How new morphologies evolve is a fundamental question in biology. Most morphological evolution involves changes in the size of organs, which is basically achieved in two ways, by changes in cell size and cell number. Recent studies have revealed a number of genes that effect cell size in organisms as diverse as nematodes, insects and mammals. However, little is known about the genetic basis of species differences in organ and cell size regulation, or how this leads to organ specific changes in morphology. We propose to investigate differences in male wing cell size between two closely related insect species in the genus Nasonia. Males of one species (N. vitripennis) have small vestigial forewings whereas in the closely related species (N. giraulti) males have 2.3-fold larger wings. This size difference is due mostly to differences in cell size. Wing cell size can be studied genetically because the species are interfertile, allowing movement of genes between them. In preliminary studies, we have introduced 5 regions with major effects on wing size from giraulti into vitripennis. One region (wsl) accounts for 40% of the wing size difference due mainly to an increase in cell size. A second (wdw) causes a dramatic 30% increase in wing width without increasing wing length; cell size is increased specifically in the medial portion of the wing. A third (sww) shortens wing length and increases width increasing the ww/wl ratio by 11%. Each of these segregates in a Mendelian fashion and therefore represents a single locus or set of tightly linked loci. They also represent unique phenotypes not described in other studies of cell size regulation. Using linked visible and lethal markers, we have reduced the size of the introgressed region around wsl to <0.5% of the genome, approximately 1.7Mb. We have also mapped several insulin-signaling genes, two of which (e.g. s6k, tor) map closely to loci involved in the wing size differences. Our specific goals are to (1) further determine the genetic basis of the interspecies wing size differences, (2) clone and molecularly characterize at least two wing cell-size genes and (3) determine how these loci contribute to the developmental control of wing cell-size differences. Marker and lethal assisted recombination will be used to reduce the size of the flanking regions and to positionally clone wing cell-size genes. Using the Nasonia BAC library and molecular markers that map within to the wing size regions, we will construct contigs within regions containing wing cell size loci. These will be used to define the size of the introgressed regions during mapping and positional cloning, and tightly linked lethals will be used for ultra-fine scale mapping of the wing cell-size genes. Developmental studies will investigate patterns of wing development and mRNA abundance in developing wing imaginal disks, using known wing patterning genes (e.g. engrailed, wingless), cell-size regulators (e.g. s6k, tor), and genes identified by positional cloning, to determine potential regulatory mechanisms of the wing cell-size differences. Nasonia is an emerging model organism, and has several features suited for genetic studies of cell and organ size evolution. These include ease of handling, short generation time, high recombination rate, small genome size, male haploidy, inter-fertile species, abundance of visible and molecular markers, and inter-specific differences in wing cell size regulation. By taking advantage of the particular features of the Nasonia system, this study will provide the first information on the genetic basis of species differences in cell and organ size, and may reveal new gene mechanisms involved in cell-size regulation.
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