During the embryonic development of fruit flies, zebra fish and humans, just a handful of molecules control cell migration, induce cell division and determine which cells form which type of tissue. A group of researchers led by Dr. Giorgos Pyrowolakis at the University of Freiburg is specifically focused on one of these so-called master regulators. How do the differently patterned BMP gradients develop in Drosophila melanogaster eggs, embryos and larvae? And how do they regulate themselves so that the wings can develop correctly? Whatever the answers are, evolution does not always reinvent the wheel. And this also has implications for us as humans.
Every cell within an organism has exactly the same genetic composition. So how are so many different types of tissue – liver, skin, brain and lung – able to develop from cells? For Dr. Giorgos Pyrowolakis from the Institute of Biology I at the University of Freiburg this is possibly the most exciting question in science. Pyrowolakis originally studied biochemistry, and during his postdoctoral studies he started working with a group of proteins that are involved in virtually all developmental processes of model organisms: the BMP superfamily. BMP is the acronym for “bone morphogenetic protein” and is known for its ability to promote bone regeneration. However, the BMP superfamily also regulates many other biological processes. BMPs that are closely related structurally and functionally have been found in fruit flies, zebra fish, worms, mice and humans. “In cell culture dishes, BMP has even been found to induce new bone growth. This can be achieved by applying the Drosophila melanogaster BMP homologue, which is known as decapentaplegic or Dpp for short, to human bone fragments, which then start growing,” said Pyrowolakis.
Pyrowolakis and his team have chosen the small fruit fly D. melanogaster as their model system for studying the evolutionary conserved molecular signalling networks involving the BMP family. Around 1,000 different fruit fly lines in which different genes were specifically manipulated are kept in a 5 m2 room in the basement of the Institute of Biology I. Researchers around the world have a huge range of genetic methods available to study and manipulate fruit flies. “Working with mice is a lot more difficult,” said Pyrowolakis. BMPs have important roles in many biological processes, and one of the best known and best studied is their role in determining the dorsal and ventral patterning of the early fruit fly embryo. BMPs are also involved in the more complex patterning of Drosophila wing imaginal discs, where the cell groups that give rise to the five wing veins are defined.
BMP, and its fly homologue Dpp are morphogens, which are necessary for the correct patterning of tissues that become specific organs and structures in the adult organism. Flies lacking components of the decapentaplegic (Dpp) signalling pathway fail to form these structures, like wings for example, correctly. On the molecular level, BMP acts as a signalling molecule: it docks to a transmembrane receptor and induces a molecular cascade inside the cell which causes specific proteins to translocate into the nucleus and regulate the transcription rate of genes. The products of these genes mediate certain cell reactions; the cell either divides, detaches from the tissue or changes its identity to become a neuron or an intestinal progenitor cell. “The big question is why this affects different genes in a target cell,” Pyrowolakis said.
A few years ago, Pyrowolakis and his team came up with a surprising finding, which showed that BMPs can activate as well as inhibit genes. They identified two different DNA structures that function like switches: an inhibitory nucleotide sequence upstream of the target gene leads to the BMP-mediated repression of this gene. Activating sequences do the opposite and activate the genes. At first sight, this discovery is intuitively understandable as the same principle is also known for other signalling systems. However, as far as the morphogen BMP is concerned, the researchers’ discovery is something special. According to Pyrowolakis, the genes that are activated or inhibited by BMP can be roughly divided into two groups: first, effectors that influence the behaviour of a cell, i.e. induce it to divide or turn it into a neuronal progenitor cell, and second, regulators that have an effect on BMP signalling – in the form of feedback loops. Such regulators are necessary in order to explain, for example, how a cell located in the wing imaginal disc of Drosophila larvae knows in which of the five wing veins it will eventually end up.
Morphogens – and this is their second important characteristic that distinguishes them from simple signalling molecules – can move freely through tissue and regulate the activity of different genes in relation to their concentration. They thus help produce different gene products that determine which part of an organ develops in each region during embryonic development. As far as wing imaginal discs are concerned, this means that a narrow stripe of cells in the region of what is later to become the wing releases BMP, which then diffuses from this stripe towards the edges of the tissue, forming a gradient. Cells that are closer to the region where BMP is produced are exposed to a higher concentration of BMP than the cells located closer to the periphery. The recipient cells can measure the BMP concentration. The genes that are either switched on or off by BMP have a different sensitivity to BMP, with the result that cells located at different distances from the BMP source use different genetic programmes. These cells then develop in different ways as the larvae grow to become mature flies. “The principle of the gradient-mediated regulation of tissue development is universal,” said Pyrowolakis, going on to add “both with regard to the different stages of embryonic development as well as with regard to the evolution of different groups of organisms.”
However, there must be regulatory mechanisms that ensure that the morphogen concentration does not fluctuate too strongly and expands as the embryo grows, i.e. in relation to the volume of the growing tissue. And last but not least, the temporal alteration of the gradient or its spatial appearance are also of great importance in patterning and growth: depending on the type of tissue that is developing, a gradient either needs to decrease asymptotically from the source or it needs to stop with a sharp edge as is the case in the development of insect body segments. So how does the gradient know what it should look like?Over the last few years, Pyrowolakis and his team have used the switches they have discovered to activate or inhibit BMP-regulated genes in the search for answers to this question. Knowing the base sequence of the switches enables the researchers to use bioinformatic methods to screen the genome of fruit flies or other organisms for the presence of genes with the upstream switches. It is assumed that these genes are regulated by BMP. And indeed, the researchers from Freiburg succeeded in identifying a gene whose product is a regulator of the BMP signalling network. The researchers called this gene “pentagone” due to the fact that it plays a key role in the correct formation of the five fly veins (mutant flies lack the fifth wing vein – “pent is gone”).
BMP inhibits the production of the protein pentagone. The protein is therefore only produced in cells that are distant enough from the region where BMP is produced. Or in other words, the greater the amount of BMP a cell is exposed to close to the region where BMP is produced, the lower the quantity of pentagone protein it produces. The BMP signal weakens as it gets farther away from the source. In a feedback loop (negative regulation), the pentagone protein itself can increase the strength of the BMP signal by inhibiting BMP-inhibiting proteins. “Pentagone can therefore be called an expander; it increases the BMP levels in cells that are at a greater distance from the source and have a lower BMP level, thereby forming a gradient towards the edge of the field,” said Pyrowolakis. Pentagone mutants have a rather shallow BMP gradient; therefore, the fifth wing vein in the periphery of the wing does not develop as a result of BMP shortage.
In theory, there are many more such regulators and the researchers from Freiburg are currently looking for them. They are also focusing on the evolutionary perspective, as the comparison of the genomes of fruit flies, mice, fish and other organisms has revealed related nucleotide sequences that have been conserved during evolution. It can further be assumed that conserved sequences have a function and that the respective genes are essential for certain biological processes. “Results obtained with other organisms are also of great clinical importance. It has been shown that defects in the genes involved in BMP signalling can lead to abnormal developments and diseases such as cancer,” Pyrowolakis said. “The better we understand the early development processes in different groups of organisms, the better we will also understand human development processes.”
Further information:Giorgos Pyrowolakis, PhDInstitute of Biology IUniversity of FreiburgHauptstrasse 179104 FreiburgTel.: +49 (0)761/ 20 38 459Laboratory: +49 (0)761/ 20 32 541Fax: +49 (0)761/ 20 32 597E-mail: g.pyrowolakis(at)biologie.uni-freiburg.de