses about these particular processes, but will also be extremely useful as tools for studies of many other aspects of vertebrate development in laboratories throughout the scientific community. The results of these studies should provide a deeper understanding of the mechanisms involved in establishing the basic body plan and the formation of limbs and kidneys. The information obtained will be relevant to understanding the mechanism of human development and the etiology of human birth defects, chronic diseases, and various forms of adult disease that originate during gestation.

[unreadable] DESCRIPTION (provided by applicant): Virtually all aspects of vertebrate embryonic development are dependent on signaling between cells in neighboring tissues. Much of this signaling is mediated by Receptor Tyrosine Kinases (RTKs). In the adult, disruptions in RTK signaling can have profound effects, resulting in various forms of disease including tumor formation. Because of its central importance in both the embryo and adult, considerable attention has been devoted to understanding the mechanisms that regulate RTK signaling. Studies in Drosophila and in mammalian cell culture have demonstrated that members of the Sprouty gene family are inhibitors of RTK signaling, and studies in vertebrate embryos have indicated that three members of the Sprouty family are expressed at various stages of development. The studies proposed here employ a genetic approach to determine the functions of the Sprouty (Spry) genes in the mouse. In the course of the current grant period, mice carrying null and conditional loss of function alleles of both Spry2 and Spry4, as well as a line of mice carrying a conditional Spry2 gain of function allele that can be activated in specific tissues by Cre-mediated recombination have been produced. Analyses of these mice have revealed that loss of Spry2 function causes a severe hearing deficit and abnormal mammary gland development, and that Spry4 null mice have abnormal forelimbs. The experiments proposed here are aimed at elucidating the functions of Spry2 in the inner ear and mammary gland, and of Spry4 in the developing limb. Additional studies are aimed at assessing the functional equivalence of the mouse Sprouty genes by generating mice that lack different combinations of Sprouty null alleles, and by producing a set of mouse lines, each carrying a different Sprouty gene conditional gain of function allele inserted into precisely the same site in the mouse genome. When activated, the Sprouty genes in these mouse lines will be identically expressed, making possible a meaningful comparison of the activities of the different Sprouty genes. Finally, experiments are described aimed at determining whether Sprouty genes function as suppressors of neoplastic cell transformation in vitro and tumorigenesis in the mammary gland. The results of the proposed studies will lead to a deep understanding of the function of the Sprouty genes, which should have important implications for preventing human congenital defects and treating disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): These studies are aimed at understanding the mechanisms that regulate tooth number and morphogenesis. We will take a genetic approach, using mice carrying mutations in Sprouty (Spry) genes, which encode proteins that antagonize signaling via Fibroblast Growth Factors (FGFs). The FGF signaling pathway plays a key role in orchestrating morphogenesis of the tooth as well as many other organs. Analyzing how alterations in FGF signaling perturb tooth development, when these alterations are caused by removing an antagonist of FGF signaling, will lead to new insights that could not be obtained by studying loss-of-function mutations in either individual FGF ligands or their receptors. Our specific goals are: 1) study mice lacking either Spry2 or Spry4 function, in which tooth buds anterior to the first molar develop into supernumerary teeth. In wild-type mice, these buds regress to yield a toothless diastema region. By analyzing gene expression, performing experiments in tooth organ cultures, and by analyzing the progeny of complex genetic crosses, we will determine how loss of Sprouty gene function enables diastema tooth buds to persist and develop into a tooth rather than regress. Our studies will lead to a better understanding of the normal mechanisms by which diastema buds are prevented from forming teeth in the mouse and by which molar development is regulated. 2) pursue our observation that inactivation of multiple Sprouty alleles has profound effects on incisor development, including development of duplicate incisors. We propose to study the normal morphogenesis of early incisors, and then examine how this process is disturbed by deletion of Sprouty gene function. 3) determine why remarkable tusk-like incisors develop in mice that are heterozygous for Spry2 and null for Spry4. We will focus on the role of FGF signaling in controlling fate decisions during embryogenesis and in regulating progenitor cell proliferation and differentiation in the adult. Results from these studies will enhance our understanding of the signaling pathways that control epithelial-mesenchymal interactions during organogenesis and will contribute to knowledge about the stem-cell niche in the adult mouse incisor. Public health implications: Through our studies we will learn more about how teeth normally develop and how this development goes awry in patients with dental abnormalities. We will also study the mechanisms that control stem cells in adult teeth, which may help to lay the groundwork for efforts to build new teeth.

The Society for Developmental Biology (SDB) has been organizing annual meetings since its foundation in 1939, missing only two during the WW II. These meetings have always been considered the major worldwide event where the latest discoveries in the field of developmental biology are presented. In 2007, together with the Latin American Society for Developmental Biology and the Sociedad Mexicana de Biologia del Desarrollo, SDB will organize the First Pan American Congress in Developmental Biology at Gran Melia Hotel, Cancun, Mexico. The time is now ripe to hold this first congress after many years of SDB's outreach and collaboration to enhance the understanding and research in the field in this hemisphere. The 43 invited speakers will report their most exciting findings in three plenary and six concurrent sessions, covering topics from early development of the plant and animal embryo to patterning of the nervous system and reproduction. Forty additional speakers, mostly junior investigators, have been chosen from submitted abstracts for short talks in the concurrent sessions and the postdoctoral symposium. All other registrants will present their recent results in poster sessions. Since education is a major emphasis in SDB's mission, a symposium on communicating science without the jargon, workshops on writing a scientific paper, on choosing of the most appropriate postdoctoral training and on funding opportunities are included in the program. Underrepresented minority students, postdoctoral fellows, as well as faculty from primarily teaching departments or undergraduate institutions will receive travel assistance to present their papers at the meeting. This First Pan American Congress follows SDB annual meetings' tradition to provide a collegial atmosphere for the participants to not only disseminate their new scientific findings, but also to forge future trainings or collaborations.

DESCRIPTION (provided by applicant): The prostate is a male accessory sex gland found in mammals that functions to produce components of the seminal fluid. Development of the prostate begins before birth and continues through sexual maturity. In adulthood the prostate can undergo pathological changes, including benign prostatic hyperplasia and prostate adenocarcinoma. As in many other organs, receptor tyrosine kinase (RTK) signaling plays a major role in regulating prostate development. This proposal is based on preliminary evidence that mutations in the Sprouty genes, which encode key modulators of RTK signaling, as well as in genes that encode components of the RTK signal transduction pathway cause abnormalities in prostate development. The overall goal of this study is to understand the normal function of RTK signaling at early stages of prostate formation and budding, which occur in the embryo, and at later stages of prostate branching and maturation, which occur after birth. This will be accomplished in studies that employ genetic, molecular, and cell biological approaches to determine the consequences of altering RTK signaling in the developing prostate. In the course of these studies we will explore the complex relationship between RTK- and androgen receptor (AR)-mediated signaling in the control of prostate morphogenesis. PUBLIC HEALTH RELEVANCE: The results of these studies will provide insight into the molecular and cell biological mechanisms the regulate prostate development and homeostasis, and thus serve as a foundation for better understanding prostatic disease.