Molecular Cell Dynamics
Life of every multicellular organism depends on the functional cellular network that enables an interaction of many different cell types. This network is formed during embryonic development by a combination of cell migration and cellular growth. Eventually the specific target cell has to be recognized and a stable adhesion is established. We are interested in elucidating the molecular mechanisms underlying these cellular processes by studying myogenesis and the migration of the primordial germ cells in the model organism Drosophila melanogaster.
We apply a range of cell and molecular biology techniques combined with genetic and transgenic approaches to identify and characterize novel gene activities involved in migration and adhesion. Drosophila is used as a model since it offers both powerful genetic tools and high-performance imaging methods to investigate the dynamics of migration in vivo. Furthermore, since morphological aspects as well as many gene functions are conserved between vertebrates and Drosophila, we anticipate that the results obtained will have a broad impact on similar processes and functions in higher organisms.
Myogenesis is an evolutionary conserved developmental process characterized by distinct cellular events such as muscle founder determination, cell fusion, myotube extension and anchorage. Each muscle fibre originates from a distinct muscle founder cell that is determined by specific combinations of transcription factors. Subsequently, the founder cells form syncitial myotubes that extend by the formation of polar growth cone-like structures while fusing with competent myoblasts. As a result, myotubes elongate and extend along the epidermis towards their apodemes at the segment borders.
Mutant studies suggest that signaling processes between the prospective apodemes and the extending myotube regulate directed extension of the myotubes. After contacting each other, myotubes and apodemes start their final differentiation, a process characterized by the expression of proteins of the contractile apparatus. The anchorage of the muscles is assured by integrins that interact with extracellular matrix proteins including Thrombospondin.
So both directed migration and functional anchorage of the somatic muscles are controlled only by the direct interaction of differentiating muscles and epidermal muscle attachment cells, the apodemes.
We characterized the apodeme-specific transcription factor Stripe and revealed that (i) stripe is required for the determination of apodemes, (ii) its activity is sufficient to transform ectodermal cells into apodemes, (iii) stripe expressing apodeme precursor cells attract myotubes, and (iv) stripe is essential for muscle attachment. We identified the regulatory elements of the stripe promotor and found that stripe expression is under direct control of converging Hedgehog (Hh) and Wingless (Wg) signaling activities.
We performed a gain-of-function screen to identify genes required for muscle attraction and repulsion. Among 4,500 genes tested, we identified more than 70 genes that cause a lethal muscle pattern defect, including syndecan (sdc). We found that the transmembrane protein Sdc has a specific function for Slit/Robo-signaling. Sdc mediates Slit reception in the responsive cells. However, unlike vertebrate Sdc, Drosophila Sdc does not act as an independent signaling receptor.
Furthermore, we characterized the function of thrombospondin. We generated a mutant and showed that Thrombospondin is a novel extracellular matrix factor directly binding Integrins, and that this binding is essential for muscle anchorage.
Recently, we complemented the gain-of-function screen by a "knock-down" screen, involving some 7,500 RNAi-expressing transgenes, to identify additional factors required for muscle guidance and attachment. The RNAi was specifically expressed within the muscle attachment sites screening for genes that are required for the formation of a functional musculature. In the future, we will characterize a subset of the identified candidate genes in detail.
Primordial germ cell migration
Germ cells are the only cells in the body that have the ability to form a new generation. These immortal cells are separated from the somatic cells very early during embryonic development and kept in an undifferentiated stage for a long period before their differentiation into sperm and oocytes. In many organisms, including Drosophila, primordial germ cells (PGCs) are formed in a specific region early in embryonic development which is enriched in maternally loaded RNA and proteins, named the germ plasm. In Drosophila PGCs are the first cells to form, separate from the early syncytial somatic tissue. However, in most animals PGCs are determined at a different position than their final destination, the gonad. The PGCs have to migrate a long distance to finally meat the zygotic gonadal precursor cells to form the embryonic gonad. In Drosophila, this cell migration starts as a passive movement with the underlying midgut anlage towards the interior of the embryo. At this timepoint the PGCs start their active migration, traverse the midgut epithelium, migrate dorsally towards the mesoderm, split into two cell groups and finally meat the mesoderm derived zygotic gonadal precursor cells to form two gonads laterally on both sides of the embryo. During this journey about half of the PGCs are lost and it is critical that those stem cells that are not embedded into their specific niche are removed as these PGCs can form juvenile cancer.
Surprisingly, both the directed growth as well as the regulation of the survival of the PGCs is controlled by an unusual signalling system that is based on the presence of phosphorylated lipids as the key regulator of both processes, wunen, encodes a lipid posphate phosphatase (LPP). The second independent attractive signal is presumable a short geranylated peptide, however in both cases only the synthesizing or the degrading enzymes are identified but not the ligands by themselves.
We became interested in the PGC migration control by two mutants we identified during our gain-of-function screen that affect both mesodermal differentiation and PGC migration. Currently, we characterize the function of these novel activities in PGC migration and muscle differentiation.