Figure 1.
Breeding strategy for the generation of G3 embryos.
C57BL/6J male mice were injected with ENU and bred with females of the inbred mapping strain, C3H/HeH (C3H). +/+ = wildtype, m = mutation, G = generation.
Table 1.
Summary of ENU-induced mouse mutants.
Figure 2.
Craniofacial and skeletal mutants.
(A–D) 11BC-5 (blood filled blisters; bfb), (E–G) 11BC-3 (cauli), (H–J) 12WT-49 (kanyon), (K–M) 12WT-9 (snoopy). bfb mutant at E13.5 (A) and E18.5 (B–D) exhibiting characteristic haemorrhagic blisters over the eye, side of the head and feet (arrows). The foot blisters can be discrete or distended as in (C) but are typically associated with digit malformations including polydactyly. The blisters over the eye are commonly associated with open eyelids (D). Cauli embryos at (E) E13.5 and (F) E16.5 present with exencephaly (asterisk) and polydactyly (arrow). (G) Fore- (FL) and hindlimbs (HL) of an E13.5 cauli embryo illustrating the variable autopod phenotype in the forelimbs. Kanyon embryos (H-J) frequently present with exencephaly (H, asterisk) and midfacial clefts. Clefts may result from a defect of frontonasal process development such that the maxillary and frontonasal processes (arrowheads) completely fail to fuse (I) or may present as bilateral cleft lip and palate (J) in mild cases. Regardless of the severity of the facial cleft, the eyes never develop normally (I, J). Snoopy embryos (K-M) present with forebrain malformation, poor eye development and mandibular hypoplasia/agnathia (arrow). (L, M) The forebrain often fails to divide into two vesicles (asterisk) and is associated with various degrees of hypotelorism (M).
Figure 3.
(A) Based on the phenotype, a targeted approach was used to establish linkage to Fras1 in the bfb strain. Polymorphic markers flanking the four previously characterised blebs genes (open boxes) were used to identify a candidate for sequencing. Strong linkage was observed with the Fras1 gene (grey box). (B) A genome-wide scan established linkage to chromosome 17 in the cauli strain. Chromosome 17-specific markers were then used to refine linkage to a 7 Mb region between rs3667809 and rs3684506 (grey boxed region) containing 218 genes. No additional informative markers were available to further refine the linkage region. (C) A genome-wide scan established linkage to chromosome 7 in the 12BCC-22a line. Subsequent analysis using chromosome 7-specific markers refined linkage to a 32 Mb region containing approximately 700 genes. Numbers to the left in each diagram denote individual mutant embryos except in (C) where both mutant and unaffected embryos are shown. Black boxes denote homozygous C57BL/6 alleles, grey boxes denote C57BL6/C3H heterozygosity, white boxes denote untested markers.
Figure 4.
(A) A Lig1 mutant (mut) with pale orange liver (arrow) compared to the red haemoglobin rich liver of a wildtype (wt) littermate. (B, C) Analysis of peripheral blood in E13.5 embryos by cytospin and Giemsa staining (x40). (B) Lig1 mutant (mut) peripheral blood shows a high proportion of nucleated erythroblasts (arrow) to enucleated erythroblasts (arrowhead) compared with (C) a wild type (wt) littermate.
Figure 5.
Testis cord defects in line 12BCC-20 at E13.5.
Immunofluorescence staining for the Sertoli cell marker AMH (red, cytoplasm) on testis sections from unaffected (XY control) and affected (XY mutant) embryos. In affected embryos, testis cords are less organized when compared to unaffected embryos. Insets: Gross-examination of testes from unaffected and affected embryos. In affected embryos, testes are smaller and testis cords are barely detectable. Arrows indicate testis cords. All scale bars are 50 µm.
Figure 6.
Urinary tract anomalies displayed by lines 12BC-19, 12BC-20 and 14BC-07.
12BC-20 (A–C) (A) Unilateral and (B) bilateral dilation of the renal pelvis (*). Kinks (A) and twisting (B) of the ureter (arrow) distal to the dilation. (C) Ureter bifurcation at the site marked with a line. Single ureter (arrow) below and bifurcated ureter (arrows) above the line. 12BC-19 (D–F) (D) Unilateral renal agenesis. (E) Bilateral hydronephrosis. (F) Bilateral hydroureter and hydronephrosis. 14BC-07 (G–I) (G) Dilated renal pelvis (*). (H) Duplicated ureter with hydroureter in one ureter leading to hydronephrosis in upper pole. (I) Unilateral renal hypoplasia (lines marking top and bottom of kidneys). a = adrenal, u = ureter.
Figure 7.
Lung anomalies displayed by lines 12BCC-013 and 12BCC-016.
Lung histology in line 12BCC-013 (A, B) and 12BCC-016 (C, D). Affected lungs in lines 12BCC-013 (B) and 12BCC-016 (D) both displayed hypercellular, thickened lung mesenchyme (m) which can be clearly seen by the increased pink/purple staining of cells and their nuclei, between the small airways (a) in B and D compared to that in the lungs of unaffected littermate embryos (A and C), respectively.