Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling Jeune syndrome

Abstract

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.

Figure 1. An Ift140 mutation is responsible for the ciliopathic phenotype observed in cauli.

Representative E13.5 wildtype (A) and mutant (B) embryos showing exencephaly (black arrowhead), open mouth (white arrowhead), polydactyly (asterisks) and caudal neural tube closure defects (arrow) in mutants. Chromatogram of cauli mutant showing the homozygous missense mutation (c.2564T>A) in the Intraflagellar Transport Protein 140 (Ift140) gene (C). IFT140 protein alignment showing the isoleucine to lysine substitution at position 855 of the protein in cauli and the corresponding amino acid across several species (D). Schematic of the IFT140 protein detailing protein domains, location of Ift140^cauli/cauli mutation and reported human mutations (E). Mainzer-Saldino (black), Jeune asphyxiating thoracic dystrophy (red), ⁺compound heterozygous, ^#homozygous ^∧no second mutation identified. Black box represents mutation reported in this paper. Primary cilia from E10.5 Ift140^+/+ (F) and Ift140^cauli/cauli (G) limb buds show a severely altered cilia morphology in the mutant.

Figure 2. Ift140^cauli/cauli embryos exhibit skeletal, somite and neural tube defects.

Morphological and expression analysis of Ift140^+/+ and Ift140^cauli/cauli embryos. Lateral view of E16.5 skull in Ift140^+/+ (A) and Ift140^cauli/cauli (B) embryos. Ift140^cauli/cauli embryos exhibit fusion of the exoccipital bone and C1/C2 vertebrae (arrow in B). Ventral view of skull base in Ift140^+/+ (C) and Ift140^cauli/cauli (D) embryos. Ift140^+/+ (E) and Ift140^cauli/cauli (F) mandibles. The normal organisation of the ribs seen in E16.5 Ift140^+/+ embryos (G) is severely disrupted in Ift140^cauli/cauli (H) with lateral branching (asterisk), thickened ossified nodules (red arrow) and abnormal costovertebral articulations (red arrowhead). (I–P) In situ hybridisation of gene expression patterns of myogenin (I–L), Msx1 (M,N) and Sox9 (O,P). Myogenin staining at E11.5 reveals the highly ordered segmental structure of a Ift140^+/+ embryo (I) while in the Ift140^cauli/cauli embryo (J) myogenin staining reveals the presence of disorganised and branched somite-derived structures (myotome; arrow). (K,L) Sections of wholemount embryos at the level indicated by the horizontal line in I and J, illustrating the loss of segmental myogenin staining and the accumulation of blood within distorted and irregular intersomitic vessels (arrowheads) in Ift140^cauli/cauli embryos. (M,N) Msx1 expression delineates the dorsal margin of the neural tube in an E11.5 Ift140^+/+ embryo (M) but highlights the disrupted neural tube structure in an Ift140^cauli/cauli embryo (arrowhead in N). In addition, the neural tube is convoluted and irregular in appearance, as shown in E12.5 embryos stained for Sox9 (arrow in P). PMX, premaxilla; MD, mandible; MX, maxilla; P, palatine; PP, palatal process; AL, alisphenoid; BS, basisphenoid; TR, tympanic ring; BO, basioccipital; EX, exoccipital; C1/C2, fused 1^st and 2^nd cervical vertebrae.

Figure 3. Ift140^cauli/caul mutants show palate defects, hydrops fetalis and malformation of the lungs and heart.

