Frank Ruddle - US grants

The studies described here continue and extend our research in the area of somatic cell genetics. We shall continue to refine gene mapping procedures, especially to increase the resolution of the physical map by gene transfer. A new aspect of our research will be the analysis of the molecular mechanisms of growth and differentiation. Using somatic cell genetic approaches, we have cloned genes tht are regulated in respect to the resting or proliferating states of cells. These are thymidine kinase, transferrin receptor, surface antigen 4F2, and surface antigen S11. The genes are being characterized structurally and in terms of their kinetics of transcriptional expression during the culture cycle and the cell cycle. We propose to determine the molecular mechanisms of transcriptional control in this gene set. Another new research program involves mouse and human DNA sequences that show a high degree of homology with the Drosophila homeotic genes Antennapedia and Ultrabithorax. We have cloned a number of such sequences and propose experiments by which their biological significance can be evaluated. If our hypothesis is correct that these genes regulate pattern formation in mammals, then we shall have a genetic approach to studying mechanisms of morphogenesis in higher organisms. In another new system (sr = segregation regulation), we shall investigate genetic factors located on the mammalian X chromosome that control chromosome segregation in hybrid cells. We postulate that this system represents a hitherto unrecognized mechanism that mediates intercellular recognition in somatic cell populations. A primary objective will be the purification and recombinant DNA cloning of these sr genes. We believe the pursuit of these projects will provide useful and significant insights into the genetic mechanisms controlling mammalian cell growth, differentiation, and morphogenesis.

This is a request for support for the Tenth International Workshop on Human Gene Mapping (HGM 10) to be held in New Haven, Connecticut, June 10-17, 1989, and a preliminary, organizing meeting of committee chairpersons to be held August 28-31, 1988, also in New Haven. The major aim of these international workshops has been the critical periodic review and updating of the extensive human gene mapping data and the compilation of a comprehensive human gene map with accompanying reports prepared by committees of experts in the appropriate areas. The continuous updating of a standard system for gene names and symbols and the clarification of mapping inconsistencies has obviated much confusion in the field. The compilation of comparative gene mapping data and the production of extremely valuable listings of the characteristics, availability, and the sources for recombinant DNA gene probes are other important objectives of these meetings. The reports from the nine prior Human Gene Mapping Workshops have been an important resource for workers in basic genetics and in human genetics, including clinical and molecular genetics, cytogenetics, oncogenetics, immunology, population genetics, and evolution. The next Workshop will play a pivotal role in summarizing the knowledge on the human gene map at the onset of a more concerted effort to fully map the human genome. A map that is as authoritative, accurate, and complete as possible is essential. The rapid rate at which data are already being collected makes it particularly critical that the information be carefully evaluated and compiled by experts in the field. The Workshop will also introduce newer methods of electronic data handling and produce a computer database, as well as the written reports, that can be widely distributed.

The aims of this proposal are both biological and technical. The biological goals relate to detailed genomic analysis of the 20-30 cM regions surrounding the homeobox complex loci on human and mouse chromosomes 7/17 and 6/11. We believe the Hox-l and Hox-2 loci on human chromosomes 7 and 17 are parologous and represent an extensive duplication which occurred before the divergence of the human and mouse lineages 60-70 My ago. We further posit that many genes in the vicinity of the homeobox loci maybe functionally interrelated and that these domains may coordinate developmental and other biological processes. The technical aims of this project focus on the development of methods that will together speed up genomic analysis by a factor of five to ten fold. These will include (1) the design of new cloning vectors capable of cloning inserts of 160 bp in bacterial hosts and in the megabase range in yeast hosts, (2) the design and synthesis of chemical reagents which will permit the site specific scission of double stranded DNA, and (3) the development of integrated methods of high resolution physical and Mendelian gene mapping methods. In sum, we believe this integrated ensemble of projects will provide new insights into current biomedical problems of significance, and to contribute new methodologies which will substantially enhance our ability to analyze complex genomes, both in germs of speed and resolution.

