- Email: [email protected]
Genomics boosts mouse molecular genetics
Research Update
TRENDS in Genetics Vol.17 No.12 December 2001
691
Genomics boosts mouse molecular genetics Carmen Birchmeier and Mathias Treier The Mouse Molecular Genetics Meeting was held at the European Molecular Biology Laboratories in Heidelberg, Germany, from 22nd to 26th August 2001.
One of the largest gatherings of mouse molecular geneticists this year, this meeting brought together 250 international delegates in Heidelberg. It was the first meeting since the release of a draft version of the mouse genome sequence, which promises a revolution for those using mice to understand gene function and to model human disease. Genomic resources and their impact on mouse genetics
Richard Mural (Celera, Rockville, MD, USA) reported on the assembly and annotation of the first draft of the mouse genomic sequence. About 98% of the mouse sequence is available, and nearly 50% of it assembles into scaffolds of more than 5 Mb. Three different strains (129X1/SV, A/J and DBA/2) were used for the sequencing, and their sequence variation has generated a wealth of information for the identification of single nucleotide polymorphisms (SNPs) used in gene mapping. The mouse genome is smaller than the human genome by 15% – not due to decreased gene number, but to the amount of noncoding sequence. The impact of the genomic sequence was already obvious at this year’s meeting, despite the fact that as yet only very few scientists in the community have access to the genome sequence. The sequence is available only to those that subscribe to the Celera mouse database. This was lamented by the many researchers whose institutions have not yet purchased, or will not pay for, the Celera licence. A letter to Francis Collins was drafted at the meeting, pleading for a speedy release by the public consortium of an annotated mouse genome sequence that will be accessible to all. Yoshihide Hayashizaki (RIKEN Center, Yokohama, Japan) reported on the RIKEN full-length mouse cDNA collection, their sequence and http://tig.trends.com
chromosomal mapping. So far, over 20 000 independent cDNA clones have been sequenced, annotated and made available to the public1. Cherry Blossom, a meeting scheduled for spring 2002, will conclude this initial effort by the annotation of 50 000 cDNA clones. The RIKEN clones can be ordered through K.K. Dnaform (http://www.dnaform.co.jp). Functional annotation is now under way, and includes the expression analysis of all cloned cDNAs in developing and adult tissues, as well as a yeast two-hybrid analysis to identify interaction partners of the encoded proteins. Characterization of other mammalian genomes is also enhancing mouse genetics. Formerly a Sisyphus enterprise, efficient positional cloning has now become feasible for mouse mutations, and it was notable that an increasing number of mouse mutants that have been available for decades are finally being characterized at a molecular level, among them the classical mutations that affect coat colour and hair development. Nancy Jenkins (NCI, Frederick, USA) presented the characterization of four genetically interacting mouse coat colour mutations, which all affect the transport of pigmentcontaining vesicles in melanocytes. Dilute encodes the melanosome transport motor MyoVa; ashen encodes Rab27a, a protein postulated to serve as the MyoVa receptor; and leaden encodes melanophilin, which has homology to known Rab effectors2. In humans, mutations in transport machinery components Myo5a and Rab27a are associated with Griscelli syndrome. The protein encoded by suppressor of dilute has an unknown role in this transport process, and is one of the first examples of the characterization of a second site suppressor in mice. Paul Overbeek (Baylor College, Houston, USA) discussed the characterization of mutants displaying hair and tooth defects, revealing how morphogenesis of hair follicles and other epithelial appendages (teeth, sweat glands) is initiated. A linear signalling cascade is encoded by the various mutant genes – tabby encodes a
TNF-like ligand, downless the receptor, and crinkled a cytoplasmic signalling molecule. The human homologue of tabby, ED1, affects also hair and tooth development when mutated (hypohydrotic ectodermal dysplasia). Advances in mapping and cloning of mutant mouse genes have encouraged the mouse community to employ forward genetic approaches (mainly N-ethyl-Nnitrosourea [ENU] mutagenesis) systematically to analyse developmental and physiological processes. Screens for dominant as well as for recessive mutations were presented. Some of these are large-scale screens, however even small-scale screens can generate enough interesting mutations to keep a laboratory busy. Maja Bucan (University of Pennsylvania, Philadelphia, USA) presented a screen for dominant mutations affecting complex behavioural traits such as circadian rhythm, sleep pattern, anxiety, neuromuscular function and the acoustic startling response. One semidominant mutation, named Earlybird, shortens the circadian period of locomotor activity. Molecular analysis revealed a mutation in a gene that had previously been characterized by reverse genetics. However, the effect of the mutation on circadian rhythm had been missed, emphasizing the importance of standardized tests to identify deviations from normal behaviour and physiology in mutant mice. Reverse genetics
