A mouse mutant was recovered that exhibited a wide range of anomalies, including: cleft lip (); pointy snout (); eye defects ranging from deeply recessed eyes (enopthalmia), small eyes (microphthalmia) to no eye (anophthalmia) (); polydactyly (); and congenital heart defects such as transposition of the great arteries (). These anomalies were accompanied by heterotaxy – the randomized left-right positioning of the heart and other visceral organs in the body (). Analyses of 23 mutants at embryonic day 10.5 (E10.5)-E11.0 showed that 11 had reversed heart looping, and approximately half of the mutants examined at later stages showed dextrocardia, some with a right-sided aortic arch (). We also observed other visceral organ situs defects, including randomized left-right positioning of the stomach (data not shown), abnormal liver situs with some exhibiting symmetric midline liver (), and polysplenia or asplenia ( and data not shown). In addition, enlargement of the kidneys was observed that, in some cases, was associated with duplex kidneys (). Histological sections revealed the presence of glomerular cysts and cysts in the kidney tubules (–D). The latter seem to be derived from the proximal tubules, because only a small number of cysts were positive for the collecting duct and ureteric bud lectin marker Dolichos biflorus agglutinin (DBA) ( and data not shown). The appearance of the liver also suggested possible cystic changes ().
Fig. 1. 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 (more ...)
Fig. 2. 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 (more ...)
Examination of the mutants further suggested a variety of skeletal anomalies. Mutants exhibited mal-alignment and fissure of the sternal vertebrae (), and fissure of the manubrium (). We also found reduced or absent ossification of the vertebral bodies of the cervical and thoracic vertebra (). In addition, craniofacial defects were observed, including a dome shaped appearance of the skull (), hypoplastic lower jaw () and cleft palate (). Polydactyly was associated with preaxial digit duplication that included an extra triphalangeal first digit (). This was most often associated with the hindlimb, whereas the forelimb frequently showed a milder phenotype with a broadened thumb ( and ). Examination of 34 near-term mutants showed 30 (88%) with polydactyly, of which 28 (82% of all examined) had preaxial digit duplication mainly associated with the hindlimb.
Fig. 3. 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, (more ...)
Identification of mutation in Mks1
To map the mutation, we intercrossed the mutant that was generated in a C57BL6 background with C3H mice to generate C57BL6:C3H hybrid offspring. The mutant embryos and fetuses obtained from these intercrosses were analyzed by genome scanning using C57BL6/C3H polymorphic DNA markers, and linkage data obtained from ten mutants localized the mutation to mouse chromosome 11. Analysis of an additional 95 mutants using six additional microsatellite DNA markers and 16 SNPs narrowed the interval to a 9.8-Mb region positioned between SNP rs13481117 and rs27099917. This map interval contained 143 genes, including Mks1. Reverse transcriptase PCR (RT-PCR) analysis of transcripts derived from genes in the interval in the mutant embryos revealed a deletion of 780 nucleotides in transcripts of Mks1 that resulted in an in-frame deletion of amino acid residues 64–323 (). In agreement with this finding, sequencing of the genomic DNA from the mutant revealed a corresponding deletion of 5226 bases that spanned intron 2 to intron 10 of Mks1 (). Subsequent genotyping analysis showed that all offspring exhibiting the mutant phenotype were homozygous for the Mks1del64-323 mutation, whereas all viable adult animals were wild-type or heterozygous (Mks1del64-323/+).
Fig. 4. 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 (more ...)
Abnormal protein localization in Mks1 mutant MEFs
To examine the functionality of the mutant Mks1 protein, we generated an antibody to Mks1 by using an epitope outside of the deleted domain, a region that is completely conserved between mouse and human MKS1 (see Methods). In addition, we generated expression constructs encoding wild-type or mutant Mks1 protein fused in frame at the N-terminus with a FLAG tag, and these were transfected into human HEK293 cells. Analysis of the transfected cells by two-color western blots showed dual detection of the expected FLAG-tagged 67-kD wild-type (upper arrow in ) and 37-kD mutant (arrowhead in ) Mks1 fusion protein bands by anti-FLAG (green) and anti-Mks1 (red) antibodies. In addition, the endogenous 64-kD Mks1 protein band was also observed in the nontransfected and transfected cells (lower arrow in ). Additional bands observed with the Mks1 and FLAG antibodies were nonspecific, because they were also found in the nontransfected cells.
