Our approach to mutagenize the X. tropicalis genome is to expose sperm to gamma rays using a Cesium-137 source. Mutagenized sperm are used to fertilize eggs, which generates F1 animals. Gamma-ray mutagenesis produces many kinds of chromosomal damage such as small to large deletions, chromosomal inversions, and other rearrangements.
Our first step was to determine a dose response curve. The Gammator that we used as a mutagen source produces approximately 300 rads/min. In zebrafish studies, a dose of approximately 300 rads was considered optimal and would result in embryonic lethality (CNS degeneration) of 25%. We exposed sperm to an increasing dose of gamma rays and quantified the embryonic lethality. In possible contrast to Zebrafish, heavy Gamma ray exposure in X. tropicalis causes a stereotypical gastrulation defect which is dose-dependent. Our results show that at a 2 min (600 rads) dose, we are getting approximately 50-70% mortality. This dose is similar to the optimized zebrafish dose. Based on these findings, we raised animals exposed to four different doses of mutagen (30s [150 rads], 1 min [300 rads], 2 min [600 rads], and 3 min [900 rads]).
Originally we had planned to screen haploid progeny from these founder females. Haploids can be produced by UV irradiation of sperm. The irradiated sperm can still activate the egg to begin development but does not contribute any genetic material. "Good" haploids are indistinguishable from diploid embryos through neurula stages. In the mid-20's, haploids develop a stereotypical "haploid syndrome." The axis is shortened and they appear stubby. By the 30's, haploids are very obvious with edema and bent tails. Although the morphology at later stages may be altered, transcripts characteristic of differentiated tissues are present. Therefore we had planned on an in situ screen of haploids to uncover potential mutants.
Unfortunately the only inbred line available, the Grainger Nigerian line, has not produced good haploids in our experience. Even control animals that received no gamma irradiation produce haploids that cannot be screened even after multiple attempts. Therefore we converted to a standard diploid F3 screen. Since this requires that an F2 generation be raised to maturity, we also chose to screen some of the F1 animals by haploids in case some of them do produce high quality haploids in the interim.
In our experience, males mature much more quickly than females (4 mo versus 7-9 mo). Therefore, we will exploit males to transmit mutations as quickly as possible. In order to identify male carriers, we backcross these males to females of the previous generation. Therefore the first mature F2 male will be mated to the F1 female to determine if the male is a carrier. If three F2 males are selected at random, then there is a >85% chance that one of them will be a carrier. Four random males increases the odds to over 93%. We intend to screen 3-4 males before abandoning a founder female.
Initially, we were using natural matings to identify phenotypes. However, because of improved husbandry, we find that our F2 clutches are larger than necessary. Therefore, we have been sacrificing three males and performing in vitro fertilization of the F1 female eggs allowing us to screen all three males at once. This three-male backcross strategy then allows us to very quickly determine if an F1 female harbors any mutations that are of interest. If a three-male backcross produces an interesting phenotype, then we begin natural matings to identify another F2 male or F2 female. We believe that this is a particular strength of X. tropicalis. Because of the massive number of eggs produced from a female, we can easily generate many hundreds of embryos for each of the males in an IVF. The hundreds of embryos produced allows us to screen not only tadpoles but fix embryos at gastrula, neurula, early and late 20’s and tailbud embryos. These fixed embryos can then be stored and used for preliminary analysis of the phenotypes.
In order to screen for phenotypes of possible mutants, we use two assays. Since the screen is a standard F3 screen with diploid embryos, we will carefully evaluate the morphology of the embryos at various stages (neurula, early 20's, late 20's, early 30's, and early 40's). We will also employ an array of molecular markers at these stages to evaluate neural induction and patterning, mesodermal patterning, and organogenesis. At Neurula stages we will use Pax6, Shh, Olig2 and Slug as a pool of neural markers. At early 20s we will use two pools: MyoD and Twist (for muscle and neural crest) and Pax3, Pax8, and Nkx2.5 (neural, kidney, heart). At late 20s we are using EphA4 and xHex (brain, kidney, and liver). Finally at early 30s, we are using Pax2, VEGF, and globin (brain, kidney, somite segments, vasculature, and blood). (See Phenotyping)