Genetic Analysis Of Vertebrate Development In Xenopus tropicalis

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Recent advances in deciphering the human genetic code have great implications for treating disease. In order to make medical use of this genetic sequence information, it is critical to understand the functions of the encoded genes, and especially how they are used in the process of assembling the developing embryo and its constituent organs. Functional data are available for only a fraction of the newly-identified genes. One of the lessons of the molecular era is the high degree to which different animals share the same genetic processes, and hence can be fruitfully used to help understand human development. This project explores using the amphibian Xenopus tropicalis as a vertebrate system in which gene functions can be studied using combinations of molecular, genetic, and microsurgical techniques. |The relatively large, externally-developing amphibian embryo has been used extensively for gain-of-function studies in which the effects of injected gene products are analysed. This projects goal is to add loss-of-function approaches, in which gene products are deleted by mutation or other methods. X. tropicalis studies allow use of both loss-of-function studies, which tell us which processes specific genes are required for, with gain-of-function approaches, which tell us which processes genes are capable of.

Technical Summary

Recent advances in deciphering the human genetic code have great implications for treating disease. In order to make medical use of this genetic sequence information, it is critical to understand the functions of the encoded genes, and especially how they are used in the process of assembling the developing embryo and its constituent organs. Functional data are available for only a fraction of the newly-identified genes. One of the lessons of the molecular era is the high degree to which different animals share the same genetic processes, and hence can be fruitfully used to help understand human biology. This project is aimed at developing the amphibian Xenopus tropicalis as a vertebrate system in which gene functions can be studied using a variety of methods. Amphibian embryos have been very useful for developmental studies due their large size and external development, which among other things permits delivery of gene products, such as mRNA and protein, by injection. Functions of which specific gene products are capable can then be analysed (gain-of-function studies). Complementing this approach are loss-of-function studies, in which gene products are deleted, by mutation or other methods, in order to identify and study the biological processes for which particular genes are required. While mice can be used for loss-of-function mutational studies, the inaccessibility of their developing embryos makes scaling up this kind of analysis difficult and expensive. X. tropicalis is suitable for gain-of-function studies, but has great potential for loss-of-function studies because unlike other lower vertebrate models (zebrafish and its close relative Xenopus laevis), it has a relatively simple genomic structure resembling that of mammals. Loss-of-function approaches currently underway include a chemical mutagenesis screen for recessive mutations in early embryonic development and patterning, and knockdown screens using antisense morpholino oligonucleotides to target genes of unknown function identified in EST sequencing projects. X. tropicalis also has a generation time about half that of X.laevis, which makes other multigeneration strategies feasible, such as studies employing stable transgenic lines. Fluorescent reporter transgenic lines are being established and used to streamline screens for effects on gene expression. Other stable transgenic approaches being developed include insertional mutagenesis, gene targeting, and binary and inducible systems for investigating effects of transgene expression on development. Transgenesis also allows directed approaches to complement random genetic screens. A principal focus of this project is dissection of the signalling pathways which pattern brain development. In addition to collecting mutations with neural phenotypes, we are studying these pathways by generating transgenic lines in which we can dial down specific growth factor signals in spatially- and temporally-controlled ways. For example, we are developing transgenic lines with which to mis-express endogenous ligand-trap molecules such as noggin and frzB, which efficiently bind and inhibit activities of the Bone Morphogenetic Protein and Wnt families of intercellular signalling factors. Two other groups in the Division of Developmental Biology at N.I.M.R., the Mohun and Logan laboratories, are also making significant contributions to this project.

Publications

10 25 50
 
Description Genetic analysis of tetrapod development (researcher influence)
Geographic Reach North America 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Xenopus White Paper
Geographic Reach North America 
Policy Influence Type Participation in advisory committee
 
Description Xenopus genetic methods training
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description PAR- Genetic and Genomic Analysis of Xenopus
Amount £196,116 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start  
 
Description R01 National Institute for Hearing and Deafness Disorders
Amount £269,666 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start  
 
Description R21 National Institute for Hearing and Deafness Disorders
Amount £43,732 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start  
 
Title Mutant frog strain lacking cardiac myosin heavy chain 
Description The 'muzak' mutation is a null allele of the lone cardiac myosin heavy chain (myh6) in developing Xenopus heart, leading to a complete absence of contractile activity, thick filaments, and myofibrils. Myh6 mutations are associated with atrial-septal defect in human. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact This mutant is unique among vertebrates in the complete absence of myosin thick filaments. Other research groups are using this model to address fundamental processes in vertebrate myofibrillogenesis. 
 
