Dach1 mutant mice bear no gross abnormalities in eye, limb, and brain development and exhibit postnatal lethality

Abstract

Drosophila dachshund is necessary and sufficient for compound eye development and is required for normal leg and brain development. A mouse homologue of dachshund, Dach1, is expressed in the developing retina and limbs, suggesting functional conservation of this gene. We have generated a loss-of-function mutation in Dach1 that results in the abrogation of the wild-type RNA and protein expression pattern in embryos. Homozygous mutants survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress. The heart, lungs, kidneys, liver, and skeleton were examined to identify factors involved in postnatal lethality, but these organs appeared to be normal. In addition, blood chemistry tests failed to reveal differences that might explain the lethal phenotype. Gross examination and histological analyses of newborn eyes, limbs, and brains revealed no detectable abnormalities. Since Dach1 mutants die shortly after birth, it remains possible that Dach1 is required for postnatal development of these structures. Alternatively, an additional Dach homologue may functionally compensate for Dach1 loss of function.

FIG. 1

Dach1 knockout strategy and genotype analysis. (A) Dach1 targeting strategy. The top line shows the wild-type 5′ Dach1 region with exon 1 (white box) and intron 1 sequences (thin line) flanked by 5′ and 3′ recombination arms (grey boxes). The middle line shows the Dach1 knockout construct designed for homologous replacement of a SacI site, exon 1, and a 5′ portion of intron 1 with PGK-Hprt sequences. The resultant Dach1 mutant allele is illustrated on the bottom line. Positions of genotyping primers p1, p2, and p3 are shown as arrows below the wild-type and mutant allele diagrams. The line below the asterisk represents additional sequences, derived from the targeting vector, inserted at the Dach1 locus which were associated with two independently isolated homologous replacement events (Materials and Methods). pBS, pBluescript; HSV tk, herpes simplex virus thymidine kinase gene. (B) PCR genotype analysis of tail DNA. An ethidium bromide-stained agarose gel containing amplification products from wild-type (+/+), heterozygote (+/−), and homozygote (−/−) tail DNA is shown. Primer combination p1-p3 detects wild-type alleles, while primers p2 and p3 detect mutant alleles (Materials and Methods). (C) Genotype analysis of newborn mice. Tail DNA was collected from 151 newborn mice (21 intercross litters) and analyzed by PCR. The numbers and corresponding percentages of wild-type (+/+), heterozygote (+/−), and homozygote (−/−) mutant animals are shown. Tails from these mice were taken less than 2 h after birth. Homozygotes may be slightly underrepresented since they may be eaten prior to detection.

FIG. 2

Targeted disruption of Dach1 exon1 is associated with abrogated RNA and protein expression. (A) Southern analysis of the Dach1 locus. Wild-type (+/+), heterozygote (+/−), and homozygote (−/−) newborn tail DNA was digested with SacI, subjected to gel electrophoresis, and transferred to a nylon membrane. The filter was hybridized to a Dach1 exon 1 probe (Fig. 1A), washed, and exposed to film. Subsequently, the filter was stripped and hybridized to a 3′ Dach1 probe (Materials and Methods). (B and C) Whole-mount in situ hybridization of E10 wild-type and homozygous mutant embryos. A Dach1 antisense riboprobe hybridizes to transcripts in the eye, limbs, neural tube, and brain in wild-type embryos (B), while this pattern is not detected in homozygous mutants (C) (11). This riboprobe corresponds to coding sequences located downstream of exon 1 and therefore is not deleted in the mutant allele. Tails were removed from the animals for PCR genotyping after color development. (D and E) Immunohistochemical analysis of E12.5 wild-type and homozygous mutant eye sections. A Dach1 antibody demonstrates protein expression within the peripheral retina, retinal pigmented epithelium, and developing cornea in wild-type eyes (D), while Dach1 staining is reduced to background levels in the homozygous mutant eyes (E). Lens and surface ectoderm staining is due to background staining of the secondary antibody.

FIG. 3

Gross and histological analyses of newborn wild-type (+/+) and Dach1 homozygous mutant (−/−) mice. (A and B) H&E staining of eye sections. The retina, optic nerve, lens, retinal pigmented epithelium, cornea, and ciliary margin are present and appear to be normal in newborn Dach1 homozygous mutant eyes. (C and D) High magnification of H&E-stained retinas. Dach1 mutant retinas exhibit a similar degree of layering and cell numbers as newborn wild-type retinas. (E and F) The external dimensions and morphology of wild-type and Dach1 mutant legs and digits are similar. (G and H) Alcian blue-alizarin red staining of wild-type and homozygous mutant skeletons does not reveal any detectable differences in either bone or cartilage morphology. The homozygote right forelimb was detached to reveal the structure of the limb. (I and J) H&E-stained coronal sections of newborn brains showing cortical layers, hippocampi, and trigeminal ganglia (arrow) in both wild-type and homozygous mutant animals. (K and L) High magnification of H&E-stained neocortex from newborn mice. The marginal zone, cortical plate, subplate, and intermediate zone of the cortex can be seen in newborn Dach1 mutant brains. (M and N) Lungs of wild-type and homozygous mutant newborn animals do not show any detectable differences in airway branching, alveolar septation, spacing, and cellular composition. (O and P) Whole-mount in situ hybridization of newborn lungs using a Clara cell-specific riboprobe, CC10. Epithelial cells lining the terminal bronchioles with similar branching patterns are found in both wild-type and homozygous mutant lungs.