We have requested funds to establish a confocal image analysis center to be shared by eight research groups in the Biology Department at Yale University. The user group has a broad range of microscopic requirements which include imaging of nuclear proteins in yeast, cytoskeletal proteins and structures in tissue culture cells, analysis of antigens involved in the patterning of vertebrate neuronal projections and a spatial analysis of cells in developing plant leaves, Drosophila, and mouse embryos. The research groups propose to use a wide range of fluorescence techniques to detect both gene transcripts and protein products. A confocal image analysis system will greatly enhance the level of detail which these studies can attain. In some cases, confocal microscopy appears essential. In comparison to conventional microscopy, confocal microscopy provides dramatic enhancements in contrast and resolution, and promises to become a standard biological technique. This is particularly true for immunofluorescence applications. Equipment of this type is not currently available at Yale.

The Amyloid Precursor Protein (APP) is a highly conserved multifunctional protein, showing expression in a broad range of tissues during the mammalian life cycle. The normal functional role(s) of the protein is poorly understood, but evidence from a variety of sources suggest an involvement in cell contact, growth, and repair processes as well as hemostasis. The abnormal expression of the protein and its multiple modified forms has been implicated in Alzheimer's Disease (AD). Heretofore, emphasis has been directed primarily to an understanding of post-transcriptional modification of the APP mRNA and protein products, but a complete understanding of APP and its role in pathogenesis requires a comparable understanding of the transcriptional control of APP gene expression, particularly within a developmental context. In the present grant proposal, we introduce a new methodology designed to rapidly produce detailed information on the control of APP gene regulation in the context of the intact organism undergoing normal ontogeny. This will be accomplished by first establishing the normal patterns of APP expression by means of in situ immunomicroscopy. Secondly, we will use APP reporter constructs in transgenic mice to determine the regulatory role of subregions of the APP gene in comparison with the normal patterns of APP gene expression. Thirdly, we will employ molecular methods to detect specific response elements, and then confirm this analysis by means of the transgenic approach. We have already established the efficacy of this approach with other genes, and we have begun to apply this analysis to the APP gene specifically in the context of the development of the CNS and the cytodifferentiation of the megakaryocyte lineage of the blood elements.

The Amyloid Precursor Protein (APP) is a highly conserved multifunctional protein, showing expression in a broad range of tissues during the mammalian life cycle. The normal functional role(s) of the protein is poorly understood, but evidence from a variety of sources suggest an involvement in cell contact, growth, and repair processes as well as hemostasis. The abnormal expression of the protein and its multiple modified forms has been implicated in Alzheimer's Disease (AD). Heretofore, emphasis has been directed primarily to an understanding of post-transcriptional modification of the APP mRNA and protein products, but a complete understanding of APP and its role in pathogenesis requires a comparable understanding of the transcriptional control of APP gene expression, particularly within a developmental context. In the present grant proposal, we introduce a new methodology designed to rapidly produce detailed information on the control of APP gene regulation in the context of the intact organism undergoing normal ontogeny. This will be accomplished by first establishing the normal patterns of APP expression by means of in situ immunomicroscopy. Secondly, we will use APP reporter constructs in transgenic mice to determine the regulatory role of subregions of the APP gene in comparison with the normal patterns of APP gene expression. Thirdly, we will employ molecular methods to detect specific response elements, and then confirm this analysis by means of the transgenic approach. We have already established the efficacy of this approach with other genes, and we have begun to apply this analysis to the APP gene specifically in the context of the development of the CNS and the cytodifferentiation of the megakaryocyte lineage of the blood elements.

