Developmental biologists have made great strides in understanding embryonic pattern formation at the genetic, molecular, and cellular levels. Much of this advancement can be attributed to the remarkable success of studies of pattern formation in model systems, such as the fruit fly Drosophila melanogaster. Identification of genes that play major roles in setting up the body plan, combined with the subsequent discovery that many of these genes are well conserved even between phyla, has also led to a renaissance in the investigation of the links between evolution and development.
Our laboratory is exploring the degree to which developmental pathways have been conserved or altered between various animals. Insights into the nature of developmental and molecular alterations help us to understand the evolutionary changes in the mechanisms of pattern formation and provide a molecular basis for analyzing the diversification of body morphologies and developmental mechanisms. Ultimately such studies will allow us to understand how evolutionary processes have generated the remarkable diversity of body forms seen in the animal kingdom.
Evolution of Segmentation Mechanisms Drosophila, like all other arthropods, has an obviously segmented body plan, and this pattern of segmentation is established very early during embryonic development. Genetic studies have revealed the molecular basis for this process, and the segmentation genes are categorized (by their mutant phenotypes) as maternal coordinate genes, gap genes, pair-rule genes, and segment polarity genes. These genes subdivide the embryo into progressively smaller and smaller domains, eventually establishing the pattern of segments. The extent of evolutionary conservation of the segmentation hierarchy within the insects, and between insects and animals from other phyla, has been a matter of considerable debate.
Our recent work has focused on the characterization of orthologs of the Drosophila maternal coordinate gene nanos, the gap genes hunchback and Krüppel, the pair-rule gene paired, and the segment polarity gene gooseberry in various animals. Our studies so far suggest that at least within insects, the genetic system for setting up the initial anterior-posterior axis using hunchback and nanos may be evolutionarily conserved. Nevertheless there are some interesting changes in these early steps of pattern formation that are consistent with what we know about the differences between the early embryology of Drosophila and the grasshopper. In Drosophila, this system is a maternal system—i.e., the early patterning is carried out via mRNA that is deposited in the egg by the mother. Our results in the grasshopper suggest that much of this system is zygotic—it is the embryo itself that sets up this pattern as it is growing. This is consistent with the notion that at least some properties of the Drosophila system are driven by selection for rapid embryonic development, hence the increased reliance on maternal systems to get pattern formation off to a quick start.
Our previous studies had failed to identify a pair-rule prepattern in grasshoppers, as is seen in Drosophila. This has led to widespread speculation on mechanisms of segmentation that are independent of pair-rule patterning. Our most recent work with homologs of the Drosophila pair-rule gene paired, however, indicate that grasshoppers appear to employ a pair-rule pattern, but clearly the components of the system differ substantially between the two insects. One of these paired homologs, pairberry 1, is expressed in a pair-rule pattern long before other molecular markers appear to reveal the future boundaries of segments. Thus, we find that there are many similarities in the hierarchy controlling segmentation between these two distantly related insects, and at the same time there are differences that can help account for their different modes of early embryogenesis. We have also developed monoclonal antibodies that recognize the products of the paired gene family in a broad range of animals and are investigating the evolutionary changes in the functions of these genes across the animal kingdom.
Increasingly, the majority of the work in my lab is shifting toward the study of pattern formation in crustaceans. Crustaceans (which include such animals as lobsters, crabs, and brine shrimp) have far more variation in their body plans than do insects. For example, there is wide variation in the number of body segments between crustacean species, while this number is relatively static within the insects. Another reason for expanding our studies to crustaceans is that they offer us a system that is more amenable to experimental and manipulative studies. Our focus has been on a marine amphipod, Parhyale hawaiensis, which also displays an extremely simple pattern of cell division that generates body segments during development. Linage analysis of the eight-cell-stage embryo of Parhyale reveals that cells are already restricted to producing progeny of only a single tissue type. Of the eight cells, one makes germline, three make the ectoderm, two produce the somatic mesoderm, one generates the visceral mesoderm, and one makes the endoderm. We are carrying out cell ablation experiments to determine how committed these cells are to their fate at various stages. In addition, we have been studying the molecular aspects of segmentation in this animal, focusing on the expression and function of the hairy and runt genes.
In the course of our studies on the evolution of segmentation, we have discovered, in collaboration with Michalis Averof (Institute of Molecular Biology and Biotechnology, Iraklio, Greece), an unusual modification in the process of arthropod segmentation. In the crustacean Triops, segmentation between the dorsal and ventral sides is uncoupled, resulting in an animal with different numbers of segments on its dorsal and ventral sides. This observation may help explain how discrepancies between segmental patterns in vertebrates might arise (such as the difference between the number of rhombomeres and branchial arches in many vertebrates). In collaboration with Derek Briggs (University of Bristol, Bristol, England), we have also made the remarkable observation that the odd body plan seen in Triops is also found in some species of arthropods from the mid-Cambrian period, suggesting that it has an ancient evolutionary history.
Regulatory Changes in Genes During Evolution
Many of our comparative studies suggest that changes in gene regulation play a major role in the evolution of patterning processes. We have begun to analyze the types of regulatory changes that occur during evolution. For these studies, we have focused on the evolution of the even-skipped stripe 2 enhancer element, one of the best understood regulatory elements in Drosophila. In response to several well-understood repressors and activators, this element directs expression in a single stripe within the early fly embryo. In collaboration with the lab of Marty Kreitman (University of Chicago), we isolated the even-skipped stripe 2 enhancer from a variety of Diptera. Our analysis has shown that, despite the fact that all of these enhancers from other species work correctly when placed into Drosophila melanogaster, there is quite a bit of evolutionary change in the enhancer sequence. We have generated chimeric enhancers and shown that these do not function properly (as assayed by reporter gene expression), providing evidence that compensatory changes within the enhancer allow for the evolutionary changes in the overall sequence. We are currently constructing transgenic animals designed to allow us to see what developmental alterations might occur when these regulatory changes are made in the context of the endogenous gene. Our long-term goal is to characterize regulatory changes between distantly related arthropods, but it is clear that these studies within the Diptera are important if we are to make sense of regulatory comparisons between more-diverged species, especially where we wish to understand the sequence changes that might be relevant to generating altered patterns of gene expression. In general, these results have major implications for genomic studies aimed at identifying regulatory elements by their conservation between either closely or distantly related species.