Reverse genetics, the dominant topic of previous mouse molecular genetics meetings, continues to yield important information on gene function and frequently generates models for human disease. An example of this was the analysis of a family of ion channels (CLC) discussed by Thomas Jentsch (ZMBH, Hamburg, Germany). These channels transport Cl− ions and function in acidification of various types of cytoplasmic vesicles. Mutation of CLC-5 in mouse unexpectedly revealed a phenotype similar to the one seen in humans with Dent’s disease, leading to the identification of mutations in the
0168-9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02516-1
692
Research Update
human CLC-5 gene. Similarly, disruption of CLC-7 causes osteoporosis in mice and human, and interferes with the acidification of the resorption lacuna in osteoclasts3. Yosuke Tanaka (Tokyo University, Tokyo, Japan) showed that a member of the kinesin family, KIF1B, functions in vesicle transport along axons and, when mutated, causes a peripheral neuropathy in mice and Charcot–Marie–Tooth disease type 2A in humans4. Complex genetic mechanisms were discussed by Peter Koopman (University of Queensland, Brisbane, Australia), who compared a targeted and a spontaneous mutation in the Sox18 gene. The spontaneous mutation (ragged) causes a premature truncation of the encoded protein and severely affects development of the skin and vasculature5. However, a targeted mutation that completely eliminates the protein has very mild phenotypes, indicating that the protein encoded by the spontaneous allele might suppress the function of other Sox proteins in trans. In general, use of double mutants, knock-in and conditional-mutant alleles has substantially increased, and the community frequently uses sophisticated reverse genetics to analyse complex processes. One example was presented by Greg Lemke (Salk Institute, San Diego, USA): the use of a knock-in of the EphA receptor to prove finally that gradients sensed by this receptor family are responsible for establishing the topographic map in the visual system6. Numb is a cytoplasmic, membrane-associated molecule that distributes asymmetrically during cell division and is implicated in Notch signalling. Using double mutants, Weimin Zhong (Yale University, New Haven, USA) presented evidence that the decision between cell renewal and differentiation in the mouse nervous system depends critically on numb and numb-like. New technologies in mouse genetics
The last day of the meeting was devoted to the discussion of novel technologies in mouse molecular genetics. Sleeping beauty, an inactive transposon identified in fish and resurrected by ‘reverse evolution’, was demonstrated by Junji Takeda http://tig.trends.com
TRENDS in Genetics Vol.17 No.12 December 2001
(Osaka University, Japan) and David Largaespada (University of Minnesota, Minneapolis, USA) to transpose with good frequency in the male germline if the transposase is provided in trans7,8. Transposons as mutational agents might thus complement chemical mutagenesis in mice. Frank Buchholz (University of California, San Francisco, USA) reported on the use of the Cre–loxP system to induce directed chromosomal translocations in the genome, a technique that will be useful in modelling human tumours. Aggregation of tetraploid embryos with ES cells results in generation of embryos that are completely derived from ES cells – previously possible only at very low efficiency. Ralf Kuehn (Artemis, Cologne, Germany) reported and confirmed that the use of hybrid ES cell lines derived from F1 embryos (C57BL/6Jx129/SvEv Tac) notably speeds up the generation of mutant mice9. Rosa Beddington lecture
