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Drosophila development Segment polarity genes and cell-cell signalling during development. [email protected] The transcription factor proteins encoded by the gap and pair rule genes divide the embryo in consecutively smaller units (segments/parasegments), ending with stripes of expression of the segment polarity genes in each segment. Up until now transcription factors were able to interact with each other [a nucleus can be influenced by two such proteins, even if neither protein was produced (as a transcript) in that nucleus], cell membranes are formed at the time the segment polarity genes become expressed. All segment polarity mutants show similar patterning defects in each segment, repeated along the trunk of the (dead) larva. Consistent with the fact that the proteins encoded by the segment polarity genes are required for patterning of the segment (evident from the mutant phenotypes), the genes are expressed repeated in each segment. In addition, in the now cellularised animal, cell-cell signalling is needed to ensure proper cell differentiation and indeed segment polarity genes encode for signalling-type proteins. Do the genetic findings tell us anything about how the system works? In a wingless mutant embryo, all cells that normally secrete naked cuticle (about half the segment) are lost or re-patterned. The wingless gene is however only expressed in a one cell wide stripe, one per segment. From such simple experiments but not necessarily knowing anything else, we can conclude that Wingless protein function is required over several cell diameters away. Indeed when the gene was sequenced it appeared that the wingless gene encoded for a secreted protein that is apparently able to travel several cell diameters from its source and influence patterning at a distance. The protein was also found to be a direct homologue of a gene already known in human and mouse to cause mammary tumours. Normal segment wingless mutant Using both molecular (looking at expression of genes in mutant backgrounds) and genetic means (combining mutants into double mutant combinations; leading to the elucidation of genetic hierarchies, a process called epistasis in genetic terms), the role of most of the segment polarity genes has been eluded. Their hierarchical interactions were thus “known” even before the proteins were biochemically characterised Pair rule gene activity is required to establish the expression of the segment polarity genes. The anterior boundary of engrailed and hedgehog expression (in same cells) is precisely put down by pair rule genes, this is the future “middle” of the segment (or also known as parasegment boundary). On the anterior side of the parasegment boundary a row of cells expresses wingless; i.e. anterior to the engrailed/hedgehog domain. It appears that two secreted signalling proteins control the patterning of the segments: these are encoded by the segment polarity genes wingless and hedgehog. Most of the other segment polarity genes encode for proteins that are required in the signalling pathways of either Hedgehog or Wingless. This was established first by genetic analysis and later (sometimes) confirmed by biochemical analysis. Both signalling pathways lead to transcriptional activation of targets. It was found that in early stages of segment polarity function, the transcriptional target of Wingless, is the hedgehog gene and vice versa. This reciprocal signalling makes sure that the segment is maintained as a unit and it also provides a reference point within the segment (i.e. the parasegment boundary). Some not so straightforward interactions between genes were found early on. For instance, the patched mutant leads to a phenotype that is almost the opposite of the hedgehog phenotype. When the hedgehog gene is over-expressed in an embryo (using transgenic flies, easy to make), a phenotype like patched is generated. This indicated that patched works to inhibit Hedgehog signalling (loss of patched = gain of Hedgehog). The double mutant of hedgehog and patched looks like patched. Patched therefore works downstream of hedgehog (see figure below). This genetic analysis has been confirmed by biochemical methods: Hedgehog protein binds to Patched protein and inactivates it. Besides genes that have been discovered as required downstream of Wingless or Hedgehog, some might be required “upstream” (for instance for diffusion of the signalling protein or secretion from producing cells). normal patched mutant hedgehog patched hedgehog The Drosophila segment can be seen as a paradigm for the patterning of fields of cells (one-dimensional in this case: anterior-posterior). A dual signalling mechanism provides a simple though reliable/stable and regenerating system. Such patterning of a group of naïve cells tells us something about for instance regeneration processes as well as giving us information on for instance stem cell biology. In both cases (Wingless and Hedgehog) a “distinct” signalling pathway has been identified through the work on the Drosophila segment polarity genes. However both hedgehog and wingless belong to large protein families that are highly conserved in evolution. Wingless was originally discovered as an oncogene. In fact, many proteins encoded by the segment polarity genes are proto-oncogenes or tumour suppressor genes in certain contexts. A lot of work has established that both the wingless and hedgehog gene families are required for patterning and development in vertebrates (humans). The signalling pathways as described in the Drosophila segment are conserved at all these sites. For instance, the sonic hedgehog gene is required for patterning at many sites. One of these is in the patterning of the neural tube. It is expressed in the notochord and patterns the ventral side of the tube (floor plate and motoneurons). Its absence leads to midline defects. However later in development required for patterning of muscle, arms,/legs, neurons etc etc. Text books: Chapter 5, Wolpert, Principles of Development; pages 150-161 (1st edition).170-181 (2nd edition). Chapter 11, Wolpert, Principles of Development, pages 346-347 (1st edition); 383-386 (2nd edition). Chapter 14, Gilbert, Developmental Biology; pages 565-569 (5th edition), 282-285 (6th edition), 283-285 (7th edition). Chapter 14, Gilbert, Developmental Biology; pages 659-660.(5th edition), 151-152 (6th and 7th edition). Literature: Ingham, P. W. (1988) Nature, 335, 25-34. Ingham and McMahon (2001), Genes and Development, 15, 3059-87. Look at website: http://www.stanford.edu/~rnusse/wntwindow.html