Fig 1.
Ift140220/220 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.
Table 1.
SBDs associated with 2 different mouse Ift140 mutant alleles.
Fig 2.
Ift140null1/null1 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.
Fig 3.
Ift140null1/null1 embryos display major anatomical defects at E9.5 and cardiac/great vessel patterning defects at E14.5.
(A–I) 3D reconstructions of E9.5 Ift140+/+ control and littermate homozygous Ift140null1/null1 embryos analyzed by episcopic confocal microscopy. In A-B note the hypertrophy of the first branchial arch (1), and hypotrophy of the other branchial arches (2–4). In C-F note neural tube abnormalities characterized by the head fold failing to close in Ift140null1/null1 embryos (E-F). In G-I note the randomization of heart tube looping characterized by normal D-looped (G), reversed L-looped (H), and midline A-looped (I) heart tubes. (J–L) Sagittal section reconstructions of Ift140null1/null1 embryos further highlight the abnormal midline A-looped heart tube phenotype (L) observed in some Ift140null1/null1 embryos compared to embryos with D-looped (J) or L-looped (K) heart tubes. (M) Quantification of the Ift140null1/null1 heart looping defects reveals the randomization of looping phenotypes compared with wild-type embryos (n = 22). (N) Measurement of the OFT length in E10.5 embryos showed significant lengthening of the OFT in the Ift14O KO embryos. Data is mean ± SD. * p = 0.0007 assessed by unpaired Student t test. (O–V) Numerous cardiac and great vessel defects were seen in E14.5 Ift140null1/null1 embryos including: small ventricles (O vs. S), AVSDs with mutant embryos displaying a complete absence of normal atrial septum (arrow P vs. T), PTA characterized by a single OFT due to OFT failing to septate into separate aorta and pulmonary vessels (Q vs. U), and TEF characterized by a single unseptated tracheoesophageal tube (R vs. V). (W) Great vessel patterning was also perturbed in Ift140null1/null1 embryos (n = 7). Besides PTA, mutants showed a combination of singular or double left and right descending aortas. *: somites; 1, 2, 3, 4: Branchial arches; A: aorta; AVSD: atrioventricular septal defect; dA: descending aorta; f: forebrain; fl: forelimb; h: hindbrain; ht: heart tube; if: inflow tract; LA: left atrium; LCA: left carotid artery; LSA: left subclavian artery; LV: left ventricle; lv: liver; m: midbrain; o: esophagus; of: outflow tract; P: pulmonary trunk; PTA: persistent truncus arteriosus; RA: right atrium; RCA: right carotid artery; RV: right ventricle; t: trachea; tn: tongue; TEF: tracheoesophageal fistula; arrowhead: ventricular septal defect; arrow: atrial septum. Scales bars: A, B, J, K, L, N–U = 0.5 mm, C–I = 0.25 mm. The data underlying this figure can be found in supplemental file S1 Data.
Fig 4.
Comparison of Ift140220 allele and Ift140null1 allele.
(A) Ift140 mRNA levels in MEFs. Levels of Ift140 transcript between exons 6 and 7 and between exons 13 and 14 was measured by qPCR. Wild type was set to 100%. For Ift140220/220, n = 1 control cell line and 2 mutant lines repeated 3 times. For Ift140null1/null1, n = 3 control lines and 3 mutant lines analyzed once each. *** p ≤ 0.001, Students t test. (B) Western blot analysis of IFT140 protein levels in MEFs from the 2 alleles. (C) MEFs from control and mutant lines stained for cilia (acetylated tubulin, red) and either IFT140 or IFT88 (green). 0% of Ift140null1/null1 cells showed IFT140 staining at the ciliary base or centrosome. 63 ± 16% of Ift140220/220 cells showed weak IFT140 staining at the ciliary base while control cells for each experiment showed 100% of the ciliated cells showing an IFT140 spot at the ciliary base. For Ift140220/220, n = 1 control cell line and 2 mutant lines repeated 3 times. For Ift140null1/null1, n = 3 control lines and 3 mutant lines analyzed once each. Scale bar is 5 microns. Arrows mark ciliary tip. (D) Percent ciliation in control and mutant fibroblast lines. For Ift140null1, n = 100 cells counted from 3 repeats of 1 control line and 1 mutant line. *****p < 0.0001 t test. For Ift140220, n = 100 cells counted from 3 repeats of 1 control and 2 Ift140220/220 mutant lines. **p = 0.0037 t test. (E) Cilia on Bowman’s capsule of the kidney stained for centrosomes (γ-tubulin, red, arrow) and IFT88 (green). Scale bar is 5 microns. (F) Scanning EM images of control, Ift140220/220, and Ift140null1/null1 embryo nodes harvested at E7.5. Arrows indicate cilia. Scale bar is 5 microns. The data underlying this figure can be found in Supporting information S1 Data. MEF, mouse embryonic fibroblast.
Fig 5.
Perturbation of hedgehog signaling associated with developmental defects in the Ift140null1/null1 and Ift140220/220 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.
Table 2.
SBD phenotypes associated with Cre targeted Ift140 deletion.
Fig 6.
Early tamoxifen deletion of Ift140 with CAGGCre-ER recapitulates the Ift140null1/null1 phenotype.
