Potential Laboratory Rotation Projects for First Year Graduate Students. These are just some suggested projects, and I am certainly open to additional experimental approaches that reflect the interests of incoming students and post-docs.
I. Evolution of Segmentation
A. Characterization of pair-rule, gap, and maternal coordinate gene orthologs in shortgerm embryos
What role, if any, does pair-rule prepatterning serve in animals that generate segments one at a time? Cloning and examining the expression pattern of pair-rule genes (such as runt) in the grasshopper might provide some insights into this issue. A major emphasis in my lab over the next few years will be to extend this analysis of segmentation to the crustaceans by analyzing pair-rule, gap, and maternal coordinate gene orthologs in Parhyale hawaiensis. These experiments can also be extended to groups outside the arthropods, such as to annelids and nematodes.
B. Role of nanos and hunchback in early patterning events
Preliminary studies in the lab have already shown that hunchback is expressed in a manner consistent with a role in early A/P patterning in the grasshopper. Also, preliminary studies indicate that nanos mRNA is localized during grasshopper oogenesis. Does nanos act to regulate the translation of zygotic hunchback mRNA? What role does hunchback protein play in grasshopper oocytes? Does nanos play a similar patterning role in crustaceans?
C. Manipulation of the “split” segmentation pattern in Triops
We have uncovered a very novel change in the body plan of the crustacean, Triops. Basically, this animal generates two different patterns of segmentation on its ventral versus dorsal sides. How is this pattern set-up and maintained? Can experimental manipulation of the cell-cycle alter this pattern? Are there other animals that do a similar thing during development? What does the fossil record suggest about this type of body plan?
II. Evolution of Limb Patterning
Development of crustacean biramous limbs
Crustaceans have branched limbs and we have also been able to show that this is also true for the limbs of insect mouthparts. We know how to manipulate dpp, wg, and hh signaling to generate branched legs in Drosophila. Do the expression patterns of any of these genes reveal how crustacean branched limbs are formed?
III. Analysis of the role of Hox genes in arthropod evolution
A. Examination of Hox gene patterns in crustaceans
Our previous studies have indicated a link between Ubx expression and the evolution of feeding appendages in the anterior thorax of crustaceans. By cloning additional Hox genes, it may be possible to also examine the diversification of other appendage types during arthropod evolution.
B. Analysis of Hox gene regulatory domains in crustaceans
What causes Hox genes boundaries to shift during evolution? One hypothesized mechanism invokes changes in the enhancers for these genes. By characterizing such enhancers from various crustaceans, we may be able to understand the mechanisms for these evolutionary changes.
C. Cloning and characterization of hunchback in crustaceans
Another mechanism for manipulating Ubx expression during evolution would be to change the expression of known trans acting factors. Hunchback is a known repressor of Ubx in Drosophila. Does it play a similar role in crustaceans? Is the changing expression pattern of hunchback responsible for the evolutionary changes in Ubx expression?
IV. Evolution of neural patterning
A. Searching for neural homologies in distantly related arthropods
Analyzing eve and engrailed expression has allowed us to identify several homologous neurons between insects and crustaceans. Do such homologous neurons exist in other arthropods?
B. Evolution of motorneuron targets
While the overall pattern of cells within the CNS is conserved between insects and crustaceans, there are striking changes in the musculature. How have motorneuron innervation patterns changed during evolution and what mechanisms have allowed for these neural pathfinding changes? In the Triops, how are motorneuron/muscle patterns maintained when their periodicities are not in synchrony.
C. Investigating changing modes of neural determination
Many homologous neurons are found between Drosophila and the grasshopper, but there appear to be differences in the mechanisms of cell fate determination. For example, genetic manipulations in Drosophila and ablation studies in grasshopper suggest some potential differences in the way sibling neuron choice is generated. How have these differences arisen? Furthermore, between Drosophila and crustaceans, there are some remarkable differences in the way that homologous neuroblasts are generated. How and why have these differences arisen? Analysis of genes such as numb, Delta, and Notch in various arthropods may help us answer these questions.
V. Evolution of Enhancer Elements
A. Evolution of eve stripe 2 enhancers in Drosophila.
Our previous experiments have focused on the evolutionary changes in the eve stripe 2 enhancer between closely related species of Drosophila. These experiments can be expanded to include reciprocal transformation of these constructs into Drosophila melanogaster and Drosophila pseudoobscura.
B. Evolution of eve enhancers in Tribolium and Schistocerca.
Cloning and characterization of eve regulatory elements in Tribolium and Schistocerca can be used to expand our understanding of enhancer evolution by looking at arthropods with patterns of eve expression distinctly different from that found in Drosophila.
C. Evolution of Prd Group III enhancers
We have cloned and characterized the expression of two homologs of the Prd GroupIII genes in Schistocerca. Examining the enhancers of these genes, and comparing them to the enhancers of prd, gsb, and gsb-n in Drosophila should shed important light on the evolution of insect segmentation.
VI. Development of gene manipulation techniques in various organisms
Evolutionary studies are hindered by the inability to carry out genetic analysis in all but a handful of model systems. For virtually all the projects described above, manipulating gene expression can help test hypotheses based on comparative gene expression data.
A. Baculovirus mediated gene misexpression
We have developed a baculovirus based gene misexpression system that has been successfully used to test wingless function in Tribolium. This system can now be used to manipulate limb patterning in a variety of insects.
B. Using transposable element vectors to characterize gene regulatory elements
We are currently testing the ability of the piggyback transposable element system for generating transformed Parhyale. Such a technique could then be used to test enhancer elements in a number of arthropods. Such a system could also be used for mutagenesis and gene misexpression.
C. Using RNAi and Morpholinos to analyze gene function
Double stranded RNA (RNAi) and Morpholinos (synthetic antisense RNAs) have proven to be useful tools for knocking out gene expression through the elimination of specific mRNAs. We are currently testing both techniques in Schistocerca and Parhyale and hope to use these methods to extend our analysis of segmentation and body patterning in these arthropods.