Full size western blots.

Top blots: Left membrane was probed for Ift140 while the right was probed for gamma tubulin. The lanes used in Fig 4B are in the white box. Middle blot: Membrane was cut (arrow) and the top half probed for Ift140 and the lower half probed for gamma tubulin. The lanes used in Fig 4B are in the white box. Bottom blot: Membrane was cut (arrow) and the top half probed for Ift140 and the lower half probed for gamma tubulin. The left image is the western blot superimposed on an image of the membrane while the right image is only the western blot. The lanes used in S2 Fig are in the boxes. (TIF)</p

<i>Ift140</i>^{<i>null1/null1</i>} embryos display major anatomical defects at E14.5.

Gross anatomical examination revealed numerous severe defects in E14.5 Ift140null1/null1 embryos (E–H) compared to controls (A–D) including significant hydrops (*), hypoplastic forelimbs (fl), hypoplastic maxillary region (mx), including reduced maxillary, medial and lateral nasal prominences resulting in bilateral cleft lip, hyperplastic mandibular region (md), missing abdominal walls and diaphragm with gastroschisis/ectopia cordis (F, G), smaller chests (D vs. H), and exencephaly with swollen neural tissue pouches surround an empty hollow cavity (‡). (I–L) 3D reconstitutions highlight the craniofacial defects (I, K) and polydactyly (J, L) found in E14.5 Ift140null1/null1 embryos. (M) Chest size was quantified by measuring chest areas that revealed that IFT140null1/null1 embryos (n = 7) displayed significantly smaller chests than age matched wild-type embryos (n = 3) (unpaired Students t test; p = 0.0065). cx: cerebral cortex; d: diaphragm; dA: descending aorta; e: eye; fb: forebrain; fl: forelimb; hl: hindlimb; hf: hair follicles; i: small intestine; ie: inner ear; ln: lung; lnp: lateral nasal prominences; lv: liver; mb: midbrain; md: mandibular region; mnp: medial nasal prominence; mx: maxillary region; ns: nasal capsule; op: otic placode; s: stomach; sc: spinal cord; t: trachea; tn: tongue; v: ventricle. Scale bars: A–I, K = 1 mm, J, L = 0.5 mm. The data underlying this figure can be found in Supporting information S1 Data.</p

Summary of mouse breeding used to generate embryos analysed in this study.

†When using tamoxifen-inducible Cre (CAGGCre-ER, Foxa2Cre-ER), tamoxifen was administered to pregnant mothers at embryonic ages listed. (DOCX)</p

SBDs associated with 2 different mouse <i>Ift140</i> mutant alleles.

SBDs associated with 2 different mouse Ift140 mutant alleles.</p

qPCR primer sequences used in this study.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.</div

Gross anatomical defects in embryos with <i>Wnt1-Cre</i> or <i>Tbx18-Cre</i> deletion of <i>Ift140</i>.

(A–C) Littermate control embryos (Ift140flox/+, Wnt1-Cre+ or Tbx18-Cre+) displayed normal embryonic anatomy. (D–F) Wnt1-Cre deletion of Ift140 resulted in a 100% penetrative phenotype characterized by significant hydrops (asterisks in D) and marked craniofacial defects including macrostomia and hypertrophied forebrain, maxillary, and mandibular regions (F). (G–M) Tbx18-Cre deletion of Ift140 resulted in severe hydrops (asterisks in G, H), but less severe cranial facial defects (H). While most embryos Tbx18-Cre experimental embryos displayed normal craniofacial anatomy (G, H), a single embryo (1/10) displayed a wide mouth phenotype reminiscent of a bird’s beak as well as marked cranial tissue hypertrophy (I). Embryos with Tbx18-Cre deletion of Ift140 displayed a number of skin protrusions located to the face (arrowhead in J) and more commonly to the abdomen (arrows in K, L). Polydactyly was also observed in Tbx18-Cre experimental embryos (M). LV: left ventricle; cx: cerebral cortex; sc: spinal cord; fb: forebrain; mb: midbrain; fl: forelimb; hl: hindlimb; e: eye; op: otic placode; hf: hair follicles; lv: liver; ln: lungs; s: stomach; i: small intestine; mx: maxillary region; md: mandibular region. All scales bars = 1 mm.</p

Perturbation of hedgehog signaling associated with developmental defects in the <i>Ift140</i>^{<i>null1/null1</i>} and <i>Ift140</i>^{<i>220/220</i>} mutant embryos.

