Richard Harland - US grants

[unreadable] DESCRIPTION (provided by applicant): Our long-term goal is to understand the signals that pattern the early vertebrate embryo. We study this problem predominantly in the frog Xenopus laevis. This animal produces large numbers of eggs that are readily manipulated by injection and microsurgery. The combination of experimental embryology and molecular manipulation provide the tools to understand embryonic signaling at the molecular level. Many of the paradigms for early developmental mechanisms in vertebrates have come from work with amphibians, and many of the signaling activities that bring about early developmental decisions in vertebrates have been identified first in amphibians. During previous grant periods, we have identified signals that act in early axis formation, mesoderm patterning and neural induction. In conjunction with the work of many other groups, this has led to a coherent picture of how the embryonic axes are established, and how a cascade of signal transductions leads to the elaborate pattern of the gastrula. Despite the progress that has been made in understanding embryonic signals, there is still only a partial picture of how the detailed pattern of the embryo emerges. The intracellular mediation of signaling is poorly understood, and although the main pathways that signal in development have been identified, the precise roles and modulation of these pathways remains to be determined. The formation of the neural plate with its elaborate patterning in both anterior-posterior and mediolateral axes poses a particular challenge, and this proposal will examine the integration of signaling pathways that induce and pattern the neural plate. Particular focus will be given to Fibroblast Growth Factor signaling in neural patterning, and in neural crest formation. The integration of signals that induce the neural crest will be studied by exploiting and comparing various manipulations that bring about neural crest development. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long term goal is to understand the signals that pattern the early vertebrate embryo, and particularly the role that BMP antagonists play in this process. We study this problem in both amphibians and mice, since each offers different experimental advantages. Xenopus laevis produces large numbers of eggs that are readily manipulated by injection and microsurgery. The combination of experimental embryology and molecular manipulation provide the tools to understand embryonic signaling at the molecular level. We also complement use of X. laevis with use of X. tropicalis, which offers the advantages of diploidy and a sequenced genome, both advantages for manipulating gene expression by Morpholino oligonucleotide-mediated knockdowns. Our work in the mouse has taken advantage of targeted mutations. The phenotypes of these mutations have suggested particular developmental contexts where BMP antagonists are crucial, and some of these contexts are particularly relevant to human developmental disorders. In the next grant period we will study how BMP antagonists implement important developmental decisions, either singly, or in overlapping combinations. These include the maintenance of neural stem cell populations and skeletal patterning in noggin mutants, and the induction and maintenance of somite differentiation in noggin/gremlin double mutants. Finally we will study the contribution of different BMP antagonists to formation of the dorsal ventral, and anterior posterior axes in the early embryo.