Patrick Tam (University of Sydney, Australia) presented the first annual lecture dedicated to Rosa Beddington, to honour the influential developmental biologist and former organizer of the Mouse Molecular Genetics Meeting who died in May this year. Tam recalled Rosa Beddigton’s seminal work on axis formation in the mouse embryo. She was the first to show that the node from a late primitive streak stage mouse embryo could induce a secondary neural axis when transplanted; the new secondary axis, however, lacked the anterior components. She then discovered that the anterior visceral endoderm of the pre-streak stage mouse embryo was important for inducing anterior neural identity, and synthesized these findings into a model for anterioraxis specification in the mouse. Tam also presented new data that show that the anterior region of the primitive streak of the mid-streak stage embryo has full axis-induction ability. His elegant transplantation studies demonstrate that the mid-gastrula organizer (MGO) is sufficient to induce a secondary axis complete with anterior neural character. He also showed that cells destined to form anterior axial mesoderm leave the MGO and migrate along the midline to reach the
prospective anterior neural plate region, facilitating inductive interactions. Tam’s new findings combined with Beddington’s pioneering observations now provide a deeper understanding of the cell fates, morphogenetic movements, and temporal inductive interactions that occur during early mouse embryogenesis to specify the anterior–posterior axis. Other excellent lectures, short presentations and the poster sessions crowded by discussants summarized the current activities of the mouse research community. All in all, it was an intense conference that has stimulated thoughts and future research. References 1 Kawai, J. et al. (2001) Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–690 2 Matesic, L.E. et al. (2001) Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defect observed in leaden mice. Proc. Natl. Acad. Sci. U. S. A. 98, 10238–10243 3 Kornak, U. et al. (2001) Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205–215. 4 Zhao, C. et al. (2001) Charcot–Marie–Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta. Cell 105, 587–597 5 Pennisi, D. et al. (2000) Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nat. Genet. 24, 434–437 6 Brown, A. et al. (2000) Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell 102, 77–88 7 Horie, K. et al. (2001) Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice. Proc. Natl. Acad. Sci. U. S. A. 98, 9191–9196 8 Dupuy, A.J. et al. (2001) Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88 9 Eggan, K. et al. (2001) Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. U. S. A. 98, 6209–6214
Carmen Birchmeier* Max-Delbrueck-Centrum, Robert-Roessle-Strasse 10, D-13125 Berlin-Buch, Germany. *e-mail: [email protected] Mathias Treier Developmental Biology Programme, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
TRENDS in Genetics Vol.17 No.12 December 2001
691
Genomics boosts mouse molecular genetics Carmen Birchmeier and Mathias Treier The Mouse Molecular Genetics Meeting was held at the European Molecular Biology Laboratories in Heidelberg, Germany, from 22nd to 26th August 2001.
One of the largest gatherings of mouse molecular geneticists this year, this meeting brought together 250 international delegates in Heidelberg. It was the first meeting since the release of a draft version of the mouse genome sequence, which promises a revolution for those using mice to understand gene function and to model human disease. Genomic resources and their impact on mouse genetics
Richard Mural (Celera, Rockville, MD, USA) reported on the assembly and annotation of the first draft of the mouse genomic sequence. About 98% of the mouse sequence is available, and nearly 50% of it assembles into scaffolds of more than 5 Mb. Three different strains (129X1/SV, A/J and DBA/2) were used for the sequencing, and their sequence variation has generated a wealth of information for the identification of single nucleotide polymorphisms (SNPs) used in gene mapping. The mouse genome is smaller than the human genome by 15% – not due to decreased gene number, but to the amount of noncoding sequence. The impact of the genomic sequence was already obvious at this year’s meeting, despite the fact that as yet only very few scientists in the community have access to the genome sequence. The sequence is available only to those that subscribe to the Celera mouse database. This was lamented by the many researchers whose institutions have not yet purchased, or will not pay for, the Celera licence. A letter to Francis Collins was drafted at the meeting, pleading for a speedy release by the public consortium of an annotated mouse genome sequence that will be accessible to all. Yoshihide Hayashizaki (RIKEN Center, Yokohama, Japan) reported on the RIKEN full-length mouse cDNA collection, their sequence and http://tig.trends.com