Double immunostaining of the transfected HEK293 cells showed that the FLAG and Mks1 protein epitopes were colocalized to the centrosome in cells expressing the wild-type FLAG-tagged Mks1 protein. By contrast, in cells expressing the FLAG-tagged mutant Mks1 protein, no FLAG tag was observed in the centrosome, indicating that the mutant protein could not be incorporated into centrosomes (data not shown). Similar results were obtained with further analysis of mouse embryonic fibroblasts (MEFs) derived from wild-type and Mks1-mutant embryos. Wild-type MEFs that were double immunostained with the anti-Mks1 antibody and antibody to γ-tubulin showed colocalization of Mks1 and γ-tubulin in punctate dots corresponding to centrosomes in 30% of the cells (). In the Mks1-mutant MEFs, although Mks1 was also observed in punctate dots, these were not colocalized with the γ-tubulin-containing centrosomes (). Together, these findings show that the mutant Mks1 protein cannot be assembled into centrosomes. Consistent with this, transfection of a full-length Mks1-GFP construct showed centrosome localization (), but this was not observed with expression of a full-length mutant-Mks1–GFP construct ().
Developmental anomalies associated with defects in ciliogenesis
To examine the role of Mks1 in ciliogenesis, we examined cilia outgrowth in wild-type and Mks1-mutant MEFs using antibodies to γ-tubulin and acetylated tubulin to delineate the centriole and cilium, respectively (). Quantitative analysis showed that, in wild-type or heterozygous MEFs, 50–60% of the cells were ciliated, but less than 20% of the homozygous mutant MEFs had cilia (). To further evaluate ciliogenesis in cells and tissues of the mutant embryos, we used scanning electron microscopy (EM) and light microscopy to examine cilia in the embryonic node, developing kidney, neural tube, cochlea and trachea.
Cilia defects in the embryonic node
Scanning EM analysis of E7.5–E8.0 embryos showed cuboidal epithelial cell morphology in the embryonic node of wild-type or heterozygous mutant embryos (), whereas, in the homozygous mutant embryos (n=4), the node had a flattened epithelial morphology (). In wild-type or heterozygous embryos, most cells in the embryonic node exhibited a single cilium that projected posteriorly. By contrast, in homozygous Mks1-mutant embryos, only a few randomly oriented abnormal short cilia-like projections were observed in a few cells in the node (arrows in ), but these were not immunostained by an anti-acetylated-tubulin antibody (data not shown). Videomicroscopy also showed no ciliary motion and fluorescent beads placed over the node showed no net nodal flow (see supplementary material Movies 1 and 2).
Fig. 5. 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) (more ...) Cilia defects in the kidney
To evaluate ciliogenesis in the kidney of E18.5 embryos, paraffin sections of the fetal tissue samples were immunostained with anti-IFT88 and anti-acetylated-tubulin antibodies. Cilia were readily found in Bowman’s capsule and the glomerulus, as well as in the epithelia of the collecting ducts (). By contrast, in Mks1-mutant embryos, very few cilia were observed and, even when present, were shorter than those seen in wild-type or heterozygous embryos (). Quantitation of the number of ciliated cells in the collecting ducts showed that more than 90% were ciliated in wild-type embryos, but less than 20% were ciliated in mutant embryos (). Immunostaining with antibodies to γ-tubulin and IFT88 showed that the centrioles were apically localized in the epithelia of the mutant kidney tubules, as observed in the control (wild-type or heterozygous) kidney epithelia (; arrows denote apical surface of opposing unilaminar epithelia in kidney tubule, with enlargement shown in panels I,J).
Cilia defects in the neural tube
Scanning EM analysis of the neural tube showed abundant cilia in the ventral groove encompassing the presumptive floor plate and also in regions dorsolateral to the floor plate. Cilia in the floor plate were generally longer than those on the lateral wall (). In the Mks1-mutant embryos, we found fewer cilia, both in the ventral groove () and along the lateral wall (). Often it was difficult to determine whether membrane projections observed in the neural tube of mutant embryos were indeed cilia ().
Fig. 6. 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. (more ...) Kinocilia and stereocilia patterning defects in the cochlea
We examined the formation of specialized cilia in the developing cochlea that are known as the kinocilia. Kinocilia play a crucial role in the patterning of actin-based microvilli, which are referred to as stereocilia, a process that is regulated by cilia-transduced PCP signaling (Jones et al., 2008
; Kelly and Chen, 2007
). In the cochlea, there are normally three outer rows [outer hair cells (OHCs)] and one inner row [inner hair cells (IHCs)] of hair cells, and, in each hair cell, a kinocilium is positioned at the tip of the ‘chevron’-shaped stereocilia bundles (). In the Mks1-
mutant cochlea, although the kinocilia were present and seemed to be of normal length and morphology, they were often misplaced relative to the stereocilia bundles (). Thus, sometimes they were found adjacent to or within the stereocilia bundles (), displaced to one side of the stereocilia bundles () or encircled by a stereocilia bundle ring ().