Acquisition of DNA Analysis Instrumentation for Molecular Studies Our goal is to establish a common core facility for DNA sequence analysis. We propose to purchase: an Automated DNA Sequencer; a molecular biology CATALYST Labstation; an Oligo Synthesizer; a PhosphorImager workstation; a Fluorimager; a Computer System; a Biomek II robot; a Thermocycler: an Automated Film Developer: a Vacuum Centrifuge: a Photodocumentation Station; and two Ultra Cold Freezers (-80 and -30). The participating faculty address a wide range of research related to biodiversity and biotechnology. Their efforts share a common theme of DNA sequence analysis and fall into two major areas: 1) Molecular genetic approaches to study gene expression, RNA processing, and molecular phylogeny, and 2) Molecular approaches in the study of Population Genetics, Ecology, and Evolution. In the area of Molecular genetic approaches to study gene expression. RNA processing. and molecular phylogeny, faculty research and research training include: Whalen's molecular genetic analyses of interactions between bacterial pathogens and plants; Marquez-Magana's investigations of the molecular mechanisms that regulate flagellin gene expression in Bacillus subtilis; and Davis's functional significance and regulation of spliced leader RNA trans-splicing in metazoa, the evolution of parasitism in flatworms, and the molecular phylogeny of flatworms and early metazoa. The focus of Ramirez' s research is to clone the RECI gene and further characterize the RPD3 (a.k.a. REC3) genes of Saccharomyces cerevisiae which are implicated in the repair of damaged DNA and in mitotic recombination. Goldman focuses on a structural unit of the eukaryotic chromosome, the chromatin domain or loop, and its potential role as a functional unit in the regulation of gene expression in mammalian X- chromosome inactivation and genomic imprinting. Perara investigates molecular approaches to the problem of protein transport across cell ular membranes, with the primary focus on a systematic comparison of the molecular mechanisms of secretory protein transport across the endoplasmic reticulum (ER) membrane and the bacterial periplasmic membrane. In the area of Molecular approaches in the study of Population Genetics. Ecology and Evolution, faculty research and research training include: Parker/Cullings' studies of plant community and evolutionary ecology, dynamics of plant recruitment, life history evolution and community turnover rates; Smith/Bayliss' investigations of evolutionary ecology and conservation biology with research focus on the role of transition zones between rain forest and savannah in speciation and their role as related to conservation of rain forest species; Arp's studies of physiological adaptation to environment, sulfide tolerance and detoxification in mudflat invertebrates, ecology and physiology of hydrothermal vent organisms; Randall's investigations of evolution of communication and social organization in desert rodents; and Rmutman' s use of modem molecular genetics to examine population structure, biogeography and intraspecific phylogeny, and the genetic architecture of complex phenotypes. Larson's research is in population biology of nearshore teleost fishes, including their behavior, feeding ecology, life histories, and population structure. Desjardin focuses on taxonomy and phylogeny of fleshy fungi, primarily Agaricales, using traditional and modern aspects of systematics, including cladistic and morphometric analysis, utilizing morphological, ecological, physiological, genetic and molecular data. Hollibaugh addresses physiological ecology of bacterial communities in geochemical cycles and foodwebs. Orrego employs the techniques of molecular genetics for the study of genetic variation in natural populations as displayed by sequences obtained via long distance PCR methods. Our proposed instrumentation will increase the amount and efficiency of our research an d training efforts and enable us to train a greater number of students. The requested equipment will provide the highest resolution available for the analysis of DNA/RNA using PCR amplification, DNA sequencing, Denaturing Gradient Gel Electrophoresis (DGGE), Single Strand Conformation Polymorphisms (SSCP), and a variety of other DNA/RNA detection and analytical methods. With the increased throughput capacity for large numbers of samples that can be processed rapidly and efficiently with longer sequence reading (>700 bp), we will be able to do new high resolution analysis on much greater numbers of samples more rapidly than in the past. For example, the ABI 377 Sequencer provides new longer gel formats that allow one to read 700 bases/run and 36 lanes/run that can be completed in 8 hour (or overnight) runs and 300-500 bp runs in 2.25 hours, allowing 3 runs in an eight hour period. Several faculty (Davis, Hollibaugh, and Parker) will collectively sequence in excess of 10,000 samples/year which represents one run of 36 samples each day for a year. While this alone clearly justifies our need, the acquisition of the proposed instrumentation would also allow us the capacity to triple this number. We will also use the equipment for faculty enhancement short courses; since 1991 we have presented 10 such courses-to over 240 faculty from 35 states.

0078912
Bargmann

Developmental biology demands the integration of cell biology,
genetics, molecular biology, and biochemistry. The exciting progress of
the past decade has depended upon bringing together people who work with a
variety of organisms, including yeast, worms, flies, and vertebrates to
learn about the molecules and principles held in common among these
seemingly diverse organisms. Despite all the excitement in the field,
there are relatively few meetings organized to emphasize the increasingly
obvious relationships among developmental events previously viewed as
distinct.

Our specific objectives are to assemble a group of scientists with broad
expertise in modern molecular analysis of development, have a series
of talks on up to date research, and have about 120 other attendees present
posters describing their research. The meeting will be held on the campus
of the University of California, Santa Cruz on July 21st to July 26th,
2000.

There are two major goals of the meeting. The first is to teach
each other. The planned program will be a superb course in advanced
developmental biology, and help those working with each particular organism
to keep abreast of exciting new developments in the study of other
organisms. This will important not just for established investigators, but
also for the many students and postdoctoral fellows who will attend the
meeting.