chromosomal mapping. So far, over 20 000 independent cDNA clones have been sequenced, annotated and made available to the public1. Cherry Blossom, a meeting scheduled for spring 2002, will conclude this initial effort by the annotation of 50 000 cDNA clones. The RIKEN clones can be ordered through K.K. Dnaform (http://www.dnaform.co.jp). Functional annotation is now under way, and includes the expression analysis of all cloned cDNAs in developing and adult tissues, as well as a yeast two-hybrid analysis to identify interaction partners of the encoded proteins. Characterization of other mammalian genomes is also enhancing mouse genetics. Formerly a Sisyphus enterprise, efficient positional cloning has now become feasible for mouse mutations, and it was notable that an increasing number of mouse mutants that have been available for decades are finally being characterized at a molecular level, among them the classical mutations that affect coat colour and hair development. Nancy Jenkins (NCI, Frederick, USA) presented the characterization of four genetically interacting mouse coat colour mutations, which all affect the transport of pigmentcontaining vesicles in melanocytes. Dilute encodes the melanosome transport motor MyoVa; ashen encodes Rab27a, a protein postulated to serve as the MyoVa receptor; and leaden encodes melanophilin, which has homology to known Rab effectors2. In humans, mutations in transport machinery components Myo5a and Rab27a are associated with Griscelli syndrome. The protein encoded by suppressor of dilute has an unknown role in this transport process, and is one of the first examples of the characterization of a second site suppressor in mice. Paul Overbeek (Baylor College, Houston, USA) discussed the characterization of mutants displaying hair and tooth defects, revealing how morphogenesis of hair follicles and other epithelial appendages (teeth, sweat glands) is initiated. A linear signalling cascade is encoded by the various mutant genes – tabby encodes a
TNF-like ligand, downless the receptor, and crinkled a cytoplasmic signalling molecule. The human homologue of tabby, ED1, affects also hair and tooth development when mutated (hypohydrotic ectodermal dysplasia). Advances in mapping and cloning of mutant mouse genes have encouraged the mouse community to employ forward genetic approaches (mainly N-ethyl-Nnitrosourea [ENU] mutagenesis) systematically to analyse developmental and physiological processes. Screens for dominant as well as for recessive mutations were presented. Some of these are large-scale screens, however even small-scale screens can generate enough interesting mutations to keep a laboratory busy. Maja Bucan (University of Pennsylvania, Philadelphia, USA) presented a screen for dominant mutations affecting complex behavioural traits such as circadian rhythm, sleep pattern, anxiety, neuromuscular function and the acoustic startling response. One semidominant mutation, named Earlybird, shortens the circadian period of locomotor activity. Molecular analysis revealed a mutation in a gene that had previously been characterized by reverse genetics. However, the effect of the mutation on circadian rhythm had been missed, emphasizing the importance of standardized tests to identify deviations from normal behaviour and physiology in mutant mice. Reverse genetics
Reverse genetics, the dominant topic of previous mouse molecular genetics meetings, continues to yield important information on gene function and frequently generates models for human disease. An example of this was the analysis of a family of ion channels (CLC) discussed by Thomas Jentsch (ZMBH, Hamburg, Germany). These channels transport Cl− ions and function in acidification of various types of cytoplasmic vesicles. Mutation of CLC-5 in mouse unexpectedly revealed a phenotype similar to the one seen in humans with Dent’s disease, leading to the identification of mutations in the
0168-9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02516-1
692
Research Update
human CLC-5 gene. Similarly, disruption of CLC-7 causes osteoporosis in mice and human, and interferes with the acidification of the resorption lacuna in osteoclasts3. Yosuke Tanaka (Tokyo University, Tokyo, Japan) showed that a member of the kinesin family, KIF1B, functions in vesicle transport along axons and, when mutated, causes a peripheral neuropathy in mice and Charcot–Marie–Tooth disease type 2A in humans4. Complex genetic mechanisms were discussed by Peter Koopman (University of Queensland, Brisbane, Australia), who compared a targeted and a spontaneous mutation in the Sox18 gene. The spontaneous mutation (ragged) causes a premature truncation of the encoded protein and severely affects development of the skin and vasculature5. However, a targeted mutation that completely eliminates the protein has very mild phenotypes, indicating that the protein encoded by the spontaneous allele might suppress the function of other Sox proteins in trans. In general, use of double mutants, knock-in and conditional-mutant alleles has substantially increased, and the community frequently uses sophisticated reverse genetics to analyse complex processes. One example was presented by Greg Lemke (Salk Institute, San Diego, USA): the use of a knock-in of the EphA receptor to prove finally that gradients sensed by this receptor family are responsible for establishing the topographic map in the visual system6. Numb is a cytoplasmic, membrane-associated molecule that distributes asymmetrically during cell division and is implicated in Notch signalling. Using double mutants, Weimin Zhong (Yale University, New Haven, USA) presented evidence that the decision between cell renewal and differentiation in the mouse nervous system depends critically on numb and numb-like. New technologies in mouse genetics
