Matthijs J. Smith

Gene expression during gonadogenesis in the chicken embryo

Genes implicated in vertebrate sex determination and differentiation were studied in embryonic chicken gonads using reverse transcription and the polymerase chain reaction (RT-PCR). Expression profiles were obtained during gonadal sex differentiation for AMH, SOX9, SOX3, the Wilms Tumour gene, WT1, and the orphan nuclear receptor genes, SF1 and DAX1. Some of these genes showed sexually dimorphic expression profiles during gonadal development, whereas others were expressed at similar levels in both sexes. The gene encoding Anti-Müllerian hormone (AMH) was expressed in both sexes prior to and during sexual differentiation of the gonads, with levels of expression consistently higher in males than in females. SOX9 expression was male-specific, and was up-regulated after the detection of AMH transcripts. SOX3 expression was observed prior to clear SOX9 expression and was up-regulated in both sexes at the onset of gonadal sex differentiation (but declined later in development). The WT1 gene was highly expressed in both sexes, whereas SF1 expression was clearly higher in developing ovaries compared to testes. DAX1 transcripts were observed in both sexes at all stages examined, but expression appeared somewhat higher in developing ovaries. These expression profiles are analysed in terms of current theories of vertebrate sex determination.

ASW: a gene with conserved avian W-linkage and female specific expression in chick embryonic gonad

Abstract Vertebrates exhibit a variety of sex determining mechanisms which fall broadly into two classes: environmental or genetic. In birds and mammals sex is determined by a genetic mechanism. In mammals males are the heterogametic sex (XY) with the Y chromosome acting as a dominant determiner of sex due to the action of the testis-determining factor, SRY. In birds females are the heterogametic sex (ZW); however, it is not known whether the W chromosome carries a dominant ovary-determining gene, or whether Z chromosome dosage determines sex. Using an experimental approach, which assumes only that the sex-determining event in birds is accompanied by sex-specific changes in gene expression, we have identified a novel gene, ASW (Avian Sex-specific W-linked). The putative protein for ASW is related to the HIT (histidine triad) family of proteins. ASW shows female-specific expression in genital ridges and maps to the chicken W chromosome. In addition, we show that, with the exception of ratites, ASW is linked to the W chromosome in each of 17 bird species from nine different families of the class Aves.

SOX14 is a candidate gene for limb defects associated with BPES and Möbius syndrome.

Abstract. Members of the SOX gene family encode proteins with homology to the HMG box DNA-binding domain of SRY, the Y-linked testis-determining gene. SOX genes are expressed during embryogenesis and are involved in the development of a wide range of different tissues. Mutations in SRY, SOX9 and SOX10 have been shown to be responsible for XY sex reversal, campomelic dysplasia and Waardenburg-Hirschsprung disease, respectively. It is likely that mutations in other SOX genes are responsible for a variety of human genetic diseases. SOX14 has been identified from a human genomic library and the mouse and chicken sequences obtained by polymerase chain reaction amplification. The SOX14 amino acid sequence is highly conserved across these species, suggesting an important role for this protein in vertebrate development. SOX14 is expressed in the neural tube and apical ectodermal ridge of the developing chicken limb. This is the only SOX gene known to be expressed in the apical ectodermal ridge, a structure that directs outgrowth of the embryonic limb bud. Human SOX14 is localised to a 1.15-Mb yeast artificial chromosome on chromosome 3q23, close to loci for BPES (blepharophimosis, ptosis, epicanthus inversus syndrome) and Möbius syndrome. Although SOX14 maps outside these loci, its expression pattern and chromosomal localisation suggest that it is a candidate gene for the limb defects frequently associated with these syndromes.

Sex Determination: Turning on sex

The orphan nuclear receptor, steroidogenic factor 1, is central to the differentiation of male and female mammalian gonads. It controls the fate of the initially bipotential gonad as well as later male-specific functions.

