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The discovery of homeotic genes

Research into the genes that cause erroneous developments in fruit flies have led to one of the most exciting discoveries in the field of developmental biology: the same type of gene that controls early embryonic development in Drosophila, also controls early embryogenesis of other organisms, including humans. These homeotic genes are lined up on the DNA in exactly the same order as they are expressed along the body axis during embryogenesis.

A small monster – a fruit fly (Drosophila) with an extra pair of wings – was crucial in the discovery of the genes that control embryonic development. Fruit flies belong to the order Diptera, members of which, as the name implies, normally only have one pair of wings. As is the case with all insects, the fruit fly thorax is composed of three segments, each of which carries a pair of legs. In addition, the second segment has a pair of wings, and the third segment has a pair of halteres, external organs involved in balance, instead of wings.

Nobel Prize 80 years after the discovery of the bithorax Drosophila mutant

Bithorax Drosophila mutant with four wings. © Max Planck Society

Around a hundred years ago, Calvin Bridges worked with Drosophila mutants that had four fully developed wings. Bridges was a student of Thomas Morgen who is famous for introducing Drosophila as model organism for genetic research. Bridges showed that when some genes (“bithorax” and “bithoraxoid”: bx and bxd) become mutated, the third thoracic segment converts into the likeness of the second, i. e. that the balancers turn into a second pair of wings. When Bridges analysed salivary gland cells of Drosophila larvae, he observed duplicated chromosomal bands and postulated that the duplication of genes, which can subsequently mutate separately and assume diversifying functions, is of huge importance for the evolution of organisms.

Edward Lewis, who continued Bridges’ work at the California Institute of Technology, showed bx and bxd to be genes that transform a particular structure into another homologous structure. As early as 1898, William Bateson had observed this phenomonen, which he named homeosis. Lewis spent his entire scientific life elucidating this phenomenon. Although many researchers subsequently became interested in homeotic processes, many decades had to pass before Lewis discovered a complex of Drosophila genes that define the appendages that are characteristic of the segments that make up the fruit fly thorax. In 1995, Edward B. Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus were awarded the Nobel Prize in Physiology or Medicine for their work on the mechanisms that pattern the body during development.

The principle of colinearity

The homeotic genes of fruit flies and mice are arranged according to the colinearity principle. © Max Planck Society
Lewis’ research on the bithorax genes led to the discovery of the colinearity principle, which has had a huge influence on the field of developmental biology. The colinearity principle refers to the phenomenon that the homeotic genes are clustered together and their order on the chromosome parallels their time of expression during development and the position of the segment on which they act. Lewis also showed that genetic regulatory functions (domains) may overlap: the first set of genes, which controls the head and thorax, becomes active before the second set of genes, which controls the abdomen, and the second set becomes active before the third, which controls the posterior parts. In addition to the bithorax gene complex, the formation and differentiation of the body segments and legs of Drosophila is also controlled by a second gene complex, which is known as antennapedia. Mutations of these genes can for example result in the development of legs instead of antennae. The genes of the antennapedia complex are also arranged in a colinear order on the same chromosome. The greatest surprise came from research which led to the discovery that the homeotic genes of Drosophila are homologous to the homeotic genes in other animals, including humans. This discovery led to a virtual revolution of the idea of how animals and their organs evolved and to the emergence of a new scientific discipline: evo-devo.

40,000 mutations and the discovery of 15 homeotic genes

Microscope image of three fluorescence-labelled homeotic genes of a blastoderm-stage Drosophila embryo: hairy: red; krüppel: green; giant: blue. The photo was awarded a prize by the journal “Biotechniques” in 1993. © Stephen Paddock

