The main goal of my lab in the next few years will be to further our understanding of the developmental basis of evolutionary change. Some of this research will follow the same experimental lines as in the past. Studies in model systems, such as Drosophila, have identified genes that govern pattern formation during development. By isolating orthologs of these genes in other animals, and examining their expression patterns during development, we may be able to learn how developmental programs have been altered during evolution. These comparative studies will be primarily centered on arthropods, but where appropriate these investigations may be extended to annelids, mollusks, nematodes, and vertebrates as well. An increased focus, however, will be placed on an experimental analysis of the function of these genes in arthropods outside of Drosophila, and on an analysis of the molecular basis for evolutionary changes in gene expression patterns. We will focus on two animals, the grasshopper, Schistocercaamericana, and the amphipod crustacean, Parhyale hawaiensis, for the majority of these future studies.
Evolution of segmentation mechanisms
In the past, our analysis of the evolution of segmentation has focused on the expression of the segment polarity gene engrailed and the pair-rule gene even-skipped. More recently, we have expanded these studies to include the pair-rule gene paired and the gap gene hunchback. These recent studies have significantly changed our thoughts on how segmentation occurs in animals that sequentially produce their body. Our data, and that of other labs, suggest that the mechanisms used by Drosophila to establish the anterior-posterior axis and subdivide the body into segments can be employed in animals that generate segments sequentially in a non-syncytial environment. There are, of course, major changes in the genetic segmentation hierarchy between different insects, and we still have yet to understand how graded information is initially specified in insects distantly related to Drosophila. We hope to expand our comparative studies to include additional members of maternal coordinate, gap, and pair-rule segmentation genes. To this end, we have now cloned and begun to characterize the expression of Krüppel andnanos in Schistocerca.
We also hope to expand these comparative studies to the amphipod crustacean, Parhyale hawaiensis. There are several reasons for expanding our studies to include crustaceans. The two most important are (1) that crustaceans display a highly organized pattern of growth (as opposed to the poorly organized and not well understood pattern of growth in most insects), which simplifies the analysis of the segmentation process, and (2) that we believe this amphipod will be amenable to various manipulations of gene expression (something for which we have had only limited success with in Schistocerca). We have already cloned Parhyale orthologs of a number of genes involved inDrosophila pattern formation. Also, we have established a robust breeding colony of these animals. Their life cycle takes about six to seven weeks and we can easily obtain embryos at the one cell stage. More importantly, we have developed techniques for injecting embryos at the one, two, or four cell stage, and have excellent survival of these injected embryos. So far we have used this injection technique to begin to establish a lineage pattern for Parhyale, but more significantly, we have shown that we can carry out gene misexpression experiments via injection of synthetic mRNA. We are currently testing whether or not it is possible to achieve “gene knockout” by RNAi or Morpholino (antisense oligo) application. Finally, we are also testing several transposable elements for their capacity to generate transgenic animals in this species. We believe that carrying out a combination of comparative gene expression studies, along with gene manipulation studies, in Parhyale will significantly advance our knowledge of how pattern formation systems evolve. We may also expand our studies outside of the arthropods if we feel that we have evidence for specific, highly conserved pathways for segmentation within the arthropods.
Depending on how successful we are with our preliminary experiments in Parhyale, we may try to develop this as a system in which we can attempt to carry out “forward genetics” and/or genome-based analyses.
Evolution of neuronal specification and limb patterning
We also plan on continuing our analysis of appendage evolution in the arthropods, and neural specification in the arthropods and annelids. Our main focus with limb evolution has been to understand the evolutionary differences between insect (unbranched) and crustacean (branched) limbs. We have examined the expression of several limb patterning genes in a variety of insects and crustaceans. Our results suggest that changes in the expression of some of these genes during the early formation of the limb primordia may be responsible for the observed changes in morphology. We are currently using a baculovirus based misexpression system which we have developed to test these ideas in a number of animals. Our current focus is to use baculovirus to misexpress the signaling moleculedecapentapalegic in beetle (Tribolium) embryos to see if we can generate branched limbs in insects that are topologically similar to the normal branched limbs of crustaceans.
Our analysis of neural evolution will focus primarily on the evolutionary differences in the way neural progenitor cells are established in different arthropods and annelids. Again, studies from Drosophila have given us a number of identified genes that participate in the process. In crustaceans, however, the mechanism by which neural precursors are established appears to be quite different than in Drosophila, and we hope to use a comparative approach to understand these evolutionary differences. Depending on what we find, we hope to expand these studies to include the manipulation of gene expression as outlined for our studies on segmentation.
Analysis of regulatory changes in genes during evolution
It is clear from the comparative work done in a number of labs that evolutionary changes in gene expression play an important role in the evolution of developmental programs. Presumably these changes in gene expression come about through changes in either trans-acting regulators of the gene or in changes in the cis-acting sequences that control the gene.
We have had an especially fruitful collaboration with Marty Kreitman and Misha Ludwig (Ecology and Evolution Dept). on the analysis of the evolution of the even-skipped stripe 2 enhancer (probably one of the best understood eukaryotic enhancers), and we hope to continue this collaboration on some future projects , as well as establish our own independent projects that relate to our specific interests. We started our analysis by making comparisons of the cis-elements from diptera closely related to Drosophila melanogaster. We will continue to expand this analysis into more and more divergent insects (Schistocerca for example) and for additional genes (such as Ubx and hairy). Much of our analysis of the eve enhancer has been carried out by placing the enhancer from other species into Drosophilamelanogaster, but we hope to take advantage of a number of new transformation vectors that may make it possible to place the same enhancer into multiple arthropod species – such an analysis will greatly expand our abilities to understand how changes in gene expression come about during evolution.