Mks1 mutants show multiple anomalies. Spectrum of Mks1-mutant phenotypes included preaxial polydactyly (A, arrow) and craniofacial defects –such as pointy snout (A,D), deeply recessed eyes (enopthalmia; A,D) and facial cleft (C). Also observed were complex congenital heart defects – such as transposition of the great arteries (B,E) and randomization of situs as seen in dextrocardia (B,E), symmetric midline liver (F), polysplenia (I) and other visceral organ situs defects. The boxed region in F is enlarged in G to show the cystic appearance of the liver. Skeletal defects were commonly found, such as mal-alignment of the sternal vertebrae (H, arrowheads), and kidney defects were commonly found, including enlarged and duplex kidneys (J). H, heart; Lg, lung; S, stomach; R, right; L, left; Ao, aorta; P (in panel E), pulmonary trunk; P (in panel I), pancreas; S, spleen; K, kidney; L1 and L2, left upper and lower liver lobes, respectively; R1 and R2, right upper and lower liver lobes, respectively. Scale bars in all panels are 2 mm, except in G, where it is 0.25 mm.

Mks1 mutants exhibit skeletal anomalies. (A–H). Skeletal preparations of Mks1 mutant (m/m; B,D,F,H) and littermate control (ctrl; A,C,E,G) newborn animals showed, in the mutant animal, dome-shaped skull and hypoplastic jaw (B), cleft palate (arrow, D), mal-aligned and split sternal vertebrae (arrows, F), and reduced or absent ossification of the vertebral bodies of the cervical and thoracic vertebrae (bracket, H). Arrowhead in F shows the fissure of sternal vertebrae. (I–L) Skeletal preparations showed Mks1 mutant animals with preaxial digit duplication with a triphalangeal first digit in the hind limb (arrows in K and L) and broadened thumbs in the forelimb (arrows in I and J), as well as extra carpal bone (arrowheads in K and L). ps, presphenoid; bs, basisphenoid; bo, basioccipital; LFL, left forelimb; RFL, right forelimb; LHL, left hindlimb; RHL, right hindlimb. Scale bars: 1 mm.

Scanning EM images of embryonic node cilia. Whereas the control (ctrl) embryo exhibited cells with cuboidal shaped (A), the mutant (m/m) node exhibited flat epithelial cell morphology (B). Cilia in the control node projected posteriorly (arrows in C) from the posterior hemisphere of nodal cells but, in Mks1-mutant embryos, node cells exhibited mostly amorphous protrusions (circle in D) and, when cilia were observed, they were shorter (10–30% of length in control node) and were randomly oriented (arrowheads in D–F). Arrows in C–F point to the roots of cilia, with the arrow aligned with the direction of cilium-projection. The two-ended arrow in panel A indicates the orientation of images in all panels, with posterior end of the embryonic node pointing down and anterior end pointing up. Scale bars: 20 μm (A,B); 2 μm (C–F).

Scanning EM shows defects in patterning of cochlear hair cells in Mks1 mutants. In newborn wild-type (ctrl) cochlea (A), three rows of OHCs were aligned in parallel rows (arrows in A) but, in the Mks1 mutant animal (m/m; B), some OHCs were mal-positioned, with abnormal orientation of the stereocilia hair bundles. Higher magnification (C–F) showed that kinocilia (black arrows) in mutant hair cells (D–F) were of normal length compared to controls (C), but they were abnormally localized relative to the stereocilia hair bundles.

Analysis of limb patterning defects by in-situ hybridization analysis. Shh, Gli3 and dHand transcripts showed normal distribution in E10.25 (Gli3, dHand; B,C,G,H) and E10.5 (Shh; D,I) mutant (m/m) limb buds. Gli1 (A,F) and PtC1 expression were lower at E10.5 (supplementary material Fig. S2) in mutant compared with control embryos. By E10.75–E11.0, PtC1 expression recovered, but was still reduced in the posterior limb in the mutants compared with controls (E,J). At this stage, a region of ectopic PtC1 expression was observed anteriorly in the mutants (arrowhead, J), and this was accompanied by the anterior expansion of Fgf8 (K,P) and Fgf4 (L,Q) (as indicated by paired arrowheads; the arrows indicate the anterior and posterior proximal limits of the limb buds), and gremlin (M,R) expression (top arrowheads). At E12.5, mesenchymal condensations, delineated by Sox9 expression (N,O,S,T), showed broadening anteriorly in the forelimb, corresponding to the region of the presumptive first digit (arrowhead in S), whereas, in the hindlimb, a more prominent extrusion could be seen, also at the position of the presumptive first digit (arrowhead, T). LFL, left forelimb; RFL, right forelimb; LHL, left hindlimb; RHL, right hindlimb. Scale bars: 400 μm.

