Draft:Madeleine Gans

Madeleine Gans (1920-2018) was a distinguished representative of the French school of Genetics having dedicated her professional life (1945-1990) to research and teaching in this discipline. Her doctoral degree, obtained in 1951 under the direction of Boris Ephrussi (1901-1979) was based on a study of classical genetics on the Drosophila melanogaster zeste mutant. From 1956 to 1970, Madeleine Gans associated herself with Georges Prévost setting up a new genetic model the mushroom Coprinus radiatus. They embarked on the genetic study of metabolic pathways thereby establishing the first large-scale genetic map of a Basidiomycete. In 1970, she switched back to the Drosophila model and initiated an original genetic methodology to identify oocyte determinants involved in embryonic development. Her discovery of the dominant female-sterile ovoD mutations and their reverting properties greatly improved clonal analysis in the female germline and, furthermore, opened up the way to the study of the regulation of transposon mobilization by small non-coding RNAs. She exerted her teaching activity from 1953, was nominated professor in 1961 at the Faculty of Sciences of Paris and from 1968 at the Université Pierre-et-Marie-Curie. In 1987, she was nominated corresponding member of the French Académie des Sciences.

Early life, education and the Second World War (1920-1945)
Madeleine Gans, birth name Madeleine David, was born on June 5th, 1920, in Pont-à-Mousson, Lorraine, France. Her father was an engineer and her mother a school teacher in mathematics. She attended high school in Pont-à-Mousson and graduated in June of 1939. Starting that same year, she pursued her education at the University of Nancy, entering both medical and scientific tracks. She showed a great interest for the physics lectures by Professor Marcel Laporte. In 1940, due to the Second World War, Madeleine Gans and her family left Pont-à-Mousson and Nancy to take refuge first in Britany (Rennes), then in Larche and Toulouse, in non-occupied south-west France. From October 1940 to September 1945, Madeleine Gans was able to continue her education in Toulouse where she obtained degrees in medicine and in science.

Doctoral years in Boris Ephrussi's laboratory at the IBPC in Paris 1945-1955
In October 1945, Ephrussi recruited Madeleine Gans in his laboratory. In February 1946, a lemon yellow eyed spontaneous mutant appeared in the Drosophila melanogaster laboratory collection. Ephrussi assigned Madeleine Gans to study this mutant, named zeste, for her training in genetics. She immersed herself with enthusiasm in this new subject and five years later, on December 21st 1951, she defended her doctoral thesis "Etude génétique et physiologique du mutant zeste de Drosophila melanogaster". Upon her arrival in the Ephrussi laboratory in 1945, Madeleine Gans was supported by an IBPC grant. Then, in 1946 she got a position as "Attachée de recherche" (Research associate) at the Centre National de la Recherche Scientifique (CNRS) which had been created in 1939. In 1952, she became "Chargée de recherche" (Research project leader). From 1953 onwards she switched to a university teaching career, first as « Chef de travaux pratiques » (Head of the laboratory courses) at the Sorbonne, then as a professor from 1961 until her retirement in 1990.

A beautiful study of classical genetics
From the beginning of Madeleine Gans’ thesis work it was clear that the Drosophila zeste (z) mutant was peculiar. In fact, its mutant phenotype (yellow eyes) is present in females but not in males, varies with temperature and shows variegation under certain conditions. Madeleine Gans performed a large and rigorous analysis, constructing numerous strains with chromosomal rearrangements and studying thousands of flies. She showed that the z mutation is localized very near the white+ (w+) gene. She studied genic dosage between z, z+, and w+, and demonstrated that the z mutant phenotype strictly depends on the presence of two doses of the w+ gene. She showed that this property is subject to position effect as the w+ gene activity is abolished or reduced when it is transferred close to centromeric heterochromatin via chromosomal rearrangements. Finally, she precisely characterized conditions leading to variegated eye pigmentation, such as external parameters (temperature) or genetic contexts. Madeleine Gans thesis work was published in French in 1953. It should be noted that it is the sole publication of Madeleine Gans’on her thesis work. Nevertheless, this work was translated in several american laboratories as a textbook case of genetic analysis. Madeleine Gans’ thesis work approaches fundamental phenomena such as position-effect, transvection, and variegation.