DESCRIPTION (provided by applicant): To pursue the aims of this 'Request For Applications" we will assess the utility of Silurana (Xenopus) tropicalis for standard genetic manipulations. To date, a limited number of organisms have been exploited for forward genetic analysis. However, it has become clear that different organisms have different strengths. Our main goal in this proposal is to establish methods that will test the utility of Silurana tropicalis as an additional useful organism for genetic manipulation. A good theoretical rationale has been established, namely that the animal has a relatively short life cycle, and is relatively easy to raise in a lab setting. The embryos develop externally, and in enormous numbers, so are available for screens of early developmental defects. S. tropicalis is a tetrapod, more closely related to humans than is the fish, and therefore more useful for examination of processes such as limb development. By comparison with Xenopus laevis, a pseudotetraploid, S. tropicalis appears not to have undergone either complete or partial genome duplications, so we do not expect multiple genes to have overlapping and therefore redundant functions. In addition, the kinds of embryological experiments that can be done in Xenopus are sufficiently different from those that can be done in the fish, that the use of mutant animals will provide new information about cell and tissue interactions. We will carry out a genetic screen, map deletions cytologically, and optimize insertional mutagenesis.

[unreadable] DESCRIPTION (provided by applicant): The goal of this research is to understand the molecular mechanisms by which congenital skeletal disorders lead to pronounced developmental and radiological bone alterations. In particular the PIs are interested in two progressive hyperosteosis disorders: Van Buchem disease (VB) (MIM 239100) and Sclerosteosis (MIM 269500). VB is a severe, autosomal-recessive bone disorder characterized by cranio-facial distortion and generalized bone overgrowth. Sclerosteosis is a skeletal dysplasia highly similar to VB but with more pronounced radiological bone alterations (jaw; long bones; gigantism) and the presence of hand abnormalities (syndactyly). Linkage analysis mapped both diseases to the same locus (17q12-q21), and Sclerosteosis patients were found to carry several coding mutations in the BMP-antagonist gene Sclerostin (Sost). In contrast, VB patients exhibit no Sost coding mutations, however displayed a 52kb noncoding deletion, 35kb downstream of the Sost transcript. Using human transgenes in mice, the PIs have demonstrated that sclerosteosis and VB are allelic, and that the 52kb deletion removes essential Sost transcriptional regulatory elements, altering the human Sost expression pattern. Also, in mice, increased levels of Sost result in severe limb abnormalities (fused and split digits), while the lack of mouse Sost leads to hyperosteosis. These initial observations suggest that Sost plays a critical role during limb development as well as throughout the adult-life controlling bone homeostasis in vertebrates. Since Sost is highly conserved from fishes to humans, the PIs are interested in elucidating the role of this molecule during skeletal development in different vertebrates. Accordingly, this grant focuses on deciphering several fundamental properties of Sost including 1) identification and characterization of Sost-specific regulatory elements, 2) determination of the role of Sost in limb patterning and adult bone homeostasis, and 3) investigation of the role of Sost across different vertebrate lineages. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Our long-term goal is to understand the signals that pattern the early vertebrate embryo. We study this problem predominantly in the frog Xenopus laevis. This animal produces large numbers of eggs that are readily manipulated by injection and microsurgery. The combination of experimental embryology and molecular manipulation provide the tools to understand embryonic signaling at the molecular level. Many of the paradigms for early developmental mechanisms in vertebrates have come from work with amphibians, and many of the signaling activities that bring about early developmental decisions in vertebrates have been identified first in amphibians. During previous grant periods, we have identified signals that act in early axis formation, mesoderm patterning and neural induction. In conjunction with the work of many other groups, this has led to a coherent picture of how the embryonic axes are established, and how a cascade of signal transductions leads to the elaborate pattern of the gastrula. Despite the progress that has been made in understanding embryonic signals, there is still only a partial picture of how the detailed pattern of the embryo emerges. The intracellular mediation of signaling is poorly understood, and although the main pathways that signal in development have been identified, the precise roles and modulation of these pathways remains to be determined. The formation of the neural plate with its elaborate patterning in both anterior-posterior and mediolateral axes poses a particular challenge, and this proposal will examine the integration of signaling pathways that induce and pattern the neural plate. Particular focus will be given to Fibroblast Growth Factor signaling in neural patterning, and in neural crest formation. The integration of signals that induce the neural crest will be studied by exploiting and comparing various manipulations that bring about neural crest development. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): To advance our understanding of ear development and the underlying molecular and genetic interactions, we are undertaking a forward genetic screen in the amphibian Xenopus tropicalis, a recently introduced genetic model animal. This pilot study reveals several loci involved in ear morphology, otolith formation, and balancing/swimming behavior. Importantly, the major events of X. tropicalis ear morphogenesis can be observed externally in the living embryos, greatly aiding screens for genetic defects. Previous work in ear development has demonstrated that underlying molecular mechanisms are well conserved from human to frog, making genetically amenable and oviparous X. tropicalis an attractive model animal in which to study the causes of hearing and balance anomalies as well as underlying molecular and genetic interactions. Previous screens for inner ear mutants have been done in zebrafish and mouse, and we expect to find both overlapping and different mutations in Xenopus. In this proposal we shall identify and map mutations affecting the ear, and link them to previously known candidate genes, or identify them as new loci involved in ear development. We shall characterize the mutant phenotypes, concentrating on the novel loci. This will establish Xenopus tropicalis as an effective new system to understand ear development and disorders. As described in the Program Announcement, the formation of the human inner ear is complex, and both the normal development and developmental disorders are poorly understood. The understanding of inner ear development will benefit from studies in model organisms such as Xenopus tropicalis, where mutant phenotypes can be used to understand the underlying mechanisms for normal development.