Mks1 mutants exhibit kidney cysts and defects in ciliogenesis. (A,B) Hematoxylin and eosin (H&E) staining of paraffin sections from E18.5 wild-type (ctrl; A) and Mks1 mutant (m/m; B) animals showed the presence of prominent glomerular and tubule cysts (‘c’) in the mutant animal kidney cortex. Arrows mark glomerular tufts. (C,D) At E18.5, immunostaining with antibody to IFT88 (green) to delineate the cilia (arrows in C,D) showed that cilia were shorter in epithelial cells of mutant glomerular capsule (D) as compared to that of control (C). γ-tubulin (red) was used to delineate the basal body. (E,F) Cilia were less abundant in E17.5 mutant (F) collecting ducts as compared to control (E). The collecting duct was stained with DBA (green) and cilia were stained with anti-acetylated-tubulin antibody (red). Note the presence of cilia (arrow) in the lumen of the control (E) but not the mutant (F) ducts. Images were maximum projections of confocal z-stacks. (G–J) Sections of the kidney from control wild-type (G,I) and mutant (H,J) embryos were immunostained with antibodies to γ-tubulin (red) and IFT88 (green). Arrows denote the opposing apical surfaces of the unilaminar kidney tubule epithelia where the centrioles are localized in the control and mutant kidney tubules. The same regions denoted by arrows in G and H are digitally enlarged in I and J. The enlarged panels show that cilia are abundant in the control sample (I), but very few cilia are observed in the mutant kidney tubule (J). (K,L) Cilia (arrow) were shorter in mutant MEFs (L) as compared with control wild-type MEFs (K). The tip and basal body were stained with IFT140 (green) and the ciliary axoneme was stained with anti-acetylated-tubulin antibody (red). (M) Quantitation of cilia observed in the MEF cultures and kidney sections showed significant reductions in the percentage of ciliated cells in the Mks1 mutant kidney (P<0.0001) and mutant MEFs (P<0.0001). Scale bars: 100 μm (A,B); 10 μm (C–H); 5 μm (I,K,L).