Coronal sections of E13.5 Ift140^+/+ (A) and Ift140^cauli/cauli (B) palates, highlighting hypoplastic palatal shelves in Ift140^cauli/cauli embryos. Transverse sections of E13.5 Ift140^+/+ (C and E) and Ift140^cauli/cauli (D and F) embryos at the level of the kidneys (C and D) and heart (E and F). Ift140^cauli/cauli mutant embryos have grossly normal kidneys but show accumulation of fluid around the kidneys and lungs (asterisks in D and F), which is not evident in Ift140^+/+ controls (C and E). The lungs of Ift140^cauli/cauli embryos are also abnormal in shape (F), unlike the cone-shaped lobes seen in Ift140^+/+ controls (E). The atrioventricular valves of the heart are well formed in Ift140^+/+ embryos (E), but both the tricuspid and mitral valves are abnormal in Ift140^cauli/cauli mutants (F). Ift140^cauli/cauli embryos appear to have ventricular hypotrophy and the interventricular septum is not well formed (depicted by dashed line in F). An irregular accumulation of blood can also be seen in the atria and ventricles of Ift140^cauli/cauli mutants (F). T, tongue; PS, palatal shelf; K, kidneys; L, lung; RA, right atrium; RV, right ventricle; TV, tricuspid valve; MV, mitral valve.

Figure 4. Molecular signalling is disturbed in Ift140^cauli/cauli embryos.

WISH analysis of the forelimbs and hindlimbs of Ift140^+/+ and Ift140^cauli/cauli embryos. Dorsal view of fore- and hindlimb buds (A–J′), where anterior is always to the top of the image. Distal view of forelimb buds (K′–M′), where dorsal side is facing to the right of the image. Arrowhead in (C) indicates ectopic Shh expression domain. Bars in (J,L and M′,N′) indicate anterior-posterior extent of Grem1 expression. Arrowhead in (K) indicates disruption in Grem1 expression in mutant forelimb. Asterisk in (W and X) indicates elevated anterior Dusp6 expression. Arrow, arrowhead and asterisk in J′ indicate a single ectopic digit, bifid ectopic digit and proximal syndactyly respectively. A, anterior; P, posterior; D, dorsal; V, ventral; FL, forelimb; HL, hindlimb. All embryos are E11.5 except G′–J′ which are E13.5.

Figure 5. Epithelial cellular architecture and levels of Ift140 are altered in Ift140^cauli/caul mutants.

Scanning electron micrographs and immunohistochemistry of epithelia from E10.5 Ift140^+/+ (A and C) and Ift140^cauli/cauli (B and D) limb buds. The rigid cellular architecture seen in wildtype limbs is highly disrupted in the mutant, as evidenced by the lack of cilia (compare arrows in A), the presence of thick, disorganised cell junctions (arrows in D) and the more diffuse E-cadherin staining in the mutant (D). Overlay of E-cad (green) and phalloidin (red) in Ift140^+/+ (C) and Ift140^cauli/cauli (D) epithelium. Cilia counts identify a significant decrease in cilia (***p = 2.05×10⁻⁷) in limb buds of Ift140^cauli/cauli when compared to Ift140^+/+ controls (E). Ift140 can be detected at the base and tip of wildtype limb bud cilia (F) but it undetectable in the majority of mutant cilia (G). Western blot analysis shows a reduction of Ift140 protein levels in Ift140^cauli/cauli tissue when compared to control and heterozygous samples (H), and a significant reduction of Ift140 transcript levels (***p = 0.0026) in Ift140^cauli/cauli mutant embryos (I). Embryos harbouring a homozygous Ift140 null allele show a complete lack of Ift140 protein by western blot (J) and exhibit identical phenotypes to those identified in Ift140^cauli/cauli embryos (K,L), including exencephaly (white arrow), open mouth (white arrowhead) and an expanded hindlimb field (asterisk). Scale bar; 2 µM (A and B), 30 µM (C and D).

Figure 6. An IFT140 mutation identified in a Jeune Syndrome patient.

Family pedigree showing the relationship between JS1-1, JS 1-2, JS 1-3 and JS 1-4 (A; red arrow indicates the proband). Whole body (B), right (C) and left (D) upper limb and feet (E) radiographs from JS1-1. Note the short ribs, short long bones with bowed humeri and femora, “trident” appearance of the acetabular roofs and metaphyseal irregularity (B), short long bones with metaphyseal cupping and bowed humerus (C and D), and metaphyseal cupping and advanced tarsal bone ossification for age (E). A, acetabulum; F, femur; H, humerus; T, tarsus. Sequence chromatogram identifying the C>T homozygous mutation in JS1-1 (F; arrow), and heterozygous mutation in immediate family members.