The last day of the meeting was devoted to the discussion of novel technologies in mouse molecular genetics. Sleeping beauty, an inactive transposon identified in fish and resurrected by ‘reverse evolution’, was demonstrated by Junji Takeda http://tig.trends.com
TRENDS in Genetics Vol.17 No.12 December 2001
(Osaka University, Japan) and David Largaespada (University of Minnesota, Minneapolis, USA) to transpose with good frequency in the male germline if the transposase is provided in trans7,8. Transposons as mutational agents might thus complement chemical mutagenesis in mice. Frank Buchholz (University of California, San Francisco, USA) reported on the use of the Cre–loxP system to induce directed chromosomal translocations in the genome, a technique that will be useful in modelling human tumours. Aggregation of tetraploid embryos with ES cells results in generation of embryos that are completely derived from ES cells – previously possible only at very low efficiency. Ralf Kuehn (Artemis, Cologne, Germany) reported and confirmed that the use of hybrid ES cell lines derived from F1 embryos (C57BL/6Jx129/SvEv Tac) notably speeds up the generation of mutant mice9. Rosa Beddington lecture
Patrick Tam (University of Sydney, Australia) presented the first annual lecture dedicated to Rosa Beddington, to honour the influential developmental biologist and former organizer of the Mouse Molecular Genetics Meeting who died in May this year. Tam recalled Rosa Beddigton’s seminal work on axis formation in the mouse embryo. She was the first to show that the node from a late primitive streak stage mouse embryo could induce a secondary neural axis when transplanted; the new secondary axis, however, lacked the anterior components. She then discovered that the anterior visceral endoderm of the pre-streak stage mouse embryo was important for inducing anterior neural identity, and synthesized these findings into a model for anterioraxis specification in the mouse. Tam also presented new data that show that the anterior region of the primitive streak of the mid-streak stage embryo has full axis-induction ability. His elegant transplantation studies demonstrate that the mid-gastrula organizer (MGO) is sufficient to induce a secondary axis complete with anterior neural character. He also showed that cells destined to form anterior axial mesoderm leave the MGO and migrate along the midline to reach the
prospective anterior neural plate region, facilitating inductive interactions. Tam’s new findings combined with Beddington’s pioneering observations now provide a deeper understanding of the cell fates, morphogenetic movements, and temporal inductive interactions that occur during early mouse embryogenesis to specify the anterior–posterior axis. Other excellent lectures, short presentations and the poster sessions crowded by discussants summarized the current activities of the mouse research community. All in all, it was an intense conference that has stimulated thoughts and future research. References 1 Kawai, J. et al. (2001) Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–690 2 Matesic, L.E. et al. (2001) Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defect observed in leaden mice. Proc. Natl. Acad. Sci. U. S. A. 98, 10238–10243 3 Kornak, U. et al. (2001) Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205–215. 4 Zhao, C. et al. (2001) Charcot–Marie–Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta. Cell 105, 587–597 5 Pennisi, D. et al. (2000) Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nat. Genet. 24, 434–437 6 Brown, A. et al. (2000) Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell 102, 77–88 7 Horie, K. et al. (2001) Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice. Proc. Natl. Acad. Sci. U. S. A. 98, 9191–9196 8 Dupuy, A.J. et al. (2001) Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88 9 Eggan, K. et al. (2001) Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. U. S. A. 98, 6209–6214
Carmen Birchmeier* Max-Delbrueck-Centrum, Robert-Roessle-Strasse 10, D-13125 Berlin-Buch, Germany. *e-mail: [email protected] Mathias Treier Developmental Biology Programme, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.