Recovery and analysis of an in-frame deletion mutation of Mks1. (A) Schematic of the mouse Mks1 gene, containing 18 exons, with the deleted genomic region highlighted, which corresponds to 5226 bases spanning exons 3–10. The sequencing trace file (not shown) from sequencing the genomic DNA (left trace file) showed that the sequence from intron 2 is contiguous with intron 10, whereas sequencing of the cDNA (right trace file) showed that the sequence from exon 2 is contiguous with exon 11 (mRNA). (B) Diagram showing the structure of wild-type and mutant Mks1 protein, with the region deleted delineated in yellow highlight. The in-frame deletion generates a truncated Mks1 protein with amino acid residues 64–323 deleted. (C) Western immunoblotting shows specificity of the anti-Mks1 antibody. Cell lysates from stably transfected HEK293 cells expressing N-terminal FLAG-tagged wild-type (Wt) or mutant (Mt) Mks1 protein were analyzed by western immunoblotting using two-color westerns with an anti-FLAG (green) and anti-Mks1 (red) antibodies. Cells expressing the FLAG-tagged wild-type Mks1 protein exhibited a 67-kD band (upper arrow) that was detected by both the anti-FLAG (green) and -Mks1 (red) antibodies (see merged panel), whereas a slightly smaller 64-kD band (lower arrow), the size expected for endogenous wild-type Mks1, was detected by anti-Mks1 antibody in nontransfected and transfected cells. In cells transfected with the mutant FLAG-tagged Mks1 construct, in addition to the expected 64-kD band, a lower 34-kD band was detected by both the anti-Mks1 and -FLAG antibodies (arrowhead), the size expected for the truncated mutant Mks1 protein. (D,E) Immunostaining of wild-type (D) and Mks1 mutant (E) MEFs with anti-γ-tubulin (green) and -Mks1 (red) antibodies showed that the mutant Mks1 protein is abnormally localized. In wild-type MEFs, γ-tubulin is localized in two punctate dots consistent with centrosome localization, with one showing colocalization with Mks1 (see arrowhead in D). However, in the mutant MEFs, Mks1 staining (red), although still localized in a punctate dot, is not colocalized with the γ-tubulin (green)-containing centrosomes (E, arrowhead). The inset in D of a wild-type MEF shows that Mks1 (red) is colocalized with γ-tubulin (green) and is in the centriole associated with the primary cilium, which is delineated with anti-acetylated-tubulin antibody staining (blue). By contrast, in the mutant MEFs, Mks1 (red) is not colocalized with γ-tubulin (green), either when the cilium is absent (left inset in E), or in rare instances where a cilium (blue)-like projection is observed (right inset in E). Panels D and E are at the same scale; scale bar in D: 5 μm. (F,G) IMCD kidney cells transfected with plasmids encoding GFP-tagged wild-type Mks1 (F) and mutant Mks1 (G) proteins. GFP-tagged wild-type Mks1 protein showed localization at the base of the cilium (F), but this was not observed with GFP-tagged mutant Mks1 protein (G). The cilia (arrowheads) were delineated with anti-IFT88 (G) or -IFT27 (F) antibodies. Panels F and G are at the same magnification; the scale bar in panel F: 5 μm.

Scanning EM shows cilia abnormalities in the neural tube. Scanning EM of an E10.5 embryo (A) was used to visualize cilia in the neuroepithelium. Magnified views (B–E) show cilia in the ventral groove (B,C) and lateral wall (D,E) of the hindbrain. Cilia in the ventral groove in control (ctrl) embryos were longer (2–3 μm; B, arrows) than those on the lateral wall (0.5–1.0 μm; D, arrows). In the Mks1-mutant (m/m) embryo (C,E), most cells showed only small globular protrusions (circles; see inset in C showing 200% enlargement of boxed region). Nevertheless, fewer cilia were observed in the mutant embryo (C, top-right arrow; E, top-right arrow) than in the control embryo (arrows in B,D). (D,E) Direction of arrows denotes orientation of cilia projection. Scale bars: 400 μm (A); 5 μm (B,C); 2 μm (D,E).

Dorsoventral neural tube patterning defects in Mks1 mutants. Expression of dorsoventral markers in the neural tube anteriorly at the level between the forelimb and hindlimb (A–G) and posteriorly, at the hindlimb level (H–N), were examined by immunohistochemistry of wild-type (ctrl) and Mks1-mutant (m/m) embryo cryosections. Note that the figure includes panels with top-bottom and left-right side-by-side placement of control and mutant embryo sections. (A–G) Anteriorly, the floor plate in Mks1 mutants was specified (A,B), even though Shh expression was markedly reduced compared with control (A,B). Thus, the floor plate marker FoxA2 (B) and also Nkx2.2 (E) were expressed ventrally, but at lower levels. As observed in the control, expression of Olig2 (C) and Pax6 (E) in the mutant embryo was separated by the floor plate. The dorsal limit of Olig2 (C) and Nkx6.1 (D) and ventral limit of Pax7 (G) were observed to shift dorsally. (H–N) At the posterior level, the floor plate was not specified and ventral neural tube exhibited an abnormal thickened morphology in mutants. Thus, neither Shh (H) nor FoxA2 (I) expression was detected in the mutants, and only a few cells expressed Nkx2.2 (M). Olig2 (J) and Pax6 (M) expanded ventrally (arrows in J and M) in mutants, whereas the dorsal limits of Olig2 (J), Nkx6.1 (K) and the ventral limit of Pax7 (N) were at almost the same level as in control (denoted by arrowhead in J, K, N). (A,B,H,I) The apical and basal surfaces of the ventral neural tube are delineated with white dashed lines. Arrowheads indicate the dorsal or ventral limits of expression of the respective markers. Scale bar: 50 μm.