March 1, 2005;
A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis.
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.
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Fig. 2. Schematic of the gynogenesis screen procedure. Eggs are harvested
from the wild-caught female, as indicated by the unfertilized egg at the top
of the figure, and undergo three different treatments (as denoted by the three
outlined columns). In the first, the eggs are fertilized by harvested sperm
and allowed to develop normally (left-hand column). In the second, haploid
embryos are created by the fertilization of the eggs by UV-irradiated sperm
(center column). In the third, after fertilization by UV-irradiated sperm, the
eggs are treated with high pressure to induce the formation of gynogenetic
diploid embryos (right-hand column). The haploid embryos have a distinct
phenotype and can readily be distinguished from their wild-type and
Fig. 3. Confirmation of the induction of gynogenetic diploid embryos. Xenopus tropicalis eggs are harvested and fertilized with sperm from males heterozygous
for a green fluorescent protein (GFP) transgene under the control of the gamma-crystallin promoter. (A) Embryos viewed under normal illumination for
morphology. (A00) Embryos viewed under UV illumination to identify GFP-expressing embryos. (A and A0) Eggs were fertilized with transgenic sperm and
allowed to develop normally. Fifty percent of the offspring express the transgene (A0). (B and B0) Haploid embryos formed from eggs fertilized with UVirradiated
transgenic sperm. There is no evidence of transgene expression in the offspring (B0). (C and C0) Gynogenetic diploid embryos formed from eggs
treated with UV-irradiated transgenic sperm and subsequently exposed to a high pressure treatment. Note there is no expression of the transgene in the
gynogenetic diploid embryos (C0).
Fig. 4. Puffy Eye. (A and C) Side and top views of stage-matched wild-type controls, respectively. (B and D) Puffy Eye mutant embryos displaying the
characteristic up-turned tail (B) and eyes that are directly adjacent to the brain tissue (D, arrow). (E) Dissected mutant (upper) and wild-type (lower) brains. The
diencephalon is reduced in size and also lacks the normal curvature. In addition, the optic tectum (midbrain) also is smaller in the mutant form. T,
Telencephalon; D, diencephalon; M, midbrain; H, hindbrain.
Fig. 5. Mixed Up. (A) Control wild-type embryo where the conotruncus of
the heart loops to the left from the right anterior aspect of the ventricle and
the gut has a right coil origin (RO) and counter-clockwise coils (CCW). (B
and C) Mixed-Up mutants displaying abnormal heart and gut looping. The
mutant tadpole in (B) exhibits a left coil origin (LO) for the gut whereas the
mutant tadpole in (C) exhibits a right coil origin (RO) for the gut. Both
mutants have clockwise coils (CW). (D and E) Mixed-Up mutant tadpoles
may display alterations in gene expression at earlier stages of development.
(D) illustrates the right hand view and E the left-hand view of three mutant
tadpoles that are stained for Nkx2.5 expression. The tadpole at the top of (D)
and (E) displays an altered expression of Nkx2.5 in the developing gut (D,
arrowhead) whereas normal gut expression is observed in its two mutant
siblings below it (E, arrowheads). This altered expression is indicative of
the fact that this tadpole has an asymmetry defect affecting the direction of
Fig. 6. Little Pitcher. (A, C and E) Wild-type tadpoles. (B, D and F) Little
Pitcher mutant tadpoles. (A and B) Arrows point to the otic vesicles in the
wild-type and mutant tadpoles. In B, the otic vesicle is significantly
enlarged in comparison to the otic vesicle of the wild-type tadpole in (A).
Note the lack of otoliths in the mutant versus the wild-type tadpole (seen as
bright white structures in the wild-type otic vesicle). (C and D) The otic
vesicle is outlined in the wild-type control and mutant tadpole, respectively.
The thin black lines in (C) and (D) represent the planes of sections viewed
below in (E) and (F). Cross-sections through the tadpole heads at the level
of the otic vesicle reveal the enlarged vesicle structure in the mutant (F) in
comparison to the wild-type structure in (E). nt, neural tube; ov, otic
Fig. 7. Rough Diamond. (A) A wild-type tadpole displaying two distinct otoliths, the saccular and utricular otoliths, within the otic vesicle (arrows). (C) Top
view of the otic vesicle with the otolith structures present at right angles to each other. (B, D) Mutant tadpoles display different degrees of severity in
perturbation of otolith structure.
Fig. 8. Balloon Head. (A and C) Top view and cross-section through the head of a wild-type tadpole. (B and D) Similar views of a mutant Balloon Head tadpole. The arrowheads in (A) and (B) indicate regions affected in the mutant. The white line in (A) and (B) demarcate the plane of section seen in (C) and (D). (E) Arrows point to areas of edema around the eye and jaw of a Balloon Head tadpole. nt, neural tube; os, optic stalk; re, retina.