A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

Abstract

In the rapidly developing, diploid amphibian Xenopus tropicalis, genetics can be married to the already powerful tools of the amphibian system to overcome a disability that has hampered Xenopus laevis as a model organism: the difficulties inherent in conducting genetic analyses in a tetraploid organism with a longer generation time. We describe here a gynogenetic screen to uncover naturally occurring recessive mutations in wild X. tropicalis populations, a procedure that is both faster and easier than conventional genetic screens traditionally employed in model organisms to dissect early developmental pathways. During the first round of our screen, gynogenetic diploids from over 160 females comprising four different wild-caught populations were examined. Forty-two potential mutant phenotypes were isolated during this round of gynogenesis. From this group, we describe 10 lines that have genetically heritable recessive mutations. A wide range of developmental defects were obtained in this screen, encompassing effects limited to individual organs as well phenotypes characterized by more global changes in tadpole body morphology. The frequency of recessive mutations detected in our screen appears lower than that seen in other vertebrate genetic screens, but given constraints on the screening procedure used here, is likely to be consistent with rates seen in other animals, and clearly illustrates how wild-caught animals can be a productive source of developmental mutations for experimental study. The development of genetic strategies for the Xenopus system, together with new genomic resources, existing technologies for transgenesis, and other means for manipulating gene expression, as well as the power of performing embryonic manipulations, will provide an impressive set of tools for resolving complex cell and developmental phenomena in the future.

Xenopus tropicalis: An Ideal Experimental Animal in Amphibia

“…When reducing the expression of a specific gene in X. tropicalis, the target is only a single gene, not two genes as in X. laevis, and identifying recessive phenotypes requires mutagenizing two independent loci, not four. More than 40 mutants were identified in X. laevis by inbreeding and gynogenesis after several decades of efforts by the early 1990s [25], while 122 potential mutations of X. tropicalis have been reported by the similar approaches, and 42 have been confirmed as genetically heritable mutations [9,12,27].…”

“…Forty-two potential mutant phenotypes were isolated by a gynogenetic screen of naturally occurring recessive mutations in wild X. tropicalis. Ten lines of the developmental mutants have genetically heritable recessive phenotypes [27]. The forward and reverse genetic screens were undertaken for chemicallyinduced mutations in X. tropicalis [9].…”

Xenopus, an ideal model system to study vertebrate left‐right asymmetry

“…The recent advent of transgenic frog technologies in Xenopus tropicalis, which has a GRP and flow just like Xenopus laevis (Blum et al, 2009), provides further options for generating and analyzing mutant lines using all of the above-mentioned techniques. The first three examples of mutants with LR defects have already been published (Noramly et al, 2005), and more should become available as mutagenesis projects proceed (Amaya, 2005;Grammer et al, 2005; http:// tropicalis.berkeley.edu/home).…”

Xenomics

“…Several groups are currently performing chemical, irradiation, and insertional mutagenesis screens, but such approaches remain in their infancy due to the lack of easily scored mutations for optimizing mutagen dose levels. However, recently two groups published the first set of naturally occurring mutations in X. tropicalis, which should help facilitate such optimization experiments (Grammer et al 2005;Noramly et al 2005). …”