0921489
Harland

This Pan-American Advanced Studies Institutes (PASI) award, jointly supported by the NSF and the Department of Energy (DOE), will take place on November 2 through 11, 2010, at the Pontifica Universidad Catolica de Chile in Santiago, Chile. Organized by Dr. Richard Harland of the University of California, Berkeley, the PASI will address the topic of concepts and model organisms in regenerative biology by exposing participants to the fundamental concepts, questions, model organisms and research tools used in regenerative and developmental biology. Laboratory workshops will provide hands-on experience to students working with different organisms that exhibit regenerative properties.

The activity will train students before entering the pathway of becoming future researchers and educators in biology and related areas. Expected outcomes in this PASI will be to: foster cooperation between senior and junior investigators and provide an open forum for discussion of current topics relevant to science policy and education in the Americas. Results of the PASI will be disseminated through a website and a digital library.

DESCRIPTION (provided by applicant): Research on Xenopus has provided numerous new insights into cell and developmental biology. The eggs are readily manipulated by microsurgery and microinjection, and with their large size and abundance, either normal or manipulated eggs provide excellent material for biochemical and cell biological analysis. In order to make Xenopus useful for the modern age of "systems biology" where proteomic and genomic analyses promise a comprehensive understanding of life's processes, a high quality assembly of the Xenopus genome is needed. A high quality genome structure will provide a comprehensive catalog of gene content and proteome, authoritative data on conservation of chromosome structure with other vertebrates, and will improve regions of mis-assembly, bringing short scaffold regions into a chromosome-scale assembly. This proposal builds on the previous high quality draft genome assembly produced at the Department of Energy's Joint Genome Institute. While the quality is good in gene-rich regions, the long-range assembly of the genome is not as good as that for other tetrapods. This proposal will bypass the previous difficulties in assembling over the long range, by avoiding cloning-based methods of sequence mapping and long-range assembly. Instead we will use high throughput DNA sequencing and statistically based map assembly to generate a physical and genetic map, incorporating newly identified Single Nucleotide Polymorphisms (SNPs) and previously identified Simple sequence length polymorphisms (SSLPs). We will provide support for genome annotation by Metazome and Xenbase and ensure that the resources are made widely available. PUBLIC HEALTH RELEVANCE: Work on model organisms has allowed the discovery of many fundamental properties of animals, and thereby allowed new insights into how human embryos develop and function. Xenopus offers large embryos that develop outside the mother, which has enabled discoveries on cell proliferation and many developmental events. The genome structure and full gene set will permit new genes and functions to be identified, functions that are highly relevant to human development and disease.