Sara Courtneidge: swimming with Srcs

Sara Courtneidge has recently been appointed vice-president of research in the California-based biotechnology company, Sugen, Inc. Her pioneering research into how the Src tyrosine kinase is involved in cellular transformation has provided the basis for understanding how members of the src gene family act in normal signal transduction pathways. In her new position, Dr Courtneidge will be trying to identify components of signal transduction pathways as targets for pharmaceutical intervention.Dr Courtneidges career began when, at the age of eight, she announced that she was going to be a scientist. Thirteen years later, she obtained an Honours degree in Biochemistry at the University of Leeds and moved to the National Institute for Medical Research (NIMR) at Mill Hill, England for her doctoral studies. There, under the supervision of Mike Crumpton and John Skehel, she studied how cells infected with influenza virus are recognized and destroyed by cytotoxic T cells. During this time, Dr Courtneidges critical and analytical skills were sharpened by the continual barrage of questions on seminars and papers from John Skehel, who worked on the lab bench opposite hers. Throughout her postgraduate studies, Dr Skehel encouraged her to think about her future scientific direction, a process that resulted in her securing a postdoctoral research position in J. Michael Bishops lab at the University of California, San Francisco.Having only ever spoken to Dr Bishop on the telephone, Dr Courtneidge was initially overwhelmed on her arrival at the lab in San Francisco. The combined Bishop and Varmus laboratories were, at that time, beginning to identify the cellular genes that had been incorporated into RNA tumour viruses and which gave them the ability to transform cells. This was, and has continued to be, a large, energetic laboratory at the cutting edge of molecular biology research and Dr Courtneidge was the youngest and newest member. However, she soon adjusted to the dynamic environment and began her work on identifying the structural and functional domains of the transforming protein isolated from Rous sarcoma virus, pp60v−src, or Src.On her return to England, she was offered a junior staff position at NIMR where, together with Alan Smith, she demonstrated that the transforming protein of polyomavirus, middle T antigen, acquired its tyrosine kinase activity by association with the cellular homologue of pp60v−src. From this work, Dr Courtneidge was inspired to examine the normal role of the Src protein in cells and discovered that the cellular Src protein is, itself, negatively regulated by tyrosine phosphorylation.In the mid-1980s, Dr Courtneidge accepted an offer to join the European Molecular Biology Laboratories (EMBL) in Heidelberg, Germany. The organization of EMBL, with several, small interactive research groups and a multi-cultural composition, provided an exciting and stimulating backdrop for Dr Courtneidge to develop her research interests. During this time, a number of groups throughout the world began to identify other cellular tyrosine kinases that were related to, and shared certain domains with, the cellular Src protein. Dr Courtneidges laboratory demonstrated that these proteins were absolutely required for the normal control of cellular growth. She also showed that members of this family were components of normal signal transduction pathways, regulating the pathways by virtue of their kinase activity. This provided a valuable means for studying how cellular growth is regulated in normal cells and also how this regulation is disrupted in transformed cells.Figure 1Figure 1Figure 1Sarah Courtneidge.View Large Image | View Hi-Res Image | Download PowerPoint SlideDespite having the rare privilege of an open-ended position at EMBL, Dr Courtneidge recently decided it was time for a new challenge. This came in the form of an offer to direct the research division of Sugen, Inc. There, her laboratory will be trying to identify components of signal transduction pathways which can act as new therapeutic targets for the treatment of diseases such as cancer. While there is no doubt that the application of her work to a commercial setting will be very challenging, Dr Courtneidge has successfully been swimming with Srcs for most of her research career and has always managed to tame them.Figure 2Figure 2Figure 2The Courtneidge laboratory at Halloween.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Tom Pollard: upwardly motile

