Prix Balzan 2002 pour la biologie du développement
The journey of a biologist – Rome, 13.11.2002 (anglais)
My journey as a biologist began when I was a small boy watching the eclosion of a marvelous butterfly from its pupal case. Immediately, I was hooked on the problem of metamorphosis and the questions of how such wonderful creatures as butterflies, develop. These mysteries have haunted me for my entire life. They provided the stimulus for my research over the last fourty years and allowed me to decipher some of the secrets of development and evolution.
The mystery of bird migration
As a high school student I became interested in bird migration. How could a small song bird find its way to Africa, how was this highly complex behavior inherited from generation to generation, and what are the celestial and terrestrial cues allowing the bird to orient itself? I joined the group of Ernst Sutter to study bird migration by means of radar at the airport of Zurich. After finishing high school I entered the University of Zurich as a Zoology student. The Zoological Institute was headed by Ernst Hadorn, one of the most outstanding biologists of his time. Hadorn became my mentor and later offered me a position as his research assistant which was a great learning experience. He allowed me to analyze the radar films that we had gathered at the airport in Zurich to carry out a study on bird migration as my diploma thesis. I found out that the migratory birds were only rarely disoriented under heavily overcast skies, when they presumably could no longer detect the sun which serves as their compass. I later continued these studies, by using a tracking radar, kindly let to us by the Swiss Army for research purposes, to study bird migration over the Swiss Alps. In a short period of time, I was able to detect the pattern of the wing beats of birds on the radar allowing us to some extent to determine which species of birds were migrating even at night. To the big surprise of the radar engineers of the army, we could not only detect small song birds on the radar screen, but even migrating butterflies.
However, there was no possibility to approach the orientation problem experimentally, since we could not afford our own radar to be optimally modified for these studies. For a genetic analysis of the inheritance of behavior, the genetic tools were simply not available at that time. Therefore, I switched to Drosophila for my doctoral thesis, a model organism which can be manipulated much more easily.
From transdetermination to flies with legs on their heads
Hadorn was one of the few developmental biologists who had recognized early that development is under strict genetic control. Famous developmental biologists like Hans Spemann had missed this crucial point almost entirely. The large majority of the early embryologists thought that the genes played only a minor role in development, for example by determining eye color, and that development is rather controlled by tissue interactions. By analyzing lethal mutations in the fruit fly Drosophila Hadorn accumulated evidence that genes control development to a large extent and that they determine the body plan in great detail. By the time I was looking for an interesting topic for my doctoral thesis, Hadorn and my fellow graduate student Theo Schläpfer had discovered a most interesting phenomenon called transdetermination, in which cells switch from one developmental pathway to another, for example from leg to wing formation. This work was based upon a method developed by Hadorn previously which involved the transplantation of imaginal discs. Imaginal discs are the primordia of the Drosophila larva, which during metamorphosis give rise to the various parts of the adult fruit fly. There are, six leg discs, two wing discs and two eye-antennal discs etc., which during metamorphosis, at the pupal stage, give rise to the legs, wings, eyes and antennae of the fly. When a leg disc, for example, is removed from one larva and transplanted into another larva, it will form during metamorphosis an additional leg in the body cavity of the host fly. Hadorn sometimes was playful in his approach and transplanted imaginal discs into an adult fly rather than a larva. This experiment led to the discovery of transdetermination: After one or more passages through an adult host in which the cells proliferate, but do not metamorphose, the disc cells can switch from the leg to the wing developmental pathway. Such cells, upon transplatation back into a host larva, now give rise to wing tissue rather than leg structures as their sister cells do. Hadorn by playing around had found a most interesting phenomenon whose mechanism still is largely elusive. I am convinced, that humans can reach their highest degree of creativity when they play, not in passionate but rather in a detached way, for example while improvising on the piano or playing around with thoughts. In any case, transdetermination became the topic of my doctoral thesis. After learning the transplantation techniques, I was able to show that transdetermination was somehow related to regulatory gene action, and that it occurs in groups of cells rather than single cells indicating that it is not due to somatic mutations but rather to cell-cell interactions leading to the activation or repression of controlling genes.