DESCRIPTION (provided by applicant): Research on the amphibian Xenopus has provided numerous new insights into cell and developmental biology. With their large size and abundance, they provide unparalleled material for biochemical and cell biological analysis of complex processes such as the cell cycle and chromosome mechanics. For embryological experiments, the embryos are readily manipulated by microsurgery and by microinjection can be subjected to gain or loss of gene function. In order to make Xenopus more useful for the modern age of systems biology where proteomic and genomic analyses promise a comprehensive understanding of life's processes, we propose here to complement the genome assembly of Xenopus tropicalis with a gene and protein level genome assembly for Xenopus laevis. The allotetraploid Xenopus laevis is in wider use than the smaller, diploid Xenopus tropicalis, because of its history, robustness, and the size and quantity of eggs that can be obtained for embryological and cell biological experiments. We propose to carry out high throughput sequencing of X. laevis, and generate a gene-scale assembly. By selecting regions complementary to the X. tropicalis sequence we will be able to assemble X. laevis genes from relatively inexpensive, short read data. The project provides some computational challenges that will need to be overcome and the approaches developed will be of wide utility in characterizing genomes of other organisms. We will provide support for genome annotation by Xenbase and deposit gene and protein collections in public databases to ensure that the resources are widely available. PUBLIC HEALTH RELEVANCE: Xenopus laevis eggs and embryos have been the material of choice for work on vertebrate experimental embryology and biochemical dissection of the cell cycle, providing insights into human biology. We propose to fill a large gap in the essential resources for this work, namely a catalogue of the gene and protein content.

DESCRIPTION (provided by applicant): Deficits in hearing or balance are common, and result from both developmental and environmental causes. In order to understand normal inner ear development, we will use the genetically tractable frog, Xenopus tropicalis, to investigate the genetic network underlying ear development. The structure and development of the inner ear shows conserved features among the vertebrates, and the development of the ear of the tetrapod is very similar to that of mammals. The relevant stages of ear development can be observed in the optically clear tadpole, making this animal ideal for genetic screens for ear mutants. Furthermore, defects in ear development lead to abnormal swimming behavior and loss of the righting response, so that defects that may not be anatomically obvious can also be scored. In response to PA-06-365, Cell Lineage and Developmental Studies in Hearing and Balance we have addressed the stated need for more comprehensive representation of model organisms systems to study ear development. We propose to advance our understanding of ear development by examining otic vesicle development in the tetrapod Xenopus tropicalis, an amphibian whose inner ear development is well-conserved with mammals. In a small-scale forward genetic screen using Xenopus tropicalis we have recovered mutants that disrupt ear morphology, otolith formation, and balancing/swimming behavior. We have isolated the affected gene in two of these that affect otoconial development and otocyst size. Thus we have shown that it is possible to screen for and recover mutants, analyze the phenotype, and map and clone the affected genes. The genome of X. tropicalis shows considerable structural similarity and synteny with mammalian genomes, with no sign of additional whole genome duplications, so we are confident that we can identify recessive mutants in a variety of genes which show conserved functions with the mammals. With recent improvements in the genome assembly and annotation, and technical advances in exon capture and high throughput sequence analysis, we are confident that other mutations can be rapidly mapped, and the affected genes isolated. In the next grant period, we propose to characterize additional mutant alleles by positional cloning, and link mutant phenotypes with the underlying molecular and cellular defects. We combine molecular approaches with classical embryological transplantations to understand the cel autonomy of mutants, and the signaling and responding tissues that interact to produce the functioning ear.