Fig. 7. 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, (more ...) Trachea epithelial cells are ciliated and motile
The respiratory epithelia in the trachea have multiciliated cells with motile cilia that move in rapid synchrony to clear mucus, bacteria and other foreign matter from the airway. These ciliated cells emerge during late fetal development, and are sparse near term but rapidly increase in number with postnatal development (Francis et al., 2009
). Examination of near-term wild-type and Mks1-
mutant fetuses showed that both had multiciliated cells with motile cilia (see supplementary material Movie 3). There was no detectable difference in the ciliary motion observed in the wild-type vs mutant airway epithelia. These findings suggest that Mks1
is dispensable for ciliogenesis in the airway epithelia.
Defects in Shh signaling and abnormalities in dorsoventral patterning of the neural tube
We further examined dorsoventral patterning of the neural tube in Mks1 mutants because this has been well described to be regulated by cilia-transduced Shh signaling. Sections were obtained from E10.5 Mks1 mutant and control embryos to examine neural tube patterning anteriorly at the region between the forelimb and hindlimb () and posteriorly at the level of the hindlimb (). Shh was detected in the notochord, both anteriorly and posteriorly, but expression levels were greatly diminished (). Shh expression was retained in the presumptive floor plate anteriorly but at diminished levels, whereas, posteriorly, Shh was not detected in the ventral neural tube. These observations suggest that the floor plate was present anteriorly but absent posteriorly. We note that, posteriorly, the ventral neural tube had an abnormal thickened morphology, not typical of the floor plate (). Consistent with loss of the floor plate posteriorly, FoxA2, another floor plate marker, was expressed anteriorly () but was entirely absent posteriorly (). In addition, expression of Nkx2.2, a marker of V3 interneurons in the ventral neural tube, was disrupted posteriorly but not anteriorly (). Overall, these findings suggest that the high-level Shh signaling required for floor plate specification was disrupted in the posterior neural tube of Mks1 mutants, whereas, anteriorly, Shh signaling persisted, but at lower level, as indicated by the reduced expression of FoxA2 and Shh in the presumptive floor plate. In addition, the dorsal limits of Olig2-expressing () and Nkx6.1-expressing () domains and the ventral limit of the Pax7-expressing () domain shifted to more-dorsal positions in the anterior but not posterior neural tube, indicating the dorsal expansion of low-level Shh signaling anteriorly (). Western immunoblotting of the neural tube spanning the region between the forelimb and hindlimb showed an increase in full-length Gli3 (Gli3-FL) but no change in Gli3R level (supplementary material Fig. S1B,D,E). We also observed an increase in full-length Gli2 protein expression level (supplementary material Fig. S1C,E).
Fig. 8. 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 (more ...)
Defects in Shh signaling in the limb and abnormal digit patterning
The finding of polydactyly in the Mks1 mutants suggested a defect in Shh signaling, because Shh is required for normal anterior-posterior patterning of the limb bud. In situ hybridization analysis showed that, in E10.0–E10.5 limb buds, Shh, Gli3 and dHand expressions were unchanged (), but Gli1 and PtC1 expressions were reduced ( and supplementary material Fig. S2C,D). At E11.0, a reduction in PtC1 expression in the posterior limb bud was accompanied by ectopic PtC1 expression in the anterior limb bud (). This was accompanied by an anterior expansion in the expression domains of Fgf8 (), Fgf4 () and gremlin (). At E12.5, the Sox9 expression domain was broadened anteriorly in the presumptive first digit of the forelimb, and in the anterior protrusion of the hindlimb bud (). Overall, these findings indicated ectopic activation of Shh signaling in the anterior limb bud. Using western immunoblot analysis, we examined Gli3 expression levels in the limb buds. Left limb buds were bisected into anterior and posterior halves, whereas the right limb buds were harvested whole. This analysis revealed a reduction in the Gli3R:Gli3-FL ratio in the anterior but not posterior halves of the fore- and hindlimb buds compared with controls. A reduction in the Gli3R:Gli3-FL ratio was also detected in the whole hindlimb bud but not forelimb bud compared with controls (supplementary material Fig. S1A,B). These results suggest that the preaxial polydactyly typically observed in the Mks1 mutant results from a relative decrease in Gli3R level in the anterior limb bud brought on by the ectopic activation of Shh.
Fig. 9. 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 (more ...)