Terminal pattern formation

Regulation of tailless occurs through relief from repression. While dorsal/ventral pattern formation is directed by a single maternal pattern forming system, anterior/posterior pattern formation relies on three distinct maternal systems, one of which is the terminal system. A crucial component of this system is the membrane bound receptor tyrosine kinase (RTK) encoded by the torso gene. Early during embryogenesis, a ligand is released at the termini of the embryo that binds and activates this receptor. Torso then activates Drosophila Ras protein, which, in turn, activates a protein phosphorylation cascade. Although the final substrates of this cascade are unknown, it must ultimately target one or more transcription factors to gener-ate gradients of transcription factor activity emanating from the anterior and posterior termini of the embryo. These transcription factors must then mediate the terminal specific expression of at least two zygotically active genes, tailless (tll) and hückebein, which are required for the development of terminal structures.

Regulation of tailless by relief from repression. Expression of tailless is controlled by a typical Ras pathway. Signaling is initiated by the terminal specific activation of the Torso transmembrane receptor tyrosine kinase (Tor RTK). This results in the serial activation of SOS, Ras, Raf, MAPKK, and MAPK. Activation of MAPK results in the inactivation of an unknown factor that works through a repression element (the Tor RE) in the tailless promoter. This allows terminal specific activation of the tailless gene as shown by the in situ hybridization on the left. The in situ hybridization on the right shows the result of a mutation that disrupts the Tor RE. In this case, communication between the Torso signaling cascade and tailless is disrupted and tailless is therefore uniformly expressed throughout the embryo.

     The signal transduction pathway utilized at the termini of the Drosophila embryo is remarkably similar to RTK-activated signal transduction cascades in a wide variety of biological systems. For example, cell proliferation directed by the vertebrate epidermal growth factor RTK and photoreceptor cell differentiation directed by the Drosophila Sevenless RTK are both mediated by series of enzymes (e.g., Ras, Raf-1, MAP kinase) similar or identical to those utilized by the Torso signal transduction pathway. These similarities engender the following question: how can the activation of these different receptor tyrosine kinases result in widely different developmental responses when they all activate the same phosphorylation cascade? One possibility is that different tissues are primed for different developmental responses by the presence within those tissues of alternative sets of transcription factor targets for the RTK pathway. Little is known, however, about the transcription factor targets of these phosphoryla-tion cascades. Thus, a major goal of our research (in a collaborative effort with the laboratory of Dr. Judith Lengyel) is to identify and characterize the targets of the Torso signal transduction pathway.

     To achieve this goal, we have carried out a detailed analysis of the cis-regulatory elements that control the expression of tll. Through this analysis we have identified regulatory elements (torso response elements or tor-RE's) that are critical for the terminal specific expression of tll (4, 8, 11). Surprisingly, mutagenesis of these elements results in uniformly high levels of transcription throughout the embryo, indicating that the transcription factor that interacts with these elements is a repressor. Presumably, terminal specific modification by the torso signaling cascade of this repressor or of proteins that interact with this repressor results in repressor inactivation. In this way, tll repression is specifi-cally alleviated at the embryonic termini. We have not yet definitively identified the factor that interacts directly with the tor-RE, but biochemical and molecular approaches have revealed a number of candidates, which are currently the subject of further investigation (8).

     Recent experiments from the Ish-Horowicz lab show that Groucho, the same repressor required for Dorsal-mediated repression, may be required for the function of the tor-RE. It is possible, therefore, that Groucho is a substrate of the torso signaling cascade, and, in accord with this possibility, we have found that MAP kinase phosphorylates Groucho in vitro. We are currently attempting to map the phosphorylation site(s) by mass spectroscopy. We will then mutagenize these sites in an effort to determine if the modification plays a role in the function of the terminal pattern forming system.