At the time when I was a graduate student, I discovered a mutation that transforms the antennae on the head of the fly into a pair of middle legs. Such mutants are called homeotic indicating that they transform something into the likeness of something else, for example, antennae into legs. Since the German poet Christian Morgenstern in one of his poems had described an imaginary animal that can walk on its nose which he named the Nasobem, I called this mutant Nasobemia. I mapped it genetically and found that it is localized close to the previously described Antennapedia gene, but at that time there were no methods available to decide whether these dominant mutations affected the same or two different, closely linked genes. In a short paper I described the phenotypic effects of the mutation carefully, presented the genetic mapping data, and in the discussion I interpreted the affected gene as a regulatory gene involved in the activation of all the genes that are required to form a leg. This was a bold conclusion, but in retrospect it was absolutely correct. Ever since I found this fascinating mutant, I have been emotionally attached to it. It was intuitively clear to me, that it provided the key to the understanding of cell fate decisions, the question of whether an antennal or a leg cell should be formed, but at that time it seemed to be impossible to get at the molecular basis and the genetic mechanism by which this gene acted.
The molecular approach
When I decided to try to understand the molecular basis of homeotic gene action, most of my senior colleagues were highly skeptical. The molecular biologists thought that a gene controlling a large number of other genes to convert an antenna into leg was much too complex as problem to solve; they were mostly focussing on single genes encoding a single enzyme. The « classical » biologists were critical of the molecular approach altogether, and thought that the molecular biologists were not asking the right questions anyway. I was not discouraged by these comments and decided to move as postdoctoral fellow to the United States, the Mekka for molecular biology at that time. I joined Alan Garen’s group at Yale to learn molecular biology. Alan Garen was trained as a biophysicist and his thinking was much more rigorous and quantitative, than that used by classical biologists. Therefore, I learnt not only molecular biology from him, but also how to design and interpret experiments more rigorously. For molecular biology, Drosophila had one major disadvantage; it was difficult to obtain enough tissue in pure form for biochemical analysis. Therefore, we first extended the studies from imaginal discs to embryos which can be obtained in larger quantities. By dissociating embryos at the early blastoderm stage, reaggregating the cells and culturing them in vivo, we could show that the imaginal disc precursor cells became determined (programmed for their future developmental pathway) as early as the blastoderm stage. Although the blastoderm appears as a homogeneous single cell layer, its cells are already programmed and an invisible fate map exists which we could later visualize by using molecular markers.
Since the work of François Jacob and Jacques Monod had shown, that genes whose function it is to regulate the activity of other genes exist at least in bacteria, Alan Garen and I speculated that homeotic genes, like Antennapedia, were also regulatory genes. Since Walter Gilbert and Benno Müller-Hill had purified the product of the lac repressor gene, the best characterized of these bacterial regulatory genes, and had shown that it is a protein capable of binding to specific DNA sequences in the bacterial genome, thereby repressing its target genes, we concentrated on DNA binding proteins. By using sophisticated methods we attempted to find differences in the pattern of DNA binding proteins in different kinds of imaginal discs, for example between leg and antennal or wing discs. But the methods available at that time were not sensitive enough to detect differences in those proteins which are present at low concentration. However, in retrospect it is gratifying to see that we were on the right track.
The advent of gene cloning: Discovery of the homeobox
The development of new techniques is extremely important in science, since it opens up new approaches to problems that could not be solved before. I faced the problem of identifying the product of the Antennapedia (or Nasobemia) gene; I was trying to find the needle in the haystack that is to isolate a single gene product among thousands of others. However, this became possible by using gene cloning. Based upon the methods developed by Stanley Cohen and Herb Boyer, my friend David Hogness and his collaborators at Stanford had isolated the first Drosophila genes. The first genes to be isolated were either repeated many times in the genome or expressed so strongly, that their gene product could be isolated biochemically. However, Antennapedia was not repetitive and we had no clue what the nature of its gene product could be, except for the speculation, that it might be a gene regulatory protein capable of binding to the DNA of its target genes. However, such proteins are usually expressed at very low abundance and were difficult to purify. At that time, David Hogness and his group developed a method called « walking along the chromosome », that allowed you to clone any gene whose position was known precisely from