Random mutagenesis of proximal mouse chromosome 5 uncovers predominantly embryonic lethal mutations

Abstract

A region-specific ENU mutagenesis screen was conducted to elucidate the functional content of proximal mouse Chr 5. We used the visibly marked, recessive, lethal inversion Rump White (Rw) as a balancer in a three-generation breeding scheme to identify recessive mutations within the approximately 50 megabases spanned by Rw. A total of 1003 pedigrees were produced, representing the largest inversion screen performed in mice. Test-class animals, homozygous for the ENU-mutagenized proximal Chr 5 and visibly distinguishable from nonhomozygous littermates, were screened for fertility, hearing, vestibular function, DNA repair, behavior, and dysmorphology. Lethals were identifiable by failure to derive test-class animals within a pedigree. Embryonic lethal mutations (total of 34) were overwhelmingly the largest class of mutants recovered. We characterized them with respect to the time of embryonic death, revealing that most act at midgestation (8.5-10.5) or sooner. To position the mutations within the Rw region and to guide allelism tests, we performed complementation analyses with a set of new and existing chromosomal deletions, as well as standard recombinational mapping on a subset of the mutations. By pooling the data from this and other region-specific mutagenesis projects, we calculate that the mouse genome contains approximately 3479-4825 embryonic lethal genes, or about 13.7%-19% of all genes.

Figure 1.

Chr 5Rw Inversion screen. Male C57BL/6J mice (generation G₀) were treated with ENU to induce point mutations (*) and mated to C3H-Rw/+ females (the Rw inversion is designated as a thick, striped bar). G₁ offspring inheriting Rw (white spotting on hindquarters) were selected and crossed to mice carrying Rw in trans to Hm, a semidominant mutation causing webbed digits. This permits visual selection of G₂ progeny that are Rw/+* (they have white spotting, but no webbed digits). G₂ animals were intercrossed to produce the three types of offspring shown. Rw/Rw embryos die in utero. Non-Rw G₃ animals are homozygous for the ENU-exposed Chr 5, and were screened for phenotypes.

Figure 2.

Phenotypes of embryonic lethal mutants. Shown are images of whole-mount (A–F,H) embryos. Except for G and H, age-matched control (wt) embryos are at left, alongside corresponding mutants (mut) at right. (B) L5Jcs2 at E9.5. The arrowhead highlights a defective placenta. (D) L5Jcs11 at E8.5. (F) L5Jcs15 at E12.5. The arrowhead is pointing to a ballooned pericardium. (H) L5Jcs32 at E10.5. The arrow is highlighting the defect in neural tube closure, and G is a hematoxylin+eosin-stained transverse section obtained from the same embryo. The black arrows on the left (dorsal) indicate the open neural tube, and the asterisks mark the telencephalic vesicle. (I,J) Alcian blue and alizarin red-stained E19.5 L5Jcs1/L5Jcs1 embryos showing overgrowth of the eighth rib attaching abnormally to the 6th sternebra. The white or black bars indicate 1 mm.

Figure 3.

Map locations of lethal mutations. The proximal region of Chr. 5 is depicted as a horizontal line, with the centromere (filled circle) on the left. The region spanned by the Rw inversion is indicated above; its breakpoints are within Dpp6 proximally and near c-kit distally. Map positions (in megabases, accordingly to mouse genome Build 33) are indicated. Microsatellite loci are abbreviated by exchanging the prefix “M” for “D5Mit.” Deletions are indicated as horizontal rectangles, either solid or striped, and are color coded with the locus at which they were induced (red) Dpp6; (blue) Hdh; (green) Qdpr. The amount of DNA known to be absent in each deletion is spanned by the rectangles. The thin lines extending from the ends of the rectangles indicate the regions in which the deletion breakpoints reside. Those deletions that have been converted into stocks of mice are represented by solid rectangles, and associated allele names are given (the bracketed text corresponds to superscripting). New deletions at the Qdpr locus existing only in ES cells are represented by the striped or unfilled rectangles. In this latter case, the number of independent ES cell lines containing a particular class of deletion (currently indistinguishable with the markers used in this study) is indicated on the right. The intervals containing certain lethal mutations (abbreviated as “L. #”) are bracketed at the bottom of the map. Those that were deletion mapped are color coded. Those that were recombination mapped are in black. The yellow-shaded “ES cell haplolethal zone” is an interval that connot be deleted in ES cells. Originally described by Schimenti et al. (2000), its proximal end has been refined as described in the text, and the distal end was also refined by further analysis of deletions centered at the Gabrb1 (data not shown). (Shh) Sonic hedgehog homolog.

Figure 4.

The Beh5Jcs1 mutant. (A) Activity level in the hole board is presented for C57BL/6J (B6) (n = 16), C3H/HeJ (n = 9), B6xC3H F1 (n = 25), nonaffected G₃ progeny (n = 118), Rw/+* (n = 5) and +*/+* test class Beh5Jcs1 littermates (n = 10). There is significant difference between nonaffected G₃ and Beh5Jcs1 +*/+* using the two-tailed t-test (P-value 0.0018). (B) Genetic mapping of the activity phenotype in 71 F₂ progeny from a Beh5Jcs1+*/+ X Beh5Jcs1+*/+ intercross using seven microsatellite markers (abbreviated by exchanging the prefix “M” for “D5Mit”) along Chr 5: M146(8.5Mb), M387(26.6Mb), M183(52.6Mb), M201(74.5Mb), M309(78.2Mb), M312(93.7Mb), M10(101.6Mb), M314(108.4Mb), and M95(123.5Mb). Black bars represent values observed in mice homozygous for C57BL/6J (B/B), white bars for homoygous C3H/HeJ loci (C/C), and striped heterozygous for the two strains (B/C). The significant P-values from the two-tailed t-test for M10 B/B vs B/C and B/B vs C/C (n = 37) are 0.0027 and 0.0006, respectively.