Ift140flox/+, CAGGCre-ER+ (Control) (A–C) and Ift140flox/null1, CAGGCre-ER+ experimental (D–F) embryos were treated with tamoxifen at E5.5 and harvested at E12.5. The experimental embryos had extensive developmental abnormalities and showed developmental delay (A, D). The experimentals recapitulated the laterality defects seen in Ift140null1/null1 embryos as characterized by reversed heart tube looping and the morphological right ventricle appearing on the embryo’s left side (B vs. E). Similar to the Ift140null1/null1 embryos, tamoxifen-driven deletion of Ift140 using CAGGCre-ER at E5.5 also caused atrial septal defects and outflow track septation defects (PTA) (C vs. F). However, head fold closure defects and exencephaly were not observed under these conditions. fb: forebrain; mb: midbrain; fl: forelimb; hl: hindlimb; e: eye; A: aorta; P: pulmonary trunk; LV: left ventricle; RV: right ventricle; LA: left atria; RA: right atria; PTA: persistent truncus arteriosus. Scales bars: A, D = 0.5 mm, B, C, E, F = 0.25 mm.
Fig 7.
Late tamoxifen deletion of Ift140 with CAGGCre-ER uncovers additional phenotypes.
Ift140flox/+, CAGGCre-ER+ (Control) (A, D, G, J, L) and Ift140flox/null1, CAGGCre-ER+ experimental (B, C, E, F, G, H, K, M, N) embryos were treated with tamoxifen at E7.5 or E8.5 and harvested at E16.5. E7.5 tamoxifen-dosed embryos show severe gastroschisis with the majority of the abdominal organs protruding from the abdominal cavity (A, B, D, E). E8.5 dosed embryos show only moderate gastroschisis with only some of the abdominal organs found outside of the abdominal cavity (A, C, D, F). Both E7.5 and E8.5 dosed embryos showed significant hydrops (* B, C, E, F), polydactyl (H, I), and hypoplastic lungs (B, C, E, F). Laterality defects were not observed in either E7.5 or E8.5 tamoxifen-dosed embryos, with all hearts displaying a normal D-looping phenotype (J, K). However, cardiac defects were observed in the experimental animals including ventricular septal defects (J, K) and AVSDs (L, M). (N) While the great vessels of both E7.5 and E8.5 tamoxifen-dosed experimental embryos displayed normally septated aorta and pulmonary trunk, approximately 50% had great artery patterning defects including: right aortic arch, interrupted aorta (arrows), hypoplastic transverse aorta (arrowhead), hypoplastic pulmonary arteries (*), and in 1 case double aortic arch with both left and right descending aortas. A: aorta; P: pulmonary trunk; LV: left ventricle; RV: right ventricle; LA: left atria; RA: right atria; t: trachea; o: esophagus; VSD: Ventricular septal defect; AVSD: atrioventricular septal defect; cx: cerebral cortex; sc: spinal cord; mb: midbrain; fl: forelimb; hl: hindlimb; e: eye; forebrain; op: otic placode; hf: hair follicles; RCA: right carotid artery; LCA: left carotid artery; LSA: left subclavian artery; LdAo: left descending aorta; RdAo: right descending aorta; RPA: right pulmonary artery; LPA: left pulmonary artery; Ao: aorta; P: pulmonary trunk; lv: liver; ln: lungs; s: stomach; i: small intestine. Scales bars: A–I = 1 mm, J–M = 0. 5 mm.
Fig 8.
Gross anatomical defects in embryos with Wnt1-Cre or Tbx18-Cre deletion of Ift140.
(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.
Fig 9.
Cardiac and great vessel defects with Wnt1-Cre or Tbx18-Cre deletion of Ift140.
(A–F) Both Wnt1-Cre and Tbx18-Cre driven Ift140 deletion (Ift140flox/null1, Cre+) produced only mild congenital cardiac defects. All experimental hearts displayed normal heart looping and the majority had normal ventricular (A, D) and atrial (C, F) septum anatomy. However, a small number of Wnt1-Cre and Tbx18-Cre experimental animals displayed perimembranous ventricular septal defects (arrowheads in B, E). (G) Wnt1-Cre driven Ift140 deletion caused defects in great artery patterning including interrupted aorta (arrows in G), with or without the development of a long hypoplastic collateral vessel linking the left carotid artery and left subclavian artery (arrowheads in G). The development of anomalous right subclavian arteries was common in Wnt1-Cre experimental embryos (Ift140flox/null1, Wnt1-Cre). These arose from either the pulmonary trunk adjacent to the pulmonary arteries (§), as a vascular sling arising from the descending aorta and wrapping behind the trachea (‡), or as a vascular ring with attachments to both the pulmonary trunk and descending aorta (*). A: aorta; P: pulmonary trunk; LV: left ventricle; RV: right ventricle; LA: left atria; RA: right atria; T: trachea; RCA: right carotid artery; LCA: left carotid artery; LSA: left subclavian artery; RSA: right subclavian artery; dAo: descending aorta; RPA: right pulmonary artery; LPA: left pulmonary artery; T: trachea. All scales bars = 0.5 mm.
Fig 10.
Hyperplasia of maxillary and mandibular prominences resulting in bone fusions with Wnt1-Cre deletion of Ift-140.
(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).
Fig 11.
Summary of the spatial and temporal roles for Ift140 during embryonic development.
(A) The timeline shows the temporal requirement for Ift140 revealed by tamoxifen mediated knock-out of Ift140 at different time points during development. (B) Overview of embryonic days Tmx was used to knock down Ift140 and the resultant phenotypes. (C) Diagram summarizing the cell lineage requirement for Ift140 revealed by Cre-driven targeted Ift140 knock-out. Mef2c-Cre: targeting cells in the anterior heart field; Tie2-Cre: targeting endothelial cells; Wnt1-Cre: targeting dorsal neural tube and neural crest; Tbx18-Cre: targeting epicardial cells, left ventricle.
Table 3.
qPCR primer sequences used in this study.