Mks1^krc mutants develop defects similar to the Meckel syndrome tetrad. Dorsal views of (A and B) e12.5 hindlimbs and (C and D) e18.5 hindlimb skeletons showing polydactyly in Mks1^krc mutants (asterisks). (E and F) Liver sections at e18.5 showing the formation of bile ducts (arrowheads) in the ductal plate flanking the portal veins [green, marked by Sox9; blue marks nuclei (DAPI)]. The ductal plate persists in mutants and multiple bile ducts form. (G and H) Posterior view of e18.5 skulls showing the mineralization of the supraoccipital bone. In 12/15 mutants, the supraoccipital bones showed reduced ossification compared with controls. (I and J) Sections through e18.5 kidneys and adrenal glands. The mutant kidney is larger than wild-type and multiple cysts are visible. In (C, D, G, H), the red staining marks bone and the blue is cartilage. ad, adrenal glands; pv, portal vein; so, supraoccipital bone. Controls and mutants are shown at the same magnification. Red scale bars = 1 mm, yellow scale bar = 0.1 mm.

Multiple segments of Mks1^krc nephrons dilate or undergo cystogenesis. (A and B) Hematoxylin and eosin-stained sections of e18.5 kidneys show dilation of the Bowman's capsule (asterisk) surrounding the glomerulus in a mutant kidney. (C and D) Large cysts (asterisks) form in the proximal tubule region (green, marked by Lotus tetragonolobus lectin, LTL) of Mks1^krc nephrons. (E and F) Collecting ducts (green, marked by Dolichos biflorus lectin, DBA) and (G, H) medullary thick ascending limbs (green, marked by Tamm–Horsfall protein, THP) are dilated in mutants (asterisks). In (C–H), blue marks nuclei (DAPI). gl, glomerulus. Controls and mutants are shown at the same magnification. Scale bars = 0.1 mm.

Mks1^krc mutants display additional Meckel syndrome characteristics. (A and B) Ventral view of e12.5 hearts. Heart looping is a marker of left–right asymmetry. When heart looping is reversed, the outflow tract is on the left and left and right ventricles are reversed (B). In Mks1^krc mutants, 32% of embryos show reversed heart looping, 41% have normal looping, 26% show partial looping (n = 87). (C and D) e18.5 hindlimbs showing shortening and bowing of the femur and tibia (campomelia; asterisks) in mutants (n = 16/16). (E and F) Dorsal view of e18.5 lungs. Mks1^krc mutants also show pulmonary hypoplasia, with variable fusions between the right lungs lobes (n = 12/12). (G and H) e18.5 female genitourinary tracts. Ovaries normally lie largely posterior to the kidneys (asterisks). In Mks1^krc mutants, ovaries are sometimes displaced (black arrow) and show abnormal morphology (n = 6/20). (I and J) e18.5 sternum and ribs. Mks1^krc mutants show a shortening of the sternum and fusions or forking of the ribs (n = 14/16). (K and L) Dorsal view of e18.5 skull vaults showing reduced mineralization of the frontal and parietal bones in Mks1^krc mutants (n = 14/14). (M and N) Dorsal view of the palate after removal of the skull vault. Mks1^krc mutants show a reduction in the size of the presephenoid bone (n = 15/15). (O and P) Ventral view of the palate after removal of the mandible. One Mks1^krc mutant (P) showed an absence of the maxilla and palatine shelves of the palate (cleft palate) and absence of the more dorsal presphenoid bone. In (C, D, I–P), the red staining marks bone and blue is cartilage. oft, outflow tract; rv, right ventricle; lv, left ventricle; fe, femur; ti, tibia; le, left lobe; cr, cranial lobe; me, medial lobe; ca, caudal lobe; ki, kidney; bl, bladder; st, sternum fr, frontal bone; pa, parietal bone; ip, interparietal bone; ps, presphenoid; ma, maxilla shelf; pa, palatine shelf. Controls and mutants are shown at the same magnification. Scale bars = 0.1 mm.