A new subject
In 1955, Georges Prévost, a new young researcher, recruited as « Chef de travaux pratiques» in the Department of Genetics at the Faculty of Sciences of Paris was encouraged by Ephrussi "to look for a new material to broaden the spectrum of research in the laboratory and could assert his originality in the exploration of a new material . The association with Georges Prévost was an opportunity for Madeleine Gans, who was 35 years old at the time, to participate to an exciting new project. They chose the Basidiomycete Coprinus for its major advantage compared to Ascomycetes, namely the long and stable dicaryotic phase during which two haploid nuclei coexist in the same cytoplasm constituting a precious tool for genetic analysis.

Coprinus research subjects
The research subjects quickly followed two main paths: isolation of mutants and analysis of their metabolism and the use of the dicaryotic phase to answer fundamental questions concerning nuclear exchanges. A collection of hundreds of mutants was constituted leading to the first large scale genetic mapping of a Basidiomycete. In 1957, Georges Prévost and Madeleine Gans left the IBPC to establish their team in the CNRS Physiological Genetics Centre in Gif-sur-Yvette. Over the years, a diversification of the subjects undertaken by the team occurred, all, nonetheless, using genetic methodology: somatic recombination , karyological study confirming the genetic mapping previously established and analysis of biosynthetic pathways using the numerous biochemical mutants isolated by G. Prévost. The work mainly focused on the study of the pyrimidine and arginine pathways. The fine structure map of the ur-1 complex locus was determined. This locus is composed of two genes belonging to a single transcription unit. These two genes, as in the yeast Saccharomyces cerevisiae, control the first two stages of the pyrimidine biosynthetic pathway. In Coprinus, as in Neurospora crassa , the results obtained led to a model whereby carbamyl-phosphate would be produced by two enzymatic complexes having different cellular localizations: mitochondrial for the enzymes of the arginine chain and cytoplasmic for those of the pyrimidine chain. However, under certain conditions, “decanalization” of the carbamyl-phosphate from arginine biosynthesis could take place. All these studies advanced knowledge on the biology of a representative species of Basidiomycetes. In addition, metabolic regulation circuits identified could be compared with equivalent circuits of relatively close species belonging to Ascomycetes or to more distant prokaryotic and eukaryotic species. In total, twenty-seven articles were published and thirteen thesis defended by sixteen searchers on Coprinus in the laboratory until the late 1980’s, the last subject concerning genetic instabilities. Unfortunately, the research topics on Coprinus radiatus have been poorly recognized internationally as nearly all papers were published in French journals, mainly in "Comptes-Rendus de l'Académie des Sciences". In addition, research on this fungus was stopped just before the emergence of molecular biology that would go on to benefit a closely-related fungus, Coprinus cinereus.

Evolution and end of the Gans-Prévost team
Besides their research activity, Madeleine Gans and Georges Prévost were also significantly involved in teaching genetics. In 1956, they participated to the creation of a new postgraduate course. In 1968, Georges Prévost was promoted professor filling the Chair of General Biology at the new Faculty of Sciences of Orsay and setting up his team in the Genetics Institute of this campus. Madeleine Gans joined the new Molecular Genetics Centre (CGM) at the CNRS in Gif-sur-Yvette, marking their separation. Subsequently, Madeleine Gans stopped working on the Coprinus model.