the corresponding mutations. The walk would start from a previously cloned DNA segment, mapping as closely as possible to the gene to be isolated, in our case Antennapedia, and progressively isolating overlapping DNA segments, step by step, until the gene to be isolated was reached. In this way David Hogness and his collaborators cloned the homeotic bithorax gene, and my group embarked upon a « chromosomal walk » to find Antennapedia. The walk was primarily carried out by Richard Garber and Atsushi Kuroiwa, two of my postdoctoral fellows. The « chromosomal walk » to isolate Antennapedia lasted for more than three and a half years, but finally it paid off. When mapping the cloned DNA segments on the physical map and comparing it to the genetic map, Richard Garber made the interesting observation that one of the DNA segments found inside the Antennapedia gene cross-hybridized with a neighboring gene, suggesting that Antennapedia and its neighboring gene shared some common DNA sequences. This was the first sign of the homeobox. As with many discoveries, only the prepared mind notices it, as Louis Pasteur has pointed out. We were in fact looking for such homologies because Ed Lewis had proposed that homeotic genes, which are clustered on the third chromosome, could have arisen by gene duplication which implied that they shared some similar sequences. The neighboring gene adjacent to Antennapedia was identified by Atsushi Kuroiwa as fushi tarazu (meaning not enough segments), a gene controlling segmentation in the embryo. The nature of the homology between Antennapedia and fushi tarazu was pinned down by William McGinnis. Interestingly it was not distributed across the entire gene, but confined to a short 180 basepair segment. Since we found this same segment also in Ultrabithorax, another homeotic gene isolated in David Hogness’ laboratory, we called it the homeobox. The homeobox encodes a specific segment of the homeotic proteins which we called the homeodomain. Homeotic proteins have a gene regulatory function and use their homeodomains to recognize and bind to their target genes in order to either activate or repress them. By using the homeobox as a probe we could isolate an entire set of other homeotic genes of Drosophila which justified the term homeobox. Later we proved definitively that homeotic genes have a gene regulatory function and encode sequence specific DNA binding proteins, as I had suspected all along. They serve as master control genes specifying the body plan. This point was clearly demonstrated by expressing the isolated Antennapedia gene allover the fly, in particular in the antennal discs of young larvae. Under these conditions the antennae are transformed into legs. This experiment was carried out by Stephan Schneuwly, one of my graduate students, and represented our first successful attempt at redesigning the fruitfly.
The fushi tarazu gene also provided some fundamental insights on how the body plan is established. At that time another major technical advance was achieved by Ernst Hafen and Michael Levine in my laboratory; they developped the method of in situ Hybridization to the point where we were able to detect the messenger RNA transcripts of homeotic genes like Antennapedia in tissue sections. When Atsushi Kuroiwa had isolated the fushi tarazu gene, he and Ernst Hafen wanted to apply this novel technique to this segmentation gene. Fushi tarazu mutant embryos lack every other body segment ending up as embryos having only half the number of segments, which is of course lethal. This suggested that fushi tarazu is normally expressed in every other segment. In order to localize the fushi tarazu transcripts (messenger-RNA) Atsushi and Ernst hybridized the radioactively labelled fushi tarazu DNA to sections across the early normal embryo. I shall never forget the moment when they called me to look into the microscope, and there were the segmental stripes, representing the body plan of the embryo at a stage when all the cells looked identical.
We then followed the Antennapedia gene from antennal legs all the way to the atomic level. In collaboration with Kurt Wüthrich we determined the structure of the Antennapedia homeodomain and its complex with the DNA target site by nuclear magnetic resonance spectroscopy at atomic resolution. It was a long journey from antennal legs to the homeodomain at atomic resolution. If the homeobox had been found exclusively in insects, it would have had little impact. However, soon after its discovery, following a lively discussion in a departmental seminar, Eddy De Robertis and I decided to find out whether vertebrates also had homeoboxes, knowing very well that vertebrates and insects have very different modes of development. Within a short period of time the first homeobox gene from the frog Xenopus was cloned by Andres Carrasco and Bill McGinnis.
In collaboration with Frank Ruddle the first mouse homeobox genes were cloned and mainly through the work of Denis Duboule it was found that, as in Drosophila, the homeotic genes of the mouse are also clustered and arranged along the chromosome in the same order as they are expressed along the anter-posterior body axis from head to tail. There is accumulating evidence that the same homeotic genes are used in both vertebrates and invertebrates to specify the body plan, and the same applies to humans. Therefore, the homeobox uncovered a universal principle and provides a unifying concept of development.