(A) To assess hedgehog signaling in cultured MEFs, RNA was isolated from cells that were left untreated or treated with 400 nM SAG for 24 h. Gli1 and Gapdh gene expression was measured by quantitative real-time PCR. For Ift140220 cells, n = 1 control and 2 mutant lines analyzed 3 times. For Ift140null1 cells, n = 3 control and 3 mutant lines each analyzed 1 time. **p ≤ 0.01; ***p ≤ 0.001; ns, not significant, assessed by one-way ANOVA. (B) Immunostaining for differentiation markers in the neural tube of E10.5 embryos revealed disturbance in Shh regulated dorsoventral patterning of the neural tube in the Ift140220/220 mutants. Ventralization is indicated with dorsal shift in expression of ventral markers OLIG2, and NKX6.1, and dorsal retraction in expression of PAX6, a dorsal marker. Arrows denote the boundaries of antibody staining. (C, D) Sagittal and Frontal views of E14.5 wild-type and Ift140220/220 mutant embryos carrying a Gli-LacZ reporter, delineating regions of hedgehog signaling. (E) In situ hybridization of limb buds from E10.5 wild-type and Ift140220/220 mutant embryos showed perturbation of Shh signaling, with expanded expression of Gremlin indicating polydactyly. (F) Ift140220/220 (left), Ptch1LacZ/LacZ (middle), and Ift140220/220:Ptc1LacZ/LacZ (right) E10.5 embryos carrying the Ptc1-LacZ knockout allele were X-gal stained to delineate regions of hedgehog signaling. The severe phenotype of the Ptc1LacZ/LacZ mutant embryo is partially rescued in the double homozygous Ift140220/220:Ptc1LacZ/LacZ mutant embryo. Scales bars: B = 100 μm, C, D, F = 1 mm, E = 0.25 mm. The data underlying this figure can be found in supplemental file S1 Data. MEF, mouse embryonic fibroblast.</p

3D reconstruction of a wild-type E16.5 embryo processed using episcopic confocal microscopy highlighting normal cardiac outflow tact development.

Ao: Aorta, dAo: Descending aorta, ASLV: Aortic semilunar valve, DA: Ductus arteriosus, LA: Left atria, LB: Left Bronchus, LPA: Left pulmonary artery, LV: Left ventricle, PA: Pulmonary artery, PSLV: Pulmonary semilunar valve, PT: Pulmonary trunk, RA: Right atria, RB: Right Bronchus, RCA: Right carotid artery, RPA: Right pulmonary artery, RV Right ventricle, T: Trachea. (MP4)</p

Hyperplasia of maxillary and mandibular prominences resulting in bone fusions with <i>Wnt1-Cre</i> deletion of <i>Ift-140</i>.

(A–C) E12.5 control (Ift140flox/+, Wnt1-Cre+ or Wnt1-Cre-) (A, B) and experimental animals (Ift140flox/null1, Wnt1-Cre+) (C). Overgrowth of the maxillary and mandibular processes (yellow outline, bottom row) is apparent in the mutant animals (C). The maxillary overgrowth is concealing the eye. (D–O) E18.5 control (D–F, J–L) and experimental animals (G–I, M–O). The Wnt1-Cre Ift140flox/null1 mutant skull and face is shortened, and smaller (G–I) than the Ift140flox/+ Wnt1-Cre+ control embryos (D–F), and have several defects in neural crest-derived bones. Laterally, the temporomandibular joint is absent resulting in the fusion between the maxilla and mandible in the experiment animals (G, L arrows). In the bird’s eye view of the skull, there are ectopic boney islands present in the mutant frontal bones (arrowhead, H), suggestive of a problem with cell migration. Remarkably, the eyes are visible in this view but are below the frontal bones and medial to the maxilla (H, arrows). The palatal view shows that the maxillary bones are displaced laterally (I, open arrowhead), the vomer is present (I, arrowhead) and the anterior, neural crest-derived cranial base is absent (I, arrows). (J–O) The mandible is missing its 3 processes (arrowhead, L). The alveolar ridge for the molars is present, but is smaller (arrowhead, M).</p

<i>Ift140</i>^{<i>220/220</i>} mutant mouse embryos display a range of developmental defects.

(A–D) Gross anatomy of E11.5 embryos reveals Ift140220/220 mutants have open neural tube defect (arrows in A and B) and defective heart tube looping (arrows in C and D). (E–H) Gross anatomy of E14.5 embryos reveals Ift140220/220 mutants display a range of developmental defects including severe craniofacial defects (F, G), anophthalmia (G), omphalocele (F, G, H), polydactyly (G), and abnormal chest skin tags (H) that may represent abnormal mammary tissue development. (I, J) Cardiac anatomy of E16.5 embryos reveals Ift140220/220 mutants display VSDs (K, L) and abnormal OFT development (M, N) including PA stenosis and aorta (Ao) dilation (N). (O–Q) Renal anatomy of E14.5 embryos reveals Ift140220/220 mutants display hydroureter (P arrow) and duplex kidney (Q arrows highlight constrictions between duplex kidneys). Scale bars: A–J, O–Q = 1 mm, K–N = 0.5 mm. OFT, outflow tract; PA, pulmonary artery; VSD, ventricular septal defect.</p