Tom Pollard is the new president of the prestigious Salk Institute for Biological Studies in La Jolla, California. Until recently, it seemed unlikely that Pollard, who has spent his research career studying how cells move, would make a move himself. He seemed settled at Johns Hopkins University School of Medicine, where he has been for the past nineteen years. But Pollard says it was time for a change, and that the chance of working at the Salk Institute — which is renowned for the high quality of its research, yet small enough to allow its president to continue his pioneering work on cellular motility — was too tempting to refuse.Pollards interest in cell motility arose from an undergraduate summer job during which he watched many hours of time-lapse film of moving cells. At this time, it was known that movement in muscle tissue was the result of actin and myosin filaments sliding over each other, but nothing was known about the biochemistry of motility in non-muscle cells.Eager to maintain his interest in research as a medical student at Harvard Medical School, Pollard worked in the laboratory of Sus Ito. Pollard recalls the extreme generosity of Ito in training him in electron microscopy and other techniques, despite the fact that he was doing research quite unrelated to the main interest of Itos laboratory. During this time, Pollard purchased some amoebae to use in an experiment that was proving difficult. Although the amoebae did not solve the experimental problem, their motility caught Pollards interest.The real breakthrough came when he heard about some experiments done by Lewis Wolpert, who had isolated cellular extracts that ‘moved’ when the chemical ATP was added. Fortunately, because he was a newcomer in the cell motility field, Pollard was not exposed to the scepticism of other workers about Wolperts work, and after several attempts he managed to repeat the in vitro motility experiment. This provided a crucial cell-free system that would allow dissection of the cellular machinery involved in cell motility. In the electron microscope, Pollard saw thick and thin filaments similar to those found in muscle tissue in these cell extracts, suggesting that cellular movement may involve the same components used in contractile tissues.After an internship, Pollard worked as a postdoctoral fellow with Ed Korn at the National Institutes for Health, where they did some of the early work on cytoplasmic actin and the association of actin filaments with the plasma membrane. They also discovered the first unconventional myosin, which they called myosin-I. The concept of a myosin different from the two-headed myosin in muscle was so unexpected that their discovery was not generally accepted for more than 10 years.Pollard returned to the Department of Anatomy at Harvard, where he continued his research on the molecular interactions involved in cell motility. He also began teaching both at Harvard Medical School and, during the summers, at Woods Hole Marine Biological Laboratory. During this time, it became clear that actin and myosin are used by a variety of cells for movement, although the architectural arrangement of these proteins is considerably more complex than in muscle tissue.In 1977, Pollard moved to Johns Hopkins where, in addition to running his research laboratory, he directed the Department of Cell Biology and Anatomy. He earned a reputation for being an exceptional teacher, if one with a regrettable taste in colourful ties. He is held in great affection by his students and postdocs, despite his expectation that they should devote their Saturdays to lab work. As he discovered the regulation of myosin-I by a cofactor protein (later shown to be a kinase) on a Saturday, he considers it a good day for doing science; one wonders if he will continue this tradition at the Salk.After surviving a potentially fatal cancer, Pollard fulfilled a lifelong dream by taking his family trekking in the Himalayas in 1988. The brush with mortality also gave him an additional impetus in a new activity — scientific public policy. Inspired both by his wife Patty, who is active in nonpartisan politics, and by a number of scientific colleagues, he began lobbying the US government for better support for biomedical research. He has gone on to chair the Commission for Life Sciences at the National Academy of Sciences, which directs studies on scientific issues of public importance such as whether environmental electromagnetic radiation affects human health (there is still no solid evidence on this).In his new position at the Salk Institute, Tom Pollard will be moving between his administrative duties — in which he will doubtless keep as firm a grip on the purse strings as ever — and the laboratory. He is determined to publish as sole author on his own experimental work within the next three years. Whether or not he achieves this goal, there is little doubt that he will continue to be upwardly motile.

