Hantel F , Liu H , Fechtner L , Neuhaus H , Ding J , Arlt D , Walentek P , Villavicencio-Lorini P , Gerhardt C , Hollemann T , Pfirrmann T .

FKZ31/06 Martin Luther University, GRK 2155 Deutsche Forschungsgemeinschaft, Deutsche Forschungsgemeinschaft, HMU Health and Medical University Potsdam GmbH

             cilium
             neural tube development
             GID complex

                neural tube defect
                microphthalmia

Xla Wt + rmnd5a MO (Fig.6.G)
Xla Wt + rmnd5a MO (Fig.7.Aa'',a1-a9)
Xla Wt + rmnd5a MO (Fig.7.Aa-b)
Xla Wt + rmnd5a MO (Fig.7.Ab)
Xla Wt + rmnd5a MO (Fig.7.Ab,Bb,Bd)
Xla Wt + rmnd5a MO (Fig.7.Ab,Bb-c)
Xla Wt + rmnd5a MO (Fig.7.Ac-c'',Be')

	Fig. 1. GID subunits colocalize to the basal body of mono-ciliated cells. (A) Schematic model of the vertebrate GID complex and its known subunits; RING domain-containing subunits are highlighted in blue, the substrate-recruiting factor GID4 is highlighted in red. (B) Schematic model of the primary cilium, composed of a basal body, axoneme and ciliary membrane. A large number of transporters, structural proteins, membrane receptors and basal body-located factors function in the primary cilium. (C,D) RMND5A localizes to the basal body of the primary cilium in NIH-3T3 cells. Cells were transfected with plasmids encoding GFP–RMND5A for 24 h and then further serum-starved (high glucose DMEM, 0.5% FCS) for an additional 24 h to induce ciliogenesis. After fixation, cells were stained for (C) acetylated tubulin (ac-tubulin) or (D) γ-tubulin to visualize the axoneme or the basal body of the primary cilium, respectively. Images were merged to identify overlapping signals (merge, yellow). Inset images show the magnification of a primary cilium and the basal body. Images shown in C and D are representative of seven and four images, respectively. Scale bars: 10 μm. (E) Rmnd5a localizes to basal bodies of motile mono-cilia of the GRP in X. laevis. mRNAs encoding GFP–Rmnd5a (green) and Centrin4–CFP (blue, centrioles/basal bodies) were injected at the four-cell stage. Embryos were fixed and stained at NF stage 17 to visualize cilia (magenta, ac-tubulin) and actin (white). Merge 1 shows a merged image of Eb and Ec; merge 2 shows a merged image of Ec and Ef; and merge 3 shows a merged image of Ee, Ef and Ec. Images are z-projections of confocal micrographs and represent five biological samples derived from one experiment. Scale bars: 20 μm (Ea), 3 μm (Eb–h).
	Fig. 2. GID complex subunits colocalize with primary cilia and centrioles. (A) Cells were serum- starved (high glucose DMEM, 0.5% FCS) for 24 h to induce ciliogenesis. After fixation, cells were stained for acetylated tubulin (ac-tubulin) to visualize the axoneme of the primary cilium. Additional antibodies were diluted 1:100 in PBS containing 3% BSA and 0.3% Tween-20 and used to visualize subunits of the GID complex (TWA1, RMND5A, MKLN1 and ARMC8). Nuclei were stained using DAPI (blue). (B) As described in A without prior serum starvation and stained for γ-tubulin to visualize the basal body of the primary cilium. Images in A and B were merged to identify overlapping signals (merge, yellow). Inset images show a cell with a primary cilium at original size at the respective magnifications. Scale bars: 20 μm. Percentage of cells with ac- Tubulin colocalization: TWA1, 75.9%, n=29; RMND5A, 60.7%, n=28; ARMC8, 96.3%, n=27; MKLN1, 93.1%, n=29. Percentage of cells with γ-tubulin colocalization: TWA1, 87.1%, n=31; RMND5A, 82.8%, n=29; n=total number of cells investigated.