Maternal-effect mutants
As of 1970, Madeleine Gans returned to the Drosophila model and immersed herself and her whole team, collaborators and students, with great enthusiasm, in the identification and analysis of mutants involved in development. The application of genetics methodology for understanding developmental processes had been clearly proposed previously. Thus, in the introduction of their 1936 paper, G.W. Beadle and B. Ephrussi wrote: "Probably the one factor which has played the most significant role in retarding progress in this field is the fact that relatively little is known from a developmental point of view about those organisms that have been studied most thoroughly from the genetic point of view, and on the other hand, little is known genetically in those organisms that have been most studied from the developmental point of view. One of the two obvious (and alternative) ways of overcoming this difficulty would be to study development in a genetically well-characterized organism. Drosophila, with its numerous mutant types, offers a favourable opportunity for a study of this kind". In his seminal 1963 paper, Ed. Lewis highlighted the strength of genetic analysis for the study of developmental pathways in Drosophila. He went on to report on the genetic analysis of bithorax mutants affecting the adult pattern of body segments along the antero-posterior axis. In France, Madeleine Gans clearly initiated the genetic approach to the analysis of development. Her work was based on what was known about Drosophila oogenesis In fact, the oocyte is a large cell polarized along the antero-posterior and dorso-ventral axis, prefiguring the same axis in the embryo, larva and adult. This polarization, present before fertilization, was postulated to result from the presence, in the cytoplasm of the oocyte, of substances deposited under control of genes acting in the female during oogenesis, the so-called “maternal-effect genes”. At this time, a few papers had described localized defects of blastoderm formation in mutants of such maternal-effect genes. In addition, in Drosophila, germ cells are the first cells to be formed at the posterior pole of the oocyte and this depends on the integrity of posterior polar plasma; removal of this polar plasma leads to flies devoid of germ cells and thus to sterile adults. A grandchildless mutant had already been described in Drosophila subobscura. Madeleine Gans and collaborators undertook several systematic mutagenesis using Ethyl-Methane-Sulfonate (EMS) to screen for such female-sterile mutations linked to the X chromosome, leading to specific anomalies in the embryo, larva or adult specifically. From more than 1000 lines examined, 93 mutants were isolated corresponding to 58 genes on the X chromosome. Mutations in 30 of these genes led to sterile females laying no eggs or eggs unable to develop. Mutations in the remaining 28 genes led to sterile females laying morphologically normal eggs whose development began normally but was arrested at a subsequent step. It was postulated that this class of mutants might affect synthesis or deposition during oogenesis of maternal products involved in the constitution and polarization of the oocyte. Subsequent analysis was performed in Gans laboratory by collaborators and students. A lot of phenotypic heterogeneity was observed in the mutants recovered with no defect affecting any specific structure. Though in most cases, development arrested before gastrulation, in some cases, it went on into organogenesis before arrest. Two grandchildless mutant lines were obtained, the mutant females giving viable but sterile adult progeny. In those grandchildless mutants, the absence of germ cells was often accompanied with other defects affecting posterior structures in the adult progeny. Only later, at the end of the 1980’s, would this absence of specificity be understood. Indeed, maternal-effect genes encode determinants distributed in density gradients each covering different broad regions of the early embryo, thereby explaining that, in mutants, anomalies affect different large regions of the embryo rather than specific organs. Madeleine Gans work was pioneering as her screens involved five steps to identify recessive female-sterile mutations and an additional one to recover grandchildless mutations. The few teams in the world also performing screens for female-sterile mutations at the same time were not implementing this additional step in their screen strategies. Subsequently, other screens for maternal-effect mutations affecting embryonic pattern formation were performed in a number of laboratories      . In parallel, systematic screens for zygotic mutations affecting embryonic pattern formation along antero-posterior and dorso-ventral axis were also undertaken by others, and the ground-breaking results obtained confirmed the strength of the genetic approach for the analysis of development. This was universally recognized with the awarding of the 1995 Nobel Prize in Physiology or Medicine to Ed. Lewis, C. Nüsslein-Volhard and E. Wieschaus.

The ovoD mutations and clonal analysis in the female germ line
Three dominant female-sterile mutants named ovoD were obtained in one of the mutagenesis screens cited above. Their striking properties were immediately recognized by Madeleine Gans. The sex-linked ovoD mutations are dominant, leading to complete sterility of females, while mutant males are entirely fertile. They are only active in the germ line but not in the somatic line of the female ovary. Further studies would go on to show that the ovoD products are implicated in the viability and sexual identity of the female germ line. Madeleine Gans understood the great advantage that these ovoD mutations offered to study the role in oogenesis of other sex-linked mutations on the X chromosome leading to zygotic lethality of X/X mutant flies. Heterozygous females such as ovoD X+/ovo+ Xm are totally sterile and do not lay eggs. However, mitotic recombination can produce twin germ-line clones, namely, ovoD X+/ovoD X+ clones and ovo+ Xm /ovo+ Xm clones, the latter being susceptible to give viable germ cells and thus possible progeny. The analysis of the phenotype of the putative progeny will give information on the possible germinal role of the X-linked mutations. Norbert Perrimon developed many of the potentialities afforded by this extremely useful genetic tool, first as a pre-doctoral student in Madeleine Gans’ laboratory, then during his thesis in Anthony Mahowald’s laboratory. Norbert Perrimon provided two major improvements to this technique. First, combining the ovoD mutation and the Flip-FRT recombination system from yeast allowed induction of germinal clones at a much higher frequency. Second, using P element transgenesis, the introduction of ovoD mutations on autosomal arms allowed the extension of germ-line clonal analysis to autosomal mutations. These genetic tools were used by all teams working on developmental genetics in Drosophila. An example of Madeleine Gans’ scientific intuition: analysis of the high frequency reversion of ovoD mutations (1983-2007)