Peter Goodfellow: sex, drugs and poetry

Peter Goodfellow has recently been appointed Senior Vice-President in the Research and Development division of the pharmaceutical company SmithKline Beecham. Dr Goodfellows most outstanding contribution to research has been the identification of genes that control the development of maleness in mammals. He has also had a continued involvement in the development of strategies currently used in human molecular genetics, and more recently has had a major role in setting up a biotechnology company. His combination of commercial experience, scientific expertise and a unique irreverence should bring a new dimension to the management of SmithKline Beecham in the next few years.Figure 1Peter Goodfellow.View Large Image | View Hi-Res Image | Download PowerPoint SlideLike many English lads, during his youth Dr Goodfellow dreamt of serving his country on the soccer pitch. At the age of eighteen, he realized that this was unlikely to happen and was granted a place in the microbiology course at the University of Bristol. Having little idea of what microbiology actually was, he read a textbook on the train to his interview and then proceeded to deliver a one hour discourse on how microorganisms could be used to extract gold.At Bristol, he discovered a natural flair for understanding and thinking within the framework of molecular biology and consequently excelled at his studies. At the end of his Honours year he was advised that eukaryotic genetics was likely to offer a more promising future than prokaryotic, and he accepted a postgraduate position in Walter Bodmers laboratory at the University of Oxford.Dr Goodfellows introduction to human molecular genetics came with his graduate project on the genetic relationship of components of the immune system. It was during this time that he began using somatic hybrid cell lines for gene mapping. In 1975 he received his PhD from Oxford University and Julia Goodfellow, his partner, became the first woman to obtain a PhD from Englands Open University. Together they set off for Stanford University, where Peter Goodfellow took up a position with Hugh McDevitt to study the serology of the mouse t locus.This was a difficult time for Dr Goodfellow as his results went against the established beliefs in the field. At the end of his time at Stanford, however, he had managed to learn some developmental biology from friends at UCSF and had also used his knowledge of somatic cell hybridization to introduce techniques for monoclonal antibody production into the San Francisco bay area. He had also been ‘gobbed on’ by Sid Vicious at the Sex Pistols final concert.He returned to London to take up a position at the Imperial Cancer Research Funds laboratories, while Julia Goodfellow took up a post in X-ray crystallography at Birkbeck College. As a newcomer to the ICRF, he had to fight for both space and acceptance in what was, then, quite a conservative establishment. This was made plain when he heard his lab — two slightly off-beat research assistants and himself — described as ‘a poof, a punk and a weirdo’.While awaiting the arrival of mouse strains from America to start one of his projects, Dr Goodfellow began using somatic cell hybrids to map the chromosomal position of genes encoding antigens that are over-expressed in cancer cells. One of these genes mapped close to the proposed testis-determining factor (TDF) gene, the locus on the Y chromosome that initiates male development. The Goodfellow lab then began a number of positional cloning strategies to isolate a particular candidate for the TDF gene but was beaten to it by David Pages group from the Massachusetts Institute of Technology.Although Dr Goodfellow began spending more time hidden under his desk, reading poetry to his staff and making them endless pots of extraordinarily strong coffee, he was not deterred. His group continued to work on the molecular genetics of mammalian sex determination, and in 1989 obtained results which suggested that the gene isolated by Pages group was not the ‘master’ male-determining gene. So the hunt for the TDF gene began again. This time, a team led by Andrew Sinclair, a postdoc in Dr Goodfellows lab, successfully isolated the correct gene and, in collaboration with Robin Lovell-Badges group at the National Institute for Medical Research at Mill Hill, demonstrated its male-determining activity.With the successful isolation of the mammalian TDF gene came a number of job offers — but an offer from an American university was withdrawn after Dr Goodfellow presented his interview seminar using the pointer as his sole visual aid, while sitting cross-legged on a table at the front of the auditorium. He eventually accepted an offer to head the Department of Genetics at the University of Cambridge, England.Figure 2Peter Goodfellow the poet.View Large Image | View Hi-Res Image | Download PowerPoint SlideDetermined to make his mark from the beginning, he immediately began reading poetry to students, rebuilding a number of the laboratories and centralizing many of the facilities. His own lab expanded considerably to include mathematicians and computer scientists as well as biologists. Dr Goodfellow continued to develop his work on gene mapping by generating somatic hybrid cell lines which contain only fragments of human chromosomes. Along with Tony Monaco, John Todd, Hans Lehrach and others he helped set up the gene mapping company Sequana, in San Diego, California. Sequana has been involved in a successful collaboration to clone the Tubby gene and has recently localized several candidate genes involved with asthma.Despite his success in numerous fields of basic research, Dr Goodfellow feels that the time is now right to apply his unique combination of scientific expertise and commercial experience to the practical development of pharmaceutical products. While a complicated three-way wager is on in Cambridge regarding the length of time his beard and pony-tail will survive divided by the number of days he will spend wearing a suit in his new position at SmithKline Beecham, Dr Goodfellow looks forward to finding out whether drugs are even better than sex.