DESCRIPTION (adapted from investigator's abstract): A major problem in developmental biology concerns the specification of identities along linear axes. Axial development frequently involves serial structures, as for example somites, which develop differentially depending on their position. Seemingly diverse developmental processes such as the formation of the cranial nerves, vertebrae, limbs, and the urogenital system all share the feature of axial specification that may involve common underlying developmental mechanisms. There is growing support for this integrative concept based primarily on the discovery of the homeobox genes which are highly conserved throughout multicellular organisms and involved in axial specification. The Principal Investigator will examine the molecular basis of homeobox gene activation associated with the specification of developmental identity. Hoxc-8 was chosen because it is representative of the medial class of Hox genes and because a wealth of information exists for this gene. Three major approaches to the analysis of Hoxc-8, all of which involve different aspects of its transcriptional control, will be taken. The first will identify enhancer motifs using reporter constructs in transgenic mice. Experience has shown this approach to be reliable, efficient, and rapid. The transgenic system will be combined with the use of YACs containing the mouse Hoxc cluster, and a novel clasper vector that permits the precise dissection of YACs by recombination. These new tools facilitate the detection of enhancer elements operating at substantial distances from the promoter, enhancer sharing between Hox genes, and genetic complementation. The second will characterize transcription factors regulating the expression of Hox genes by interaction with the defined enhancer motifs. Attention will be given to the combinational interaction of these proteins, a possibility underlined by their preliminary findings. The third will investigate functional aspects of Hoxc-8 enhancers at the molecular level using in vivo ligation-mediated PCR footprinting, using embryonic tissues, transgenic mice, and transformed cell populations.

The use of transgenic mice has been instrumental in the characterization and understanding of the genetic basis of mammalian development and physiology. The genetics group at Yale University in particular has utilized transgenic mice to study the evolution of the HOX genes that are essential for normal development, the role of the transcription factor AP-2 in vertebrate morphogenesis, and the role of T-cells in immune system function and development, among other projects. This proposal requests funds to augment the transgenic mouse facility at Yale University. The facility presently has one old transgene injection set-up and one modern set-up for blastocyst injection of stem cells. The transgene apparatus is now 20 years old and despite mechanical wear and tear is used at capacity. This project proposes to add a new transgene injection station, to repair and upgrade the existing apparatus, and to add ancillary equipment that will generally support the overall program in transgene research and education. As transgene techniques continue to expand, the facility comes under even greater use by the faculty and students at Yale. The sheer volume of experimental studies necessitates the acquisition of new instrumentation. New instruments will enhance the efficiency of both research and teaching activities. Additional equipment will also make possible the development of new techniques such as the controlled expression of transgenes, fluorigenic reporter constructs, and the modification of endogenous genes by "knock-in" procedures. The requested equipment will be accessible to a broad spectrum of investigators and students both within and outside of Yale University. Access if facilitated by housing the equipment in an Animal Research Facility managed by a consortium of faculty investigators rather than within a single investigator's group. The facility is self- contained and integrates both animal care and housing and transgene laboratory space.

This grant application describes a new experimental approach to the analysis of axis formation in vertebrates. Anterior-posterior axis formation is governed by the Hox genes that are organized into four clusters in mammals. Each cluster contains subsets of thirteen primordial cognate genes organized within a span of approximately 100 kb. The clusters are estimated to have existed over a period of at least 500 million years, suggesting an intimate and biological adaptive interplay among the clustered genes. The Ruddle laboratory has studied the role of the Hoxc8 gene intensively with respect to its contributions to axis formation in the brachial and thoracic regions of the developing mouse embryo from molecular, genetic, developmental, and evolutionary points of view. The PI has identified enhancers that regulate the transcriptional activity of Hoxc8 and regulate its expression temporally, control its spatial distribution on the A/P axis, and target its expression in spedtic organ rudiments. One of these enhancers, the early enhancer, has been analysed in considerable detail. It is highly conserved in amniotes, extends over a 200 bp domain, and contains at least ten transcription factor binding motifs. The PI has assembled evidence for the identities of the transcription factors interacting with this regulatory unit and have shown that the factors interact cooperatively in a manner similar to that of an enhanceosome. Much of this work has been accomplished using normal and mutated forms of the early enhancer in reporter constructs that are introduced into the early embryo by transgenesis. While highly informative, reporter experiments do not provide direct knowledge of enhancer behavior in the context of the intact gene cluster. The PI now seeks to specifically modify the endogenous enhancer element by homologous recombination using embryonal stem cell methodologies. In this way, he expects to obtain unambiguous information on the influence of individual enhancer motifs on developmental patterning, the effect of the early enhancer on cis regulation of genes throughout the Hoxc cluster, and the interplay between the early enhancer and other enhancer elements in the Hoxc8 region of the C cluster. The PI believes that this new line of analysis will greatly deepen understanding of the means by which the Hox genes regulate pattern formation.