High frequency reversion of ovoD mutations
In the course of her thorough analysis of the germ-line clone progeny using ovoD mutations, Madeleine Gans uncovered an unexpected phenomenon: some viable flies among the progeny did not result from mitotic recombination but rather from phenotypic reversion of ovoD mutations in the female germ line. The reversion event occurred with high frequency, up to 6% of ovoD/+ females producing such revertant clones. Moreover, this frequency depended on the genotype of the mothers giving ovoD/+ females. In these strains, the ovoD mutations were shown to be converted into ovo0 mutations behaving like loss of function mutations associated with recessive female sterility. Also, these reversions were often accompanied with other lethal or morphological mutations, arising either near the ovoD mutation (4%) or elsewhere on one of the two X chromosomes (5%). The reversion events never occurred in the male germ line.

From high frequency reversion to transposon mobilization
These properties, namely the high rate of ovoD reversion and the simultaneous occurrence of numerous lethal and morphological mutations, immediately suggested the mobilization of a transposable element. The authors proposed that the ovoD mutations corresponded to classical point mutations which were, in certain conditions, the target for the insertion of a transposable element. In 1989, Gans and collaborators showed that the mobilization of gypsy and copia transposons was able to induce ovoD reversions. The analysis was pursued in collaboration with Alain Bucheton and Alain Pélisson’s team and the conditions of the transposon mobilization were more precisely characterized. As suggested in the princeps paper, this mobilization depends on the genotype of the mothers of ovoD/+ females. The genetic element involved, that was named flamenco, is localized in the heterochromatin of the X chromosome. In most strains, gypsy is stable and does not transpose, and it was postulated that the presence of an allelic restrictive form of the flamenco locus, flamR, permits the repression of gypsy. In some strains, however, flamenco is present as an inactive permissive form, flamP, which cannot repress gypsy: gypsy is unstable and transposes at a high frequency in the germ line of the daughters of flamP/flamP homozygous females. A. Bucheton and A. Pélisson’s team as well as V. Corces’ team, were able to further characterize the conditions of gypsy mobilization.

The control of transposon mobilization by piRNAs: an up-to-date story
Where was the flamenco locus exactly and how did it function precisely? A lot of studies were performed to answer these questions by a relatively small community of scientists. The subject then took on an international dimension when it merged with the extensive small RNA world The first data concerning the locus were published by A. Bucheton and A. Pélisson’s team and Chantal Vaury’s team. They showed that the flamenco locus (also named COM for « Centre Organisateur de Mobilisation ») was involved in the control of many other transposons, besides gypsy and copia, such as ZAM and Idefix. They localized it to a 130kb region in the B pericentromeric heterochromatin of the X chromosome. In 2007, G.J. Hannon’s team published a systematic analysis of the genomic localization of piwiRNA (piRNA) sequences recovered from Drosophila ovaries. They identified the loci corresponding to the production sites of piRNAs, the major site being the flamenco/COM locus, composed of repetitions of fragments of retro-transposon sequences from several families, extending over a region of 179kb. From their analysis, the authors proposed that the flamenco locus is a platform generating long antisense precursor transcripts, which are then cleaved into small RNAs and charged with Piwi protein. Hybridization of the small RNAs to the transcripts of active transposons present in the genome will lead to transposon silencing. In conclusion, the story of the analysis of ovoD mutations illustrates how the mastery of the genetic methodology by Madeleine Gans, and her scientific intuition, opened the promising paths offered by those mutants, both for the development of clonal analysis in the female germ line and for the discovery of small RNA control of transposon mobilization. Therefore, she laid the foundations for studies still active today, several decades after the initial work.