	Fig. 3. GID deficiency interferes with SHH signaling. (A) Schematic representation of primary cilium-dependent SHH signaling in the ‘on state’. After binding of SHH ligand to PTCH1, it relieves the inhibition of smoothened (SMO). Then, GLI transcription factors are activated (GLIA), turning on expression of downstream genes, such as Gli1 and Ptch1. (B–D) qPCR of two SHH signaling markers (Gli1 in B,D; Ptch1 in C) during RMND5A KO (B,C) or knockdown (D). Cells were cultured under cilia-induced conditions (high glucose DMEM, 0.5% FCS) with or without SAG (100 nM) treatment for 24 h and harvested for further analysis. ns-siRNA, control siRNA; siRmnd5a, RMND5A knockdown. Mean±s.e.m., n=3. *P<0.05; **P<0.01 (two-tailed, unpaired Student’s t-test). (E) Western blot of GLI3 (same blot and exposure time). WT and KO cells were cultured under cilia-induced conditions (high glucose DMEM, 0.5% FCS) with or without SAG (100 nM) treatment for 24 h and harvested. A protein that cross-reacted with the GLI3 antibody was used as a loading control. (F,G) Quantification of western blots as in E showing relative protein level of GLI3 full-length form (F) and of GLI3 repressor form (G) in NIH-3T3 WT and RMND5A KO cells. Signal intensities of all lanes were measured on the same western blot with same exposure times and are normalized to the WT −SAG condition. WT −SAG, GLI3-FL/GLI3-R=1.51; KO −SAG, GLI3-FL/GLI3-R=1.4. Data are mean±s.e.m. of n=3. (H) qPCR of Rmnd5a mRNA during RMND5A knockdown. Cells were cultured under cilia- induced conditions (high glucose DMEM, 0.5% FCS) with or without SAG (100 nM) treatment for 24 h and harvested. Data are mean±s.e.m. of n=3. ***P<0.001 (two-tailed, unpaired Student’s t-test). Knockdown efficiency showed an 85% reduction of Rmnd5a mRNA.
	Fig. 4. Reduction of SHH signaling is independent of the AMPK–MTOR signaling axis. (A) Western blot analysis of NIH-3T3 WT and RMND5A KO cell extracts with the markers of the MTOR–autophagy pathway RPS6, p-RPS6 and LC3. Cells were cultured under cilia-induced condition (high glucose DMEM, 0.5% FCS) with or without SAG (100 nM) treatment for 24 h. ACTB (β-actin) was used as a loading control. LC3, microtubule-associated protein 1A/1B-light chain 3; LC3-I, unlipidated cytosolic form of LC3; LC3-II, PEI-conjugated LC3 protein. (B,C) Quantification of western blotting as in A, showing the relative ratio between p-RPS6 and RPS6 (B), and between LC3-II and LC3-I (C). Data are mean±s.e.m., n=3. *P<0.05 (two-tailed unpaired Student’s t-test). (D) Western blot analysis of NIH-3T3 WT and KO cell extracts with the markers of the MTOR–autophagy pathway RPS6, p-RPS6 and LC3. Cells were cultured under non-starvation conditions (high glucose DMEM, 10% FCS) with or without rapamycin (50 nM or 100 nM) treatment for 24 h. ACTB was used as a loading control. Blots are representative of three individual experiments. (E) qPCR of relative mRNA levels of the SHH signaling marker Gli1 during rapamycin treatment. WT and KO cells were cultured under cilia-induced condition (high glucose DMEM, 0.5% FCS) with SAG (500 nM) and rapamycin (1 μM) treatment for 24 h as indicated. Mean±s.e.m., n=5. **P<0.01; ***P<0.001; ns, not significant (two-tailed unpaired Student’s t-test). (F) qPCR of relative mRNA levels of the SHH signaling marker Gli1 during compound C treatment. WT and KO cells were cultured under cilia-induced condition (high glucose DMEM, 0.5% FCS) with SAG (100 nM) and compound C (10 μM) treatment for 24 h as indicated. Mean±s.e.m., n=6. **P<0.01; ns, not significant (two-tailed unpaired Student’s t-test).
	Fig. 5. Reduction of GID complex function results in aberrant protein homeostasis in primary cilia. (A) Representative microscope images of GLI2 localization. WT and KO cells were treated with cilia-inducing medium (high- glucose DMEM with 0.5% FCS) for 24 h with or without SAG and then fixed. Axonemes were stained with anti-acetylated TUBA4A (ac-tubulin) antibody (green); anti-GLI2 antibody (red) was used to visualize GLI2 protein, and DAPI (blue) was used to stain DNA. Inset images show magnification of a primary cilium. Scale bars: 20 μm. (B) Relative ciliary area of WT and KO. Relative cilia area was measured