Defective cilia formation in Mks1^krc mutants. Sections through (A and B) e11.5 hindlimb mesenchyme, (C and D) e18.5 cortical kidney regions and (E and F) e18.5 brains demonstrate a dramatic reduction in the number of cilia (green) in Mks1^krc mutants. In limbs (A and B), cilia are associated with basal bodies (red) in controls but this is reduced in mutants. Cilia are located on the apical surface of kidney tubules (C) and lateral brain ventricles (E) but the number and length of cilia is reduced in mutants (D, section through a cyst). (G and H) Scanning electron micrograph of e8.25 mouse nodes show a reduction in the number and abnormal morphology of Mks1^krc mutant nodal cilia. Insets show that the morphology of the node is normal in mutants. A few cilia are found that are nearly normal in length, but most cilia are reduced in size and some have bulges at their distal tips. In contrast to MKS1 siRNA knockdown (8), we do not see a noticeable difference in microvilli formation. (I–L) Basal bodies (red) localize to the apical side of polarized epithelia. (I and J) e12.5 lung epithelium. At this stage, the lung epithelial cells are monociliated. The white line indicates the basal side of the cells, and the apical side is to the right. (K and L) e11.5 neural tubes. The apical side of the cell faces the lumen (center, dashed line). In both of these tissue types, basal bodies are apically localized but there is a reduction in the number of cilia in mutants. (M and N) The lumens of e18.5 bile ducts (asterisks) show similar numbers of cilia in Mks1^krc mutants compared with wild-type despite the persistence of the ductal plate and increased number of bile ducts. (O and P) e18.5 multiciliated lung airway cells. Mks1^krc mutant cells form multiple, long cilia similar to controls. In all panels except (G and H) green marks cilia (Arl13b, A–F, I–L; or acetylated α-tubulin, M–P). In (A, B, I–L), red marks basal bodies (γ-tubulin). Purple (C–F, O, P) marks F-actin (phalloidin). In (M and N), red marks bile duct cells (Sox9). Controls and mutants are shown at the same magnification. White scale bars = 0.1 mm, red scale bars = 2 μm. (A–D, I–L, O, P) These are confocal X–Z projections.

Mks1 is required for normal Shh-dependent patterning in the neural tube. (A and F) Lateral views of e10.5 embryos. Mks1^krc mutants have a similar size and morphology as wild-type. (B–J) Immunofluorescent images of sections through e10.5 wild-type (B–E) and mutant (G–J) neural tubes at the level of the forelimb. HB9 marks motor neurons, NKX6.1 labels V2, V3 and motor neuron progenitors and DBX1 marks V0 progenitors. (B, C, G, H) PAX7 and PAX6, markers of dorsal and intermediate progenitor cells are expressed in similar domains in mutants and wild-type. In Mks1^krc mutants, motor neurons (I) expand dorsally and approach the V0 progenitor domain (marked by Dbx1; bracket). Additionally, the dorsal boundary of Nkx6.1+ cells (J) is expanded dorsally (brackets). Controls and mutants are shown at the same magnification. White scale bar = 1 mm. Yellow scale bars = 0.05 mm.

Mks1 acts downstream of Ptch1 and Smo in neural tube patterning.(A–X) Immunofluorescent images of sections through e10.5 wild-type (A–D) Mks1^krc (E–H), ptch1 (I–L), krc;ptch1 (M–P), smo (Q–T) and smo;krc (U–X) neural tubes at the level of the forelimb. FoxA2 marks floor plate cells, Nkx2.2 marks V3 interneuron progenitors, HB9 marks motor neurons, Nkx6.1 labels V2, V3 and motor neuron progenitors. In Mks1^krc mutants, floor plate cells are reduced in number or absent (H) compared with wild-type (D). (G) V3 interneuron progenitors are found more ventrally in mutants than in controls (C) and (F) motor neurons expand ventrally to the midline. Additional ventral progenitors largely form normally in mutants (E). ptch1 Mutants fail to close the neural tube and show a gain of Shh signaling including an expansion of floorplate markers (L), V3 and other ventral progenitors (K, I). Note: ptch1 mutants are delayed compared with wild-type and have not formed significant numbers of motor neurons at this stage. Mks1^krc;ptch1 double mutants look more similar to Mks1^krc mutants than ptch1 mutants: only a few floor plate cells are specified (P), V3 progenitors are largely found at the ventral midline and motor neurons and other ventral progenitors are ventrally restricted (N, M). smo Mutants show a complete loss of Shh signaling in the neural tube and ventral cell types such are not specified (Q–T). smo;Mks1^krc double mutants fail to specify the most ventral cell types (W, X) but some motor neurons (V) as well as Nkx6.1+ (U) and V0 progenitors (data not shown) are specified. Note: ptch1 (I–L) and smo (Q–T) mutant sections are shown at twice the magnification of the other sections. Scale bars = 0.1 mm. The scale bar in (A) is valid for all panels except (I–L; Q–T). See (I and Q) for appropriate scale bars.

Shh signaling is altered in Mks1^krc mutant limbs. (A, C, E, G) Dorsal views of e11.5 wild-type forelimbs showing posteriorly restricted (A) Shh, (C) Grem1, (E) Gli1 and (G) Ptch1 expression. (B, D, F, H) Mks1^krc mutant limbs show strong posterior expression of Shh and its target genes. (D) All Mks1^krc mutant mutants assayed showed an anterior expansion of Grem1 (n = 12). (F) In a subset of mutants, Gli1 is ectopically expressed on the anterior side of the limb (n = 2/12) despite the lack of ectopic Shh. Arrowheads indicate ectopic expression in the anterior limb. Controls and mutants are shown at the same magnification. The scale bar in (A) is valid for all panels (0.5 mm).