Paul Nurse: cycling up the hill

Combining primary research with the administrative demands of a senior position in science is a tricky balancing act. But Paul Nurse, who has recently been appointed Director General of the UKs prestigious research organization, the Imperial Cancer Research Fund (ICRF), certainly has no intention of retiring behind a desk. His promotion from Director of Laboratory Research promises to bring exciting times to the ICRF. With his background in the fundamental research that has revolutionized our understanding of the cell cycle, and his commitment to continuing his primary research, Nurse will be directing the ICRF into the next century clutching a petri dish in one hand.Nurse was brought up in a North London working-class household and was the only one of four children to carry on education beyond school. After completing secondary education, he worked for a year in the Guinness Brewery laboratories, where he was first introduced to large-scale screening procedures. This consolidated his interest in biology, which he went on to study at Birmingham University. After graduating with Honours, he moved to the University of East Anglia for his doctorate, to study the intracellular localization of molecules during cell division. A large part of this work involved baby-sitting a temperamental amino-acid analyzer, which often required attention into the small hours of the night. During one of these nocturnal vigils, he recalls reading a paper by Lee Hartwell describing how genetics could be used to study the cell cycle in budding yeast. Nurse was inspired by the elegant use of genetics to study a fundamental process and wanted to try a similar approach in fission yeast. But there were a couple of problems: he knew not a great deal about yeast and even less about genetics.Nurse contacted Murdoch Mitchison, a prominent fission yeast physiologist in Edinburgh, who agreed that this was a very exciting approach but one that would require Nurse to learn some yeast genetics. Nurse was given funding for six months from the Royal Society to learn these techniques from Urs Leupold in Bern, Switzerland. In 1974, Nurse joined Mitchisons lab and began screening for mutated yeast strains that divided abnormally. The first mutant was soon identified and isolated on the basis of its small size during cell division (with a dash of his infamous humour, he wanted to christen it wee1, ‘wee’ being Scots for tiny). Nurse continued to expand his collection of mutant strains and used genetic techniques to identify the network of genes controlling the fission yeast cell cycle. Mitchisons lab soon became a meeting point for other cell cyclers, such as Kim Nasmyth, who did his doctoral research with Nurse, and Peter Fantes and Pierre Thuriaux, who were interested in the regulation of cell-size control.In the late 1970s, techniques were developed for transforming budding yeast with exogenous DNA. Unfortunately, this technology — which would have allowed Nurse to isolate the actual genes mutated in his yeast strains — was not available for fission yeast. Never one to fight shy of a problem, Nurse moved to the University of Sussex to establish methods for transforming fission yeast. This was a long and arduous task but, with the help of David Beach, he got the technique working and used it to isolate the cdc2 gene from one of his strains.Figure 1High flyer Paul Nurse, ready for take-off.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn 1984, Nurse joined the ICRF laboratories in London. Although the ICRF was initially interested in the use of yeast purely as a eukaryotic gene expression system, Nurse soon convinced them of the importance of studies in yeast for understanding the mammalian cell cycle. Using DNA-hybridization techniques, he began hunting for mammalian homologues of his yeast cell-cycle genes, with little success. Nurse recalls thinking it was worth one last shot, and in 1986 he and postdoc Melanie Lee attempted to functionally complement one of their yeast mutants with a library of human cDNAs. Remarkably, it worked, and soon they had isolated the human cdc2 homologue.Despite his success and the abundant facilities available to him at the ICRF, Nurse took up the post of Iveagh Professor of Microbiology at Oxford in 1987, in a move largely inspired by the better lifestyle Oxford offered his family. His lab grew in size, partly because of an influx of American postdocs attracted by both the excellence of the science and the deserved reputation of ‘the wee one’ for fairness and frivolity. Then, in 1993, Nurse returned to the ICRF in London as the Director of Laboratory Research. Although taking a position with such heavy administrative responsibilities seemed like a surprising move for a ‘hands on’ scientist, the organizational structure of the ICRF has allowed him to spend up to half his time in the laboratory. When Nurse moves into the Director-Generals chair this month, he will continue to cycle his energies between the lab and the office.