Haploid and gynogenetic diploid S. tropicalis and X. laevis. 

A series of useful techniques have been developed in amphibians for manipulating the ploidy of embryos.  Haploids develop fairly well before arresting at mid-tadpole stages.  By using UV-irradiated sperm to produce haploid embryos and then immediately restoring diploidy, it is possible to generate embryos that are homozygous over some or all of their genome in a single generation.  These techniques are likely to be especially useful in conjunction with transgenic procedures, to rapidly create lines of frogs that are homozygous for an introduced reporter locus or gene trap.
 We assayed gynogenesis by fertilizing albino X. laevis eggs with UV-irradiated wildtype sperm; at tadpole stages, haploid embryos remain unpigmented in contrast to embryos fertilized with untreated sperm (Fig. 5A), demonstrating that UV-inactivation of the paternal genome was effective.  Haploid embryos develop a distinct phenotype, usually including axis kinking and ventral edema, but at early tailbud stages are virtually indistinguishable from diploid embryos (Fig. 5B) and may be used as a background on which to uncover recessive mutations affecting embryogenesis.
 Restoration of diploidy to haploid amphibian embryos was accomplished by "early pressure" application of ~5000 lbs/in2 5-10 minutes after fertilization, which is thought to suppress the formation of the second polar body.  Application of early pressure to haploids does not neccessarily result in embryos that are completely homozygous, due to meiotic crossover, but loci that are not distal to the centromere are likely to have been rendered homozygous.  We have not attempted to suppress first cleavage using "late pressure", which has been used to generate completely homozygous X. laevis embryos.  Early pressure successfully rescued haploid X. laevis (Fig. 5C; ) and S. tropicalis in a high percentage of embryos (Fig. 5F) (compare control diploid and haploid embryos in Fig. 5D and E, respectively).
 

A. Diploid (Dip.) and haploid (Hap.) X. laevis tadpoles.

B. Diploid (Dip.) and haploid (Hap.) X. laevis embryos at the tailbud stage.

C. Early pressure used to rescue X. laevis haploid embryos.

D. Wildtype S. tropicalis tadpoles.

E. Haploid S. tropicalis tadpoles at a stage similar to those in D.

F. Early pressure used to rescue S. tropicalis haploid embryos.