Phenotypes Arising from ENU Mutagenesis
The preliminary gynogenetic screening of F1 offspring of ENU mutagenized males has been been successful in producing fifteen confirmed mutations and twenty-seven putative mutations. Well over 150 F1 animals have been subjected to gynogenesis, but only 54 of those animals were discerned screenable. In five animals, the first screen revealed one putative phenotype and subsequent screens revealed a different and unrelated phenotype each time. The remainder of the animals did not produce eggs or produced eggs of low quality.
(Pirate) Eye degeneration
The pirate mutant appears to be the first dominant mutation isolated from any of our gynogenetic screens. It is unique in that the phenotype appears after the onset of metamorphosis and is not detected in early embryological or tadpole development. The outcross population was raised because the first round of gynogenetic offspring exhibited a truncated tail and the potential founder died shortly after gynogensis due to disease. When the outcross reached maturity, approximately fifty percent of the population were missing or had a severely reduced eye(s). The phenotype entails a small pit where the eye should be located or a drastically reduced eye when compared to a wild type. The frequency of the phenotype occured more on the left side than the right, leading one to believe a symmetry defect may also exist. A small fraction of the mutants were missing both eyes. The outcross was tested gynogenetically and eye development appeared normal (e.i. no reduction in size, eye morphology normal) in the developing tadpoles. The putative truncated tail phenotype was also not detected. A female member of the outcross population exhibiting the missing eye phenotype was bred to a wild type male and the offspring raised to maturity. In this cross, the mature offspring once again exhibited the missing/reduced eye at approximately a 60% rate.
(Bleary Eyed) Improper Cornea Formation
The homozygous mutants exhibit a defect in the formation of the cornea, as detected by sections of the mutant's eye around stage 42-45. The gross morphology of the eye observed in the mutants indicated a misshape of the eye, wherein the lens or cornea appeared enlarged and incorrectly shaped. In addition, the cornea appears pigmented and not completely transparent or translucent. The phenotype is most easily observed at stage 45, but probably manifests earlier in development at some undetermined time. The phenotype has been observed in several members of a wild type outcross of the female founder and at least one male has been isolated as a carrier through brother-sister matings using cryopreserved sperm from the male.
(Moon Ears) Crescent shaped otoconia
Also isolated from an F1 offspring of a male mutagenized with 2 treatments of ENU (ENU 2 x 100). This phenotype is unique; it is rarely seen as background in natural matings. In animals exhibiting this phenotype, the otoliths are crescent shaped instead of the normal inverted “L” shape. The eyes are also more dorsal than in wild type animals and the brain seems to be condensed, although it does not appear that any portion of the brain is missing. The head is slightly misshaped, appearing to be a rounded triangle instead of the normal oval shaped. This mutation is not lethal. The phenotypic gynogenetic tadpoles were raised to maturity and outcrossed and the phenotype subsequently confirmed. They are slow to grow, however, and did not reach sexual maturity until approximately a year after birth.
(Monstro) Spina Bifida and Pronounced ventral edema
These animals were isolated from an F1 offspring of the ENU 2x100 male. The phenotype includes a truncated and sinuous tail that is divided. These animals also show a specific U-shaped ventral edema extending from the most anterior part of the ventral side to the maximum vertex below the heart and ending just behind the gut. The animals do not live long enough for the gut to completely differentiate. The presumed carriers have succumed to disease and this line no longer exists.
(Ethereal) Decreased Neural Crest Pigment
This phenotype, also isolated from the 2 x 100 ENU male offspring, could be a matter of subjectivity and natural variation. The tadpoles appeared normal except they appeared to have very few neural crest pigment cells that were very light in color. In a few cases, the neural crest pigment cells appeared to be absent. The mutants did not survive, but that could be related to husbandry. The wild type outcross is a small population that should be sexually mature very soon.
(Heartthrob) Heart Arrhythmia
Also isolated from the ENU 2 x 100 male. This phenotype has recapitulated three times and we have a large wild type outcross growing to maturity, although it will take several more months. The animals exhibiting this phenotype had an erratic heart rhythm and the heart would stop for several counts. The heart would then start beating again, but in a completely different rhythm than before. A milder form of this heart phenotype may have been observed in the form of an enlarged heart that had a regular heartbeat. Some portion of the animals with the erratic rhythm also had an enlarged heart.
(Heartache) Improper heart formation/beat
The heartache mutant was confirmed in the founder animal twice via gynogenesis, but few outcross offspring survived to confirm the germline transmission of the mutation. The founder subsequently died shortly after the second gynogenesis procedure due to disease, and only a few members of the outcross population survived to maturity due to low numbers of animals and perhaps disease and/or improper care. Most of the homozygotes exhibit a morphological defect of the heart wherein the heart chambers are not properly shaped and possibly the heart is not in the correct position within the tadpole thorax cavity. The heart capacity also appears smaller than normal wild type animals. Presumably due to the shape and size, the heart also exhibits weaker contraction than wild type animals. Nearly twenty mutants have been described in zebrafish in which the heart function (beat) was compromised. (See Chen, N. et al., 1996).
Mandibular cartilage malformation
The mandibular cartilage is tetrahedral shaped instead of semi-circular which results in a depression between the boundaries of the cartilage. The ceratohyal cartilage appears to be completely missing and the distal portions of the palatoquadrate seem to be inverted, which also appears to lead to a compression and misshape of the Meckle's cartilage. The shape of the head is also amiss; it is square like in appearance instead of oval shaped. The phenotype has been confirmed in several females of an outcross population, but few animals remain due to prolific disease death.
(Semistoned) Disorganized otoconia
The saccular and utricular otoliths are not discreetly formed and appear somewhat randomly formed. The phenotype shows variability in that some phenotypic animals may be missing a portion of the saccular otolith, while others may be missing a part of the utricular otolith, whilst others still may be missing some portion of both.
The animals suffering this phenotype show gross edema of the entire body. They are missing the telencephalon and perhaps part of the diencephalon. These animals are always cyclopic and are missing most if not all the anterior structures such as mouth and nasal placodes. They do not appear to have otic vesicles and certainly the otoconia are missing. The gut is mostly differentiated, but the heart is only tube like with one bulbous chamber. The tail is truncated and the animal suffers from scholiosis. The kidney vesicles are enlarged. No part of this animal seems to be normal, not even the anus which appears to be closed off.
Appears the brain did not divide into the correct hemispheres. Phenotypes range from mild midline fusion in which the eyes and nasal placodes are closer to the midline than normal, moderate midline fusion in which the optic stalks are fused, to the severe form in which the eyes and nasal placodes are completely fused and in many cases the telencephelon is missing. This appears as background in many natural matings but has been isolated in gynogenetically in several of the F1 offspring of the male mutagenized with two treatments of ENU (ENU 2 x 100 pit tag #500F1D5F4F). There is a possibility that this phenotype also has a heterozygous form exhibited in the wild type outcross of one such female. These potential heterozygous animals appear to have some sort of sensory deficiency that causes them to swim into the walls of the aquarium, have trouble finding their food and they are unresponsive to external stimuli, such as a hand moving towards them, until they are physically touched. I've tried to test the heterozygous females and all the offspring had severe gastrulation defects.
Pinhead (incomplete division of brain, missing telencephalon)
Also isolated from an F1 of the ENU 2 x 100 male. The head is diamond shaped instead of oval. The eyes are very close in proximity or fused. A proboscis like structure is extending from the brain region directly above the eyes. This maybe similar to oep or cyc in zebrafish or may be related to a condition called holoproencephaly caused by a mutation is shh in humans. The founder animal died prior to the creation of a proper outcross carrier population.