A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

Abstract

Background: ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome.

Results: We isolated 11 lethal lines that map to the region of chromosome 4 between D4Mit117 and D4Mit281. These lines form 10 complementation groups. The majority of lines die during embryonic development between E5.5 and E12.5 and display defects in gastrulation, cardiac development, and craniofacial development. One line displayed postnatal lethality and neurological defects, including ataxia and seizures.

Conclusion: These eleven mutants allow us to query gene function within the distal region of mouse chromosome 4 and demonstrate that new mouse models of mammalian developmental defects can easily and quickly be generated and mapped with the use of ENU-mutagenesis in combination with balancer chromosomes. The low number of mutations isolated in this screen compared with other balancer chromosome screens indicates that the functions of genes in different regions of the genome vary widely.

Figure 1

Chromosome 4 (117–281) Balancer Screen. A. The balancer regions used in this screen (red) and in a previously published screen (dark gray) are shown. Genotyping microsatellite markers are shown in red. Base pairs refer to Ensembl v51. B. The mating scheme used for the chromosome 4 (117–281) balancer screen is shown. Red or "m" indicates a new ENU-induced lesion and black indicates the balancer chromosome. Males are depicted with squares and females are depicted with circles. Diamonds represent either sex. C. The mouse chromosome 4 balancer is depicted with a red line. Microsyntenies, representing 50–800 bp fragments and averaging 100–200 bp, were blasted and aligned to full-length human chromosome 1, depicted with a blue line. Light blue lines connect each hit of microsynteny conservation between the two chromosomes. Two large areas of linkage conservation are evident on the human chromosome. D-G. Homozygous mutants, heterozygous balancer animals, and homozygous balancer animals are all easily distinguished by coat color and the presence or absence of eye pigment at several different stages. A homozygous lethal embryo (no eye pigment) is pictured at E15.5 (D, right) next to a control littermate (D, left). An albino mutant pup lacking eye pigment is easily distinguished from control littermates at P3 (E, far right). Homozygous mutants (white, no eye pigment), heterozygous balancer animals (light brown), and homozygous balancer animals (dark brown) are shown at both the P11 (F) and adult stages (G) and can easily be differentiated by coat color.

Figure 2

Chromosome 4 (117–281) Lethal Mutants. Mutant embryos are pictured on the right and littermate controls are pictured on the left. All of the images of mutants and control from the same line were taken at the same magnification except D in which the mutant was photographed at a higher magnification in order to better visualize the allantois. A. Hematoxylin and Eosin stained sections of a presumed l4Jus29 homozygous mutant (right) and control littermate (left) are pictured at E7.5. The mutants die prior to gastrulation. Because of lack of embryonic tissue, these mutants were not genotyped but were identified by abnormal histology. B. l4Jus26 at E8.5. Mutants are smaller than control littermates and exhibit developmental delay. C. l4Jus25 at E10.5. Mutants display cardiovascular defects and are smaller than control littermates. Pericardial edema is evident in the mutant and is indicated with a white arrow. D. l4Jus28 at E9.5. Mutant embryos have not turned and the allantois grows but does not fuse with the chorion, shown with a white arrow. E. l4Jus30 at E11.5. Mutant embryos are smaller than control littermates and exhibit growth defects, as well as abnormal telencephalon development.

Figure 3

l4Jus31 mutants are smaller than control littermates. A. The homozygous mutant is confirmed by the white coat color and is pictured on the right. The heterozygous littermate, with a light brown coat color, is pictured on the left. The picture was taken at postnatal day 28. B. l4Jus31 homozygous mutants are significantly smaller than their control littermates at P14 (Student's t-test, n = 5 mutants, 4 control animals, p < 0.001).