Naturally Occurring Mutations
Putative Mutants found in Xenopus tropicalis gynogenetic screens:
Wild Caught (Naturally-occuring mutations)
Several populations of wild-caught Xenopus tropicalis, dubbed Populations A, B, C, and F, were screened using the gynogenesis procedure utilizing hydrostatic pressure. These wild-caught animals were purchased from Pacific Biological. Females from Population A yielded the most phenotypes. For the entire screen, 160 animals were initially screened. Out of the 160 animals, 57 females exhibited some phenotype.
Out of fifty-seven potential female carriers:
10 genetically heritable phenotypes confirmed (eleven total females)
15 phenotypes did not recapitulate upon second bombing
31 females died prior to secondary screening
Several confirmed females exhibited the same phenotype but it is not known whether the phenotype is related to the same mutation. We suspect that these females actually carry mutations in different genes, but that these mutations manifest themselves in the form of the same phenotype. At this early stage in the screen, many of these mutants have not been scrutinized with an in depth analysis. A closer inspection of these mutants might reveal subtle (or drastic) differences beyond morphology.
The Puffy Eye mutant was one of the first mutants isolated in the gynogenesis screen and exhibited the same phenotype in the gynogenetic offspring when the female was rescreened a second time as well as in the offspring of the F1 outcross. The homozygous phenotype is characterized by an extensive edema of the gut and head and is lethal between Days 5-7 of development. It is first recognized during Day 3 of development (ie Stages 40-41) where the tail tip is seen to start to curl upwards. Soon after, the edema sets in and there appear to be circulatory problems especially evident along the dorsal and ventral lengths of the body and tail region. Sections through the embryos show that by Stage 41-42 there is necrosis of the brain tissue followed soon after by a generalized necrosis of body tissue. Dissection of whole brains at this stages show microencephaly where the forebrain and possibly the midbrain regions are reduced in size. Interestingly the head is also narrower in shape and the eyes are closely associated with the neighboring brain tissue. This phenotype seems to be very similar to a zebrafish embryonic lethal mutant called aquabat , also characterized by the tail curling up, brain necrosis followed by generalized necrosis and a reduced circulation (Brand et al. 1996a) . bent tail (bt) , a Xenopus laevis mutant, exhibits a similar phenotype (Droin et al 1970). Whole mount in situ hybridization analysis was carrried out on Stage 22-30 tadpoles using the following gene probes Xbf-1, Fgf-8, Rx, Nkx2.1, Otx2, Emx1 and Pax2. No significant difference was seen in the expression patterns between mutants and controls.
In the Gut coiling 5 /31#1 mutants there is heterotaxy i.e. randomization of the symmetry of the internal organs. The wildtype Xenopus tropicalis embryo will have normal heart where the conotruncus loops to the left from the right anterior aspect of the ventricle, a right coil origin for the gut (RO class) and counter-clockwise coils (CCW class)(see Branford et al, 2000 for definitions/descriptions). All seven possible mutant combinations of the 3 above phenotypes are represented in these animals. There are mutants that in addition to the above 3 phenotype combinations exhibit a defect where the small intestine instead of travelling posteriorly down one side of the embryo before crossing over ventrally to form the coils on the other side, crossed over immediately forming a 45 degree angle across the ventral part of the embryo. When examining younger mutant embryos, the gut looping defects can first be seen at Stage 39 when the duodenum starts to curve. The aberrant heart looping is apparent by Stage 35-36. Whole mount in situ hybridization studies using various gut, heart and other internal organ markers have been conducted on Stage 33-47 tadpoles and results indicate that the organs themselves are morphologically normal. Two other wild-caught females showed similar phenotypes in their offspring – combo gut heart 6/9#3 and gut coiling 8/25#7. Interestingly, no left-right asymmetry mutants have been reported in Xenopus laevis.
These homozygous mutants exhibit an enlarged ear vesicle and is missing both saccular and utricular otoliths. This phenotype is first visible during Stages 28-30. They are lethal in the homozygous state and tadpoles die after 6-7 days of development. The behavioral phenotype exhibited later in development includes trouble orientating and balancing themselves at rest as well as swimming in circles - both in horizontal planes and vertical loops. Similar phenotypes have been seen in zebrafish mutants - helter-skelter, keinstein and backstroke (Malicki et al, 1996; Whitfield et al, 1996) . The phenotype is strikingly similar to that of a described Xenopus laevis mutant – otolithless (otl) which has no otoliths and an enlarged ear. Droin (1967) also describe a slight dorsally curved shape. We also observe that a curvature of the spine develops but it is likely that it is due to the erratic swimming habits of the tadpoles. Three wild-caught females gynogenetic offspring showed this phenotype, though only one line survives.
Rough Diamond (Otolith)
In these gynogenetic diploids, different degrees of otolith perturbation were observed. For example, some tadpoles may be missing a substantial potion of the normal otolith structure and in others, the otoliths may be abnormally placed and/or shaped. Their swimming and balancing abilities are similarly variably affected. A number of zebrafish mutants with perturbed otolith formation have been isolated (Whitfield et al., 1996 and Malicki et al., 1996) and a Xenopus laevis mutant Turner (tr) has also been described (see Droin 1971).
Balloon Head (Head misshape)
The phenotype exhibited by these tadpoles seems similar to that described for the Xenopus laevis bubblehead (bh) mutant (Krotoski et al 1985). The embryos develop an edema of the tissues surrounding the head and die shortly hereafter. Likewise, bubblehead develops edema at stage 42 which results in death by stage 45.
Like the left-right asymmetry mutants, no Xenopus laevis heart mutants have been previously reported. The heart of this particular Xenopus tropicalis mutant, when viewed under anethesia, beats less vigorously than wild-type hearts and the composition of either the heart wall tissue or blood is different from normal rendering the heart less red than normal and slightly smaller in size.
Somersault (Swimming in Circles)
This phenotype was produced in a brother-sister mating of putative left-right asymmetry gut coiling mutants. Upon isolating the population and screening the females gynogenetically, this phenotype recapitulated in several females. Tadpoles exhibiting this phenotype were observed swimming in erratic vertical loops and horizontal planes between stages 45-47. Attempts to raise the mutant have been fruitless, but a small wild type outcross exists. Morphology of the ear and brain appear to be normal in these tadpoles and only their swimming orientation is defunctory
The lens of the tadpole appears opaque on one or both sides of the embryo. The gross morphological features indicated a congenital cataract, but paraffin sections of the eye showed an abnormal shape, size and placement of the lens within the eye, as well. The phenotype manifests six to eight days post fertilization and was still visible twelve days after fertilization.