Title Mutant frog strain lacking nephronectin gene 
Description homozygous mutants of this strain abrogate forelimb formation, but form hindlimbs normally 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2010 
Provided To Others? Yes  
Impact This mutation identifies a step in tetrapod limb subtype initiation upstream of the earliest known limb field marker, tbx5. 
 
Title Mutant frog strain model for Menkes Disease 
Description Menkes Disease in human is caused by defects in the copper transporter ATP7a. We have shown that the kaleidoscope mutation in X. tropicalis is caused by a splicing mutation in ATP7a, and shares defects in pigmentation and cartilage formation with the human disease. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact The frog strains have been deposited in the European Xenopus Resource stock centre. 
 
Title Xenopus tropicalis mutant database 
Description Online collection of phenotypic descriptions of induced mutations in Xenopus tropicalis 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Research community open access to phenotypic analysis 
 
Title mutant frog strain lacking smad4 gene 
Description This mutation deletes a gene required for transducing certain intercellular signals involved in blood formation and other processes 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact The smad4 gene is involved in a number of cancer processes, as well as normal developmental events. This mutation will permit the study of these processes in an accessible lower vertebrate embryo. 
 
Description Bio-electrospray of Xenopus embryos 
Organisation University College London
Department Mechanical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team generated the embryos and performed molecular and morphological analysis of development following bio-electrospray of embryos ("inkjet"-like manipulation).
Collaborator Contribution The Jayasinghe group at Mechanical Engineering, UCL, developed the bio-electrospray method and machinery, making it possible to test this approach for manipulation of complex tissues.
Impact Demonstration that this direct cell engineering approach can be succesfully adapted to complex tissue environments including living embryos Published in PMID 19305925
Start Year 2008
 
Description Genetic Analysis of Inner Ear Development in Xenopus tropicalis 
Organisation University of California, Berkeley
Country United States 
Sector Academic/University 
PI Contribution Histological characterization of inner ear development, generation, phenotypic characterization, and mapping of mutations.
Collaborator Contribution Sharing resources and results, coordinating genetic mapping efforts and characterization of normal inner ear development. Collaboration has also resulted in a successful grant application (NIH R21) with funding from 2010.
Impact Successful application for NIH R01 support (funding from 2011).
Start Year 2008
 
Description Genetic analysis of Haematopoiesis in Xenopus tropicalis 
Organisation University of Oxford
Department Oxford Stem Cell Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated the mutations, characterized their phenotypes and mapped them genetically
Collaborator Contribution The collaborators characterized mutant phenotypes in haematopoiesis by in situ hybridization.
Impact This collaboration has resulted in detailed phenotypic characterization of mutations affecting embryonic and definitive blood formation, including the identification of one mutation in the smad4 gene.
Start Year 2008
 
Description Gynogenetic mapping of Xenopus centromeres and mutants 
Organisation Carnegie Institution for Science (CIS)
Country United States 
Sector Charity/Non Profit 
PI Contribution Our group developed the overall strategy, performed assays to locate centromeres genetically, evaluated recombination interference, and demonstrated the method for mapping mutations to chromosomes.
Collaborator Contribution The Harland lab at UC Berkeley performed assays to help locate the centromeres of Xenopus tropicalis chromosomes.The Houston group generated the genetic map of the Xenopus tropicalis genome upon which centromere location was based.Joe Gall at the Carnegie Institution performed cytological analysis of Xenopus tropicalis oocytes, providing fundamental information about how genetic recombination operates in this species.The Macha group performed fluorescence in situ hybridization analysis to confirm by cytology centromere locations obtained by genetic means.
Impact Published in PMID 19441086
Start Year 2007
 
Description Gynogenetic mapping of Xenopus centromeres and mutants 
Organisation Charles University
Country Czech Republic 
Sector Academic/University 
PI Contribution Our group developed the overall strategy, performed assays to locate centromeres genetically, evaluated recombination interference, and demonstrated the method for mapping mutations to chromosomes.
Collaborator Contribution The Harland lab at UC Berkeley performed assays to help locate the centromeres of Xenopus tropicalis chromosomes.The Houston group generated the genetic map of the Xenopus tropicalis genome upon which centromere location was based.Joe Gall at the Carnegie Institution performed cytological analysis of Xenopus tropicalis oocytes, providing fundamental information about how genetic recombination operates in this species.The Macha group performed fluorescence in situ hybridization analysis to confirm by cytology centromere locations obtained by genetic means.
Impact Published in PMID 19441086
Start Year 2007
 