DESCRIPTION (provided by applicant): Research on the amphibian Xenopus has provided numerous new insights into cell and developmental biology. With their large size and abundance, they provide unparalleled material for biochemical and cell biological analysis of complex processes such as the cell cycle and chromosome mechanics. For embryological experiments, the embryos are readily manipulated by microsurgery or microinjection, and can be subjected to both gain or loss of gene function. In order to make Xenopus more useful for the modern age of systems biology where proteomic and genomic analyses promise a comprehensive understanding of life's processes, we propose here to continue improvement of both the short range, and long range (chromosome level) assemblies of Xenopus tropicalis and Xenopus laevis. To achieve new insights into the function of both coding and non-coding DNA in the Xenopus clade, and add new information from outgroups, we will assemble the pseudotetraploid genomes of X. mulleri, X. epitropicalis, and X. borealis. These allotetraploid Xenopus species will provide new insights into the evolution of tetraploid genomes and aid in annotating non-coding sequences of Xenopus. In addition, we will assemble genomes for other outgroups, including the direct developing frog Eleutherodactylus coqui, the Tungara frog Engystomops, and the spadefoot toad, Spea, all of which are models for developmental, neurobiological or behavioral studies. These will also provide outgroup sequences for comparison and annotation, as well as enabling molecular approaches for the communities who study these frogs for their developmental or neurobiological advantages. We will provide genome assemblies and automated annotation of the comparative information to Xenbase and deposit gene and protein collections in public databases to ensure that the resources are widely available.

ABSTRACT Autism spectrum disorder (ASD) is a devastating neurodevelopmental disorder of undetermined etiology and without effective therapeutics. Recent advances in genomic approaches have led to the identification of over 65 ASD risk genes. Despite the genetic heterogeneity of ASD, several lines of evidence suggest that these genes share common molecular underpinnings. Therefore, we hypothesize that these genes will converge upon shared phenotypes when inhibited in model organisms and that these phenotypes will be the most central to the neuropathology of ASD. Identifying such convergent phenotypes requires a high-throughput system for modifying many ASD genes in parallel and assaying their effect(s) on embryonic brain development. Here we propose to leverage the diploid vertebrate tetrapod, Xenopus tropicalis, and the CRISPR/Cas9 system to identify convergent phenotypes among ASD genes during brain development. By injecting Cas9 protein and a single guide RNA (sgRNA) against an ASD gene at the two-cell stage, animals will be generated in which exactly half the body (separated by the midline) is mutant, allowing for direct comparison of mutant and control cells in the same animal. This will be performed for approximately 65 ASD risk genes, and the effects of ASD gene loss will be assayed by imaging neurons throughout embryogenesis using fluorescent reporters and by in situ RNA hybridization for neuronal cell fate specification markers. By employing fluorescence activated cell sorting (FACS), next generation RNA sequencing, and weighted gene co-expression network analysis (WGCNA), convergent transcriptional signatures of ASD gene loss will be characterized. The biological pathways indicated by these convergent signatures will be validated through targeted manipulation of key genes. Importantly, the validated biological pathways may provide clues about the observed convergent phenotypes. By combining the high-throughput capability of the CRISPR/Cas9 system and a tractable vertebrate model organism with a reliably-associated set of ASD genes, this study aims to understand the neuropathology of ASD at a critical developmental period, which should provide critical insights into the etiology of this disorder.

Abstract Xenopus is a widely-used model organism for basic biomedical discovery. This application requests funds to support the 17th International Xenopus Conference. Held every two years since 1984, this biennial meeting?s prime objective is to provide a forum for information exchange and interaction amongst researchers using Xenopus as a model organism for biomedical research. The last iteration of this meeting (in the United States) was attended by over 350 researchers from all over the world. In addition to serving as a platform for information exchange, this meeting will also provide 1) a venue for introducing younger scientists to more established members of the field, 2) an opportunity for the Xenopus community to learn about the latest technologies and to coordinate its infrastructure, and 3) an opportunity to introduce this powerful model organism to students from under-represented groups. This meeting will have a significant impact because Xenopus is widely used in developmental biology, cell biology, molecular biology, and neurobiology, and many of the most exciting discoveries now are coming from the interfaces between these disciplines. By bringing together researchers with divergent research interested, but a common model system, the meeting should foster outstanding interdisciplinary thinking. Modern biology demands that we be well-informed, broad-based, organized, and collaborative in our approaches, and the International Xenopus Conference will facilitate exactly that style of science.