At the time when Lewis was investigating the genes involved in homeosis in Drosophila, Christiane Nüsslein-Volhard and Eric F. Wieschaus were carrying out a search for genes that affected the segmentation pattern in fertilised Drosophila eggs. From 1978 to 1981, Christiane Nüsslein-Volhard and Eric F. Wieschaus led two research groups in a tiny laboratory at the European Molecular Biology Laboratory (EMBL) in Heidelberg. When they were awarded the Nobel Prize in Medicine or Physiology in 1995, Nüsslein-Volhard and Wieschaus spoke about these three years as the most stimulating and productive in their entire scientific lives. They exposed female Drosophila to chemicals to induce random mutations and systematically isolated specific genes that are responsible for the embryo’s early development. After testing around 40,000 mutations, Nüsslein-Volhard and Wieschaus found fifteen genes that, if mutated, would cause defects in the flies’ segmentation pattern. Since their pioneering work, 25 such genes have been discovered. Mutations in these particular genes lead to defects and errors in the flies’ body pattern.

The homeotic genes were classified into three functional types based on their effects on segmentation:

1. “gap genes" – these genes control the body plan along the head-tail axis. The loss of a gap gene results in a reduced number of body segments. The gene “krüppel” is a gap gene.

2. “pair rule genes" – these genes affect every second body segment. Loss of the “even-skipped” gene results in an embryo consisting only of odd numbered segments. 

3. “segment polarity genes" – these genes affect head-to-tail polarity of individual segments, which means that the anterior and posterior ends of an individual segment differ. The gene “hedgehog” is a segment polarity gene.

The three gene types are expressed in three consecutive waves, which in Drosophila last only a few hours. These genes reflect the increasing refinement in the developmental programme of Drosophila.

Nüsslein-Volhard and Wieschaus published the major results of the work they carried out at EMBL in the journal “Nature” in 1980. The paper had a great impact on future scientists and is regarded as a milestone in developmental biology. Generations of young scientists are still focusing on the elucidation of these genes.

Homeodomain and Hox genes

A few years after this sensational publication, two groups of scientists discovered independently from each other that many of these homeotic genes had a DNA sequence of 184 nucleotides in common: Michael Levine and William McGinnis were working in Walter Gehring’s lab in the Basel Biocentre; Matthew Scott was working with Thomas Kaufman at Indiana University in Bloomington, USA. This sequence, named homeobox, encodes a 60 amino acid long protein domain (homeodomain). Most homeobox genes encode transcription factors. The products of homeotic genes (Hox genes) that have a homeodomain are referred to as Hox proteins. However, there are also genes with a homeobox that are not homeotic genes; such genes encode adhesion proteins, receptors and components of signalling chains.

Unexpectedly, the researchers found a virtually identical sequence in many genes in almost all living organisms, including vertebrates. In addition, these sequences turned out to be components of homeotic genes that are arranged on the chromosomes according to the aforementioned principle of colinearity. Vertebrates have four such clusters, totalling around 40 genes. As in the fly, the order of the genes in each cluster relates both to their time of action and to the body part on which they act. Nowadays, there is no doubt whatsoever that these genes have arisen by way of gene duplication as was postulated by Calvin Bridges 80 years ago.

The most surprising observation came from a comparison between the sequence of bases for genes in similar positions in different clusters of the same animal (the mouse for example) with those in similar positions in the single cluster of the fly. For each position, the genes in the mouse and the fly are more similar than those between two genes of any cluster within the same species.

This means that the same type of genes control early embryonic development in flies and mice (and humans) and that similar genetic control mechanisms have been conserved during evolution – at least since the late Precambrian 650 million years ago when insects and mammals diverged. In her speech at the Nobel Banquet in December 1995, Christiane Nüsslein-Volhard highlighted that “it has become apparent that at least some of the fundamental principles we learned from the fly apply to higher vertebrates as well, including humans” and quoted from Goethe’s poem “Metamorphosis of Animals”:

All its organs are formed according to laws that are timeless, even a form very rare will hold to its type, though in secret.

Subsequent research in mice showed that the normal function of these genes is to inform embryonic cells of their position. According to which of the 40 genes are active, a cell can tell how far from the head and from the tail it should be located. If all the genes in a particular position in all four clusters are silenced, one part of the body is converted into the likeness of another, a true vertebrate analogy of Bateson’s homeosis.

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