Description Gynogenetic mapping of Xenopus centromeres and mutants 
Organisation University of California, Berkeley
Country United States 
Sector Academic/University 
PI Contribution Our group developed the overall strategy, performed assays to locate centromeres genetically, evaluated recombination interference, and demonstrated the method for mapping mutations to chromosomes.
Collaborator Contribution The Harland lab at UC Berkeley performed assays to help locate the centromeres of Xenopus tropicalis chromosomes.The Houston group generated the genetic map of the Xenopus tropicalis genome upon which centromere location was based.Joe Gall at the Carnegie Institution performed cytological analysis of Xenopus tropicalis oocytes, providing fundamental information about how genetic recombination operates in this species.The Macha group performed fluorescence in situ hybridization analysis to confirm by cytology centromere locations obtained by genetic means.
Impact Published in PMID 19441086
Start Year 2007
 
Description Gynogenetic mapping of Xenopus centromeres and mutants 
Organisation University of Houston
Country United States 
Sector Academic/University 
PI Contribution Our group developed the overall strategy, performed assays to locate centromeres genetically, evaluated recombination interference, and demonstrated the method for mapping mutations to chromosomes.
Collaborator Contribution The Harland lab at UC Berkeley performed assays to help locate the centromeres of Xenopus tropicalis chromosomes.The Houston group generated the genetic map of the Xenopus tropicalis genome upon which centromere location was based.Joe Gall at the Carnegie Institution performed cytological analysis of Xenopus tropicalis oocytes, providing fundamental information about how genetic recombination operates in this species.The Macha group performed fluorescence in situ hybridization analysis to confirm by cytology centromere locations obtained by genetic means.
Impact Published in PMID 19441086
Start Year 2007
 
Description Mapping mutations in Xenopus tropicalis 
Organisation University of Houston
Country United States 
Sector Academic/University 
PI Contribution Our group generated the mutation, characterized its phenotype, and performed the genetic mapping studies. We continue to collaborate to improve the genetic map of X. tropicalis by additional high-resolution mapping studies
Collaborator Contribution Collaborators helped create and refine the genetic map for the region of the genome containing a mutation which ablates functional myofibrils in the developing Xenopus heart
Impact This resulted in the first mapping to sequence lesion of a mutated gene in Xenopus tropicalis. The mutation is significant since it allows the study of myofibrillogenesis in the absence of myosin thick filaments as well as cardiac development in the absence of beating and blood flow. Published in PMID 1976995
Start Year 2007
 
Description Response to chytrid fungal infection in Xenopus tropicalis 
Organisation Imperial College London
Department Department of Infectious Disease & Epidemiology
Country United Kingdom 
Sector Academic/University 
PI Contribution Our group controlled for uninfected specimens, and provided tissue and live animals for infection studies.
Collaborator Contribution Collaborators performed infection and expression profiling analysis on Xenopus frogs
Impact These studies provided a critical understanding of the transcriptional response to infection by this fungal pathogen, which is thought to be a major contributor to worldwide amphibian declines and extinctions. Publication (accepted) 'Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachychytrium dendrobatidis', PLoS One
Start Year 2007
 
Description Reverse genetic resource for Xenopus tropicalis 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution conducted mutagenesis, grown animals, sampled tissue for reverse genetic analysis, analyzed phenotypes
Collaborator Contribution Collaborators have provided assistance with genetic and genomic analysis, including microarray and sequencing for gene expression profiling of mutants.
Impact determined molecular rates of mutagenesis for mutagenized populations of animals; established a large population to scan by sequence; developed methods for high-throughput identification of specific mutated genes; successful identification of >100 new mutations carried by specific lines.
Start Year 2008
 
Description Sex determination in Xenopus tropicalis 
Organisation University of Jaen
Country Spain 
Sector Academic/University 
PI Contribution Designed experiments to genetically map the sex determining locus, generated the pedigrees/animals and tissue, and performed genetic manipulations.
Collaborator Contribution Our collaborators have characterized early events in gonadal differentiation and helped to genetically map the sex determining locus in Xenopus tropicalis
Impact We have determined that X. tropicalis uses a ZW form of sex determination, and mapped the sex locus to a 15cM region.
Start Year 2007
 
Description Training secondary school students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Supervised secondary school students for 4-6 week research projects in the lab.

Students received exposure to and training in molecular and developmental biology, and produced written and oral reports for the scheme, which in 2010 won awards. Several students have gone on to biomedical courses at university.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011
 
Description visits of schools to MRC institute 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presented demonstrations on studying vertebrate development with Xenopus embryos at open house for school students

Exposure of students to developmental genetics increases interest in health sciences and adds to pool of students participating in summer research projects at NIMR
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013