IFT27 links the BBSome to IFT for maintenance of the ciliary signaling compartment

Vertebrate hedgehog signaling is coordinated by the differential localization of the receptors patched-1 and Smoothened in the primary cilium. Cilia assembly is mediated by intraflagellar transport (IFT), and cilia defects disrupt hedgehog signaling, causing many structural birth defects. We generated Ift25 and Ift27 knockout mice and show that they have structural birth defects indicative of hedgehog signaling dysfunction. Surprisingly, ciliary assembly is not affected, but abnormal hedgehog signaling is observed in conjunction with ciliary accumulation of patched-1 and Smoothened. Similarly, Smoothened accumulates in cilia on cells mutated for BBSome components or the BBS binding protein/regulator Lztfl1. Interestingly, the BBSome and Lztfl1 accumulate to high levels in Ift27 mutant cilia. Because Lztfl1 mutant cells accumulate BBSome but not IFT27, it is likely that Lztfl1 functions downstream of IFT27 to couple the BBSome to the IFT particle for coordinated removal of patched-1 and Smoothened from cilia during hedgehog signaling

Late tamoxifen deletion of <i>Ift140</i> with <i>CAGGCre-ER</i> 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.</p

<i>Ift140</i>^{<i>null1/null1</i>} 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.</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

Excel sheet containing the raw data used to generate all graphs found in the main figures.

Excel sheet containing the raw data used to generate all graphs found in the main figures.</p

3D reconstruction of a wild-type E16.5 embryo processed using episcopic confocal microscopy highlighting normal chest/lung development.

dAo: Descending aorta, D: Diaphragm, E: Esophagus, L: Liver, LB: Left Bronchus, LL: Left lung lobe, RB: Right Bronchus, RL(SL): Right lung (Superior lobe), RL(ML): Right lung (Middle lobe), RL(IL): Right lung (Inferior lobe), RL(PCL): Right lung (Post-caval lobe). (MP4)</p

Early tamoxifen deletion of <i>Ift140</i> with <i>CAGGCre-ER</i> recapitulates the <i>Ift140</i>^null1/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.</p

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

Ao: Aorta, dAo: Descending aorta, DA: Ductus arteriosus, LA: Left atria, LV: Left ventricle, MV: Mitral valve, PA: Pulmonary artery, PT: Pulmonary trunk, RA: Right atria, RV Right ventricle, TV: Tricuspid valve. (MP4)</p

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>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