This collaborative project between the laboratories at Yale (Ruddle and Wagner) and Boston University (Amemiya) seeks to understand the evolutionary diversification of the vertebrate body plan in molecular and developmental terms. Key organisms such as the lamprey, horned shark, and the primitive bony fish Bichir will be major subjects for research. A primary approach to the problem of the vertebrate radiation will be to isolate and sequence the Hox gene clusters that govern pattern formation on the primary and secondary body axes. In particular, the investigators will be interested in determining copy number of the clusters in the selected species, the identification of conserved non-coding elements that may serve as regulatory elements, the history of Hox cluster duplications and their possible role in vertebrate evolution. Finally, the investigators want to test the neutralist theory with respect to the duplication and diversification of gene complexes governing body plan.

DESCRIPTION (provided by the applicant): Williams Syndrome (WS) is an autosomal dominant genetic condition characterized by an ensemble of physical, cognitive, and behavioral traits. The syndrome has been mapped to 7ql1.23, where genetic causation is attributed to a microdeletion of approximately 1.5 Mb in length. To date, 17 genes have been identified in the haplo-insufficiency region, which serve as specific candidates for the multiple features of the condition. While the 1.5 Mb deletion occurs most commonly, smaller more informative deletions occur at a lower frequency and facilitate the presumptive identification of genes that are causal to specific cranio-facial and neurological attributes of WS. Currently, deletion mapping implicates genes near the telomeric terminus of the deletion, as most critical in phenotype causation. Three genes are viable candidates. These are CLIP-115, BEN, and TFII-I. CLIP-115 is a cytoplasmic linker protein, while TFII-I and BEN are closely related helix-loop-helix transcription factors. We have recently isolated the BEN gene in mice in a search for factors that bind to the early enhancer of the developmentally important Hoxc8 gene. This implicates BEN and TFII-I as candidate developmental factors, deficiencies of which may be expected to generate the symptomology of WS. In an effort to establish the molecular basis of WS, we will use chromosome engineering and other transgenic methodologies to simulate a haplo-insufficiency for these three candidate genes in mice. The mutant mice will be examined for physical, biochemical, and behavioral phenotypes that are typical of persons with WS. In this way, we hope to implicate definitively the three candidate genes singly or in combination as casual factors in WS. This will represent the first step in establishing the molecular genetic basis of WS. The second step will involve the discovery of downstream genes regulated by the transcription factors BEN and TFII-I. We believe certain genes in this category may be profoundly deregulated in the WS haplo-insuficiency condition, and are therefore most probably the immediate causal factors in WS. The establishment of the developmental genetic basis of WS is important beyond the understanding it brings to WS itself. The identification of genes that regulate behavior allows further investigation of genetic polymorphisms of these genes that may be causal to less severe behavioral conditions or to variations in behavior within a range considered normal.