Life in Madeleine Gans’ laboratory, a landscape of genetic research in France from the seventies to the nineties
From 1970 onwards, Madeleine Gans’ laboratory expanded considerably. Several students who defended their thesis on the Coprinus projects followed Madeleine Gans in her conversion to the Drosophila model and established independent groups thereafter. Numerous doctoral and postdoctoral students joined her laboratory in the following years. Madeleine Gans’ notoriety attracted brilliant young researchers such as Eric Wieschaus who spent several months in her laboratory in 1975 on an EMBO short-term fellowship to study fs(1) mutants and Norbert Perrimon who started the ovoD clonal analysis during his 1983 pre-doctoral rotation with her. Madeleine Gans welcomed foreign researchers with the aim to enrich her own research with their particular expertise. They constituted active groups in her laboratory. Marco Zalokar was there from 1969 to 1984, bringing his expertise on nuclear transplantation and cell manipulation. Pedro Santamaria, a student of Antonio Garcia-Bellido, brought both the clonal analysis methodology and the study of homeotic genes. He formed an independent group from 1975 to 2008 publishing numerous articles (about 30) and directing a number of thesis. Patricia Simpson spent five years from 1976 to 1981, bringing her expertise on clonal analysis, as well as topics around cellular growth and division, and actively collaborating with foreign researchers, namely R. Nöthiger, G. Morata, and P.A. Lawrence. As it was the period of the emergence of molecular biology, Madeleine Gans attracted two developmental biochemists from Belgium, Hermann Denis and Maurice Wegnez. They entered the CGM in 1975 but chose to pursue work on amphibian models rather than Drosophila and thus constituted independent groups. Madeleine Gans exerted a non-hierarchical direction of her team. She never showed any obvious career ambition. Her sole motivation was science, experiments, and discoveries. She spent a lot of time at the bench, crossing and examining flies, with her famous slow-burning long-ash cigarette despite the presence of ether used to put the flies to sleep. She had a special and quick intelligence for genetics, able to follow the results of several-generation complicated cross schemes directly in her mind, leaving her students far behind. When experiments went well, she sang, " à tue-tête" (at the top of her lungs), « Toreador » for a good result and « C’est la lutte finale » for an exceptional result. Surely, it was the latter she sang when she discovered ovoD mutations! She was a very cheerful and gay person, transmitting her enthusiasm to every person working with her, researchers, students, technical assistants, thus conferring a congenial ambience in the lab. Madeleine Gans recruited many women, perhaps because she got along better with them, or possible, because girl, more than boy, students preferred a laboratory directed by a woman. She spoke very approximate English but this never prevented her from establishing fruitful interactions with English-speaking researchers. However, she did not like to put herself ahead preferring to send her students to international meetings rather than going herself. Also, she possessed high ideals for ethical and humanist considerations. She was always very aware of the personal difficulties that her students or researchers might be undergoing. It is worth to place Madeleine Gans’ research in the larger context of scientific politics in France. From the end of fifties, the French government invested in scientific activity, especially in the biological sciences, creating positions and opportunities for young people. The CGM in Gif-sur-Yvette was created in 1967 as a CNRS institute. It was first directed by Boris Ephrussi until 1971, then by Piotr Slonimski, a yeast geneticist, until 1991. As the director of the CGM, Slonimski constantly supported Madeleine Gans’ research (Fig. 4). They had been acquainted since a long time, since they were both students of Ephrussi at the IBPC. They mutually appreciated each other, despite their very different characters. Piotr Slonimski had a real admiration for Madeleine Gans. She told us that, against her will, he had submitted her candidature to the French Académie des Sciences. She was elected as a corresponding member in 1987 but she confessed that she had never participated to assemblies. Madeleine Gans retired from teaching in 1990 and from the laboratory in 1994. She spent the remaining years of her life in a small pleasant house full of plants and flowers in Gif-sur-Yvette, not far from the laboratory. She died on April 18th 2018 at 97 years of age. Some months later, in the new Integrative Biology Institute replacing the CGM in Gif-sur-Yvette, a warm symposium focused on "Developmental genetics: the impact of Drosophila" was held in her memory. Several generations of students and eminent biologists who had been marked by their interaction with Madeleine Gans presented the recent advances of paths she had forged.

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Awards and honors
in 1987 Gans was named a correspondent of the French Academy of Sciences.