Master control genes in eye development
In a control experiment my graduate student Rebecca Quiring quite accidentally cloned a Drosophila gene that is homologous to the mouse Pax-6 gene. The mouse Pax-6 gene was isolated by Claudia Walther and Peter Gruss and the corresponding human gene by Ton and collaborators. The cloned genes correspond to the mutations small eye in the mouse and to Aniridia in humans. In homozygous condition, with both copies of the gene defective, the mutant embryos lack eyes, noses and show serious brain damage, so that they die. Pax-6 contains two boxes, a homeobox and a paired box encoding two different DNA binding domains in the same protein. The finding of a Drosophila homolog of Pax-6 was not unusual, but the surprise was, that the cloned Drosophila gene as shown by Uwe Walldorf corresponds to the mutation eyeless in Drosophila. This was a total surprise, since all the textbooks tell you that the compound eye of insects and the camera-type eye of vertebrates have evolved separately, and that the various eye-types found in different animal phyla originated independently in evolution, that is polyphyletically. To the contrary, our findings suggested to me that the various eye-types might share basically the same genetic program and that Pax-6 might be the universal master control gene for eye development. When I presented this idea at our Drosophila workshop in Crete my colleagues were highly sceptical. I proposed to induce the expression of the Pax-6 gene in other regions of body in order to find out whether this single gene can induce the formation of an eye. I convinced two of my collaborators Patrick Callaerts and Georg Halder to try this crazy experiment, and to express the Drosophila Pax-6 gene in other body regions of the embryo and larva. The results of this experiment made the front page of the « New York Times » and the journal « Science ». To everybodies surprise a single master control gene (Pax-6) is capable of inducing an entire cascade of some 2000 genes required for eye morphogenesis leading to a complete and functional compound eye on the antennae, wings or legs of the fly.
New perspectives on eye evolution
Even the mouse gene Pax-6, introduced into Drosophila is capable of inducing an eye, of course a Drosophila eye, since the mouse Pax-6 gene is only the main switch turning on the entire gene cascade which is provided by Drosophila. More recently we succeeded also in the reciprocal experiment of inducing additonal eye structures in the frog by injecting the messenger RNA from the Drosophila Pax-6 gene into frog embryos. This indicates that the insect and mammalian Pax-6 genes are interchangeable. Since we also found a Pax-6 gene in flat worms, round worms, molluscs, and a large number of other animal phyla, we are convinced that the various eye-types are of monophyletic origin. This solves the old problem of eye evolution that was raised by Charles Darwin in « The Origin of Species » in which he freely confesses that « to suppose that the eye with all its inimitable contrivances for adjusting the focus to different distances, for admitting different amounts of light, and for the correction of spherical and chromatic aberration could have been formed by natural selection, seems absurd to the highest degree ». However, he then finds a way out of the dilemma by postulating a prototypic eye, « the simplest organ which can be called an eye consists of an optic nerve » (photoreceptor), « surrounded by pigment-cells and covered by translucent skin, but without any lens or other retractive body ». Such prototypic eyes consisting of a single photoreceptor and a single pigment cell are found in certain flat worms and annelid worm larvae. Darwin argues that « if numerous gradations from a simple and imperfect eye to one complex and perfect can be shown to exist, each grade being useful to its possessor… » then the theory of natural selection may provide a valid explanation. Our data on the monophyletic origin of the eyes fully support Darwin’s view.
Neodarwinists like Salvini-Plaven and Ernst Mayr have proposed that the eyes of the various phyla have evolved independently 40 – 60 times, which is incompatible with Darwin’s theory. Natural selection can only work as a driving force once the prototypic eye has evolved. Therefore, evolution of the prototype is a rare stochastic event, and the probability for prototype formation is very small. Since Pax-6 is a transcription factor which basically can regulate any gene which has the appropriate regulatory sequences, there is no functional necessity that Pax-6 controls eye development in all the phyla tested so far; the reason for Pax-6 controlling eye development must be historical, i.e. Pax-6 has been involved in the development of the prototypic eye and was conserved in all the various eye-types which have originated from this single prototype. Real new « inventions » are rare in evolution and the diversity of life is generated by what François Jacob has called evolutionary tinkering.
At the end of this exposé I would like to give a brief outlook into my future research. The fruitfly Drosophila has allowed us to study eye development in considerable depth and the results obtained in Drosophila can in most cases be extrapolated to humans. Therefore, I would like to use the knowledge that we have acquired in Drosophila for medical applications. Retinal degeneration is a very common disease in a large fraction of ageing people, including my mother, and can lead to blindness. Therefore, we have joined forces with ophthalmologists in trying to develop a possible treatment for this disease based on our knowledge acquired in Drosophila. The friendly little fruitfly has brought us very far on our journey. The funds provided by the Balzan Foundation will provide the necessary support for this ambitious project.
W. J.Gehring: Master Control Genes in Development and Evolution. Yale University Press, 1998.