The Hox and the Distalless (Dlx) gene complexes play a major role in establishing pattern formation over the anterior/posterior axis (A/P) of the body and over the proximal/distal (P/D) axis of the limbs in mammals. The Hox genes regulate A/P axis patterning from the tail to the hind brain/midbrain junction, while the Dlx genes regulate patterning from the hindbrain to the forebrain. The Hox and Dlx clusters are linked chromosomally with a one megabase interval separating the two clusters. The expression of the Hox and Dlx genes is regulated by transcription factors and morphogens such as retinoic acid that bind enhancers and other protein binding sites in non-coding domains within the gene clusters. The protein binding sites have been shown to exert important cis regulatory control with respect to the orderly activation and display of Hox and Dlx genes over the A/P body and (P/D) limb axes. Little is known about the number, location, composition, and function of these control domains. Our previous reports on the early enhancer (EE) of the Hoxc8 gene provide the most detailed information of one such site, but much remains to be learned even about the EE, not to mention the myriad of additional sites. In this grant application, we describe our plan to investigate in greater detail the stucture and function of Hox and Dlx cis- regulatory sites. We will concentrate on two domains with which we have had experience, namely the Hoxc8 (40 kb) and Dlx3/7 (80 kb) regions. New methods of our design will be used to locate, describe, and mutate control motifs in these domains. An important new aspect of our research is the identification and functional analysis of transcription factor proteins that bind to control motifs. We view enhancer elements as the ultimate target of developmental signaling pathways. The enhancer in its complexity can be viewed as a decoding device to integrate multiple signals and to elicit appropriate gene expression responses in competent cells. Knowledge regarding enhancer sequence, protein binding domains, and the specific transcription factors binding the enhancers will contribute to our understanding of the epigenetic function of these motifs. We believe an in depth investigation of non-coding control elements in the Hoxc8 and Dlx3/7 domains will provide insightful information relevant to the entire clusters with respect to their control of developmental patterning and their role in the evolutionary diversification of body plans.

0321470
Ruddle

The interconnection between development and evolution (Devo-Evo) was recognized in the mid-1800's by Darwin and Haeckel, though it has not been until very recently that the mechanistic basis for this crucial link has been readily addressable to experimental biology. In this collaborative project a question concerning the developmental evolution of the vertebrate bauplan is addressed: what are the developmental and evolutionary consequences of gen(om)e duplication during vertebrate phylogeny? It has been known for a long time that the evolution of the vertebrates was associated with a major expansion of genome size, either by gene or genome duplications. The present project focuses on the Hox gene clusters, a complex of genes that encode transcription factors known to be involved in a wide range of biological activities, including the development of the body axis, fins and limbs and many other organs. Vertebrates have the peculiar tendency to duplicate and retain Hox clusters, while no such tendency is apparent in invertebrates. It is not known why vertebrates have this tendency and whether these duplication events had an influence on the evolution of the vertebrate body plan. The goals of this project are to understand the extent and history of Hox cluster duplications among the vertebrates (specifically focusing on the teleosts), and to determine whether such duplications have facilitated evolutionary change both functionally and morphologically. We will use the ray finned fish radiation, in which the most recent Hox cluster duplication has been documented, as our experimental focus.

The experimental approach involves: (1) tracing the patterns of molecular evolution of the Hox clusters and their genes, with the aim to determine whether the teleost Hox cluster duplication was coincident with the teleost radiation; (2) using Bacterial Artificial Chromosome technology and computational genomics methods in order to detect changes in the organization of clusters as well as identify changes in the pattern of non-coding sequence conservation; (3) employing BAC-reporter transgenesis as a comparative tool to draw inferences with respect to the effects of gene duplication on expression patterns; and (4) employing statistical tests to both coding and non-coding regions of Hox clusters to draw inferences with regard to the evolutionary forces (selection/drift) acting on duplicated genes and clusters.

This investigation represents a part of an ongoing collaboration between three investigators of diverse backgrounds: Frank Ruddle (Yale University) has expertise in mammalian developmental biology, mouse transgenesis and homeobox genes; Gunter Wagner (Yale University) has an extensive background in quantitative-mathematical approaches to evolution and developmental evolution; and Chris Amemiya (formerly Boston University and now at Virginia Mason Research Center) has expertise in vertebrate zoology, genetics, and genomics. Each investigator is focussing on different aspects of the project but with the same overall goals in mind. This consortial approach has proven effective in the past and considerable progress has been made since the previous funding cycle.

The broad impact of this proposal is measured in two ways: (1) by employing a highly interdisciplinary and novel approach for evaluating a complex problem in biology; and (2) by providing a wealth of research and educational resources. These training opportunities, including new summer traineeships for teachers and undergraduates of underrepresented minorities.