Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice

Abstract

We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48 Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.

Keywords: Dnaaf1/Lrrc50/Oda7; ENU mutagenesis; Lrrc48/FAP134/Drc3; motile cilia; primary ciliary dyskinesia.

Figure 1

Mutations in Dnaaf1 and Lrrc48. (A) A chromatogram from Sanger sequencing showing a T > C mutation (asterisk) at the junction of exon 4 and intron. (B) RT-PCR result showing amplification of a fragment corresponding to exon 3–6 of Dnaaf1 (lanes 1 and 2). Multiple bands with incorrect sizes were detected in Dnaaf1^m4Bei, indicating a splicing defect. Cloned RT-PCR products revealed abnormal transcripts with exon 4 skipping (lane 4, insert size 279 bp) or intron 4–5 insertion (lane 5, insert size 617 bp). The largest band (nonspecific) was not a Dnaaf1 transcript. (C) A chromatogram from Sanger sequencing showing a T > C mutation (asterisk) causing an amino acid change (Leu89Pro). (D) Multiple protein sequence alignment adapted from NCBI HomoloGene search result, including conserved leucine-rich repeat domains near the mutation. Leucine residues in the domain are highlighted in yellow. The leucine residue substituted in Lrrc48^m6Bei mutants is shown in red (asterisk). Other conserved residues are marked in blue. (E) RT-PCR results using three different primer sets spanning the Lrrc48 transcript. All amplified weaker bands from the mutant. Actb (Beta-actin) was used as a control. NCBI, National Center for Biotechnology Information; NTC, no template control; RT-PCR, reverse transcription polymerase chain reaction.

Figure 2

Hydrocephalus phenotype. Nissl-stained images of coronal sections are shown. Enlarged lateral ventricles are marked with asterisks. Enlargement of the third ventricles is also evident in both mutants. Dotted lines in Dnaaf1^m4Bei mutant images indicate disruption in ependymal lining. Double-headed arrows indicate transependymal edema. Scale bars, 1 mm.

Figure 3

Situs inversus phenotype. The position of the heart (H), lung lobes (L, Cr, M, Ca, and A), stomach (St), and spleen (Sp) shows a laterality defect. Arrows indicate the angle of pulmonary trunk. A, accessary lobe; Ca, right caudal lobe; Cr, right cranial lobe; L, left lobe; M, right middle lobe.

Figure 4

Mucus accumulation in the sinuses. (A–D) Coronal sections of the maxillary sinuses (ms) from mutant and littermate wild-type mice are stained using hematoxylin and eosin. Higher magnification images of the boxed region (a–f for wild-type and a’–f’ for mutants) are shown in lower panels. e and e’ are from more posterior paranasal cavity sections. Severe mucus accumulation and infiltration of neutrophils (c’) were obvious in a Dnaaf1^m4Bei mutant at P7. Lrrc48^m6Bei mutants show milder phenotype that is undetectable until later (16 week-old). Scale bars: A–D, 1 mm; a, a’, b, b’, d’, e, and e’, 100 μm; c, c’, f, and f’, 25 μm.

Figure 5

The presence of cilia in the mutants. Cilia in ependyma and choroid plexus of the brain, upper airway, and lung were visualized by anti-ARL13B immunostaining (gray or green). The nuclei were stained using Hoechst (blue). Red arrows indicate representative cilia. Scale bars: ependyma, choroid plexus, and lung, 10 μm; upper airway, 50 μm.

Figure 6

Normal localization of DNAH9 in the mutants. Anti-ARL13B (green, middle) and anti-DNAH9 (red, right) coimmunostaining was performed. DNAH9 is distally localized in the ciliary axoneme of the motile cilia in the upper airway. The nuclei were stained using Hoechst (blue). Scale bars, 5 μm.