in the area demarcating the axoneme (green) in WT and KO cells, and the WT average set to 1. Mean±s.e.m.; n=90 (WT), n=90 (KO). ns, P>0.05 (two-tailed, unpaired Student’s t-test). (C) Relative ciliary area of WT and WT+SAG. Relative cilia area was measured in the area demarcating the axoneme (green) in WT and WT+SAG cells, with the WT average set to 1. Mean±s.e.m.; n=30 (WT), n=30 (KO). ns, P>0.05 (two-tailed, unpaired Student’s t-test). (D) Relative ciliary GLI2 protein level. GLI2 (red) and ac- Tubulin (green) fluorescence intensity was measured in the area demarcating the axoneme (green) in WT and KO cells with or without SAG (100 nM). Intensity was normalized to the level of ac-tubulin. Mean±s.e.m.; n=30 (WT), n=30 (WT+SAG), n=30 (KO), n=30 (KO+SAG). ****P<0.0001; **P=0.0038 (two-tailed, unpaired Student’s t-test). (E) Relative nuclear GLI2 protein level. GLI2 fluorescence intensity (red) was measured in the area demarcating the nucleus (blue) in WT and KO cells with or without SAG (100 nM) and normalized to the signal intensity of DAPI. Mean±s.e.m.; n=30 (WT), n=30 (WT+SAG), n=30 (KO), n=30 (KO+SAG). ****P<0.0001 (two-tailed, unpaired Student’s t-test). (F) qPCR of relative Gli2 levels in WT and KO cells. Cells were cultured under cilia-induced condition (high glucose DMEM, 0.5% FCS) with or without SAG (100 nM) treatment for 24 h, as indicated, and harvested for further analysis. Mean±s.e.m., n=6. ns, P>0.05 (two-tailed, unpaired Student’s t-test). (G) Western blot analysis of cytosolic GLI2 in WT and KO cells. Cells were cultured under cilia-induced condition (high glucose DMEM, 0.5% FCS) with or without SAG (100 nM) treatment for 24 h. GAPDH was used as a loading control. (H) Quantification of western blot signals as in G, showing the relative ratio between cytosolic GLI2 and GAPDH. Mean±s.e.m., n=3. ns, P>0.05 (two-tailed, unpaired Student’s t-test).
	Fig. 6. GID genes are expressed in ciliated organs during X. laevis development. Spatial analysis of expression of GID complex subunits rmnd5a (A) and mkln1 (B) in X. laevis. WMISH of WT X. laevis embryos at developmental stage 34 (St. 34; lateral views in Aa and Ba) and the corresponding transverse (b,e,d) and sagittal (c) sections (ov, otic vesicle; pe, prosencephalon; ba, branchial arches; kd, pronephric kidney; gc, ganglion cells). Images are representative of three experiments. Images in Ab and Bb are the result of tiling multiple fields of view. Scale bars: 300 μm in Aa–c and Ba–c; 100 μm in Ad, Ae, Bd and Be. (C) RT-PCR analysis of rmnd5a expression in distinct tissues of X. laevis. RT-PCR of odc1 (odc) is shown as an RNA input control (bottom). Data are representative of three experiments. (D) Quantification of relative mRNA levels of human RMND5A in various tissues. cDNA was reverse transcribed from the indicated RNA samples. β-actin (ACTB) levels were used as the qPCR internal control. RMND5A mRNA of different tissues is compared to its corresponding housekeeping gene (ACTB) in percent. In most tissues, RMND5A is expressed in an amount equivalent to more than 5% of the housekeeping gene. Mean±s.e.m., n=3. (E–H) Rmnd5a alteration is associated with ciliopathy-like phenotypes. (E) rmnd5a-mo-injected embryos (NF-St. 32, NF stage 32; is, injected side; nis, non-injected side) were used for in situ hybridization with the pax6 marker. The red arrow in images b and c indicates the eye of the injected side of the morpholinos. Scale bars: 300 μm. (F) Quantitative representation of rmnd5a-mo (rmnd5), negative control standard-mo (strd) and rescue (co-injection with rmnd5a-mo and synthetic Rmnd5a-encoding RNA) phenotypes, presented as a bar graph (embryos with phenotype as a percentage of the total number of embryos scored; black, phenotype; gray, no phenotype). n=number of injected embryos analyzed for the respective marker. ***P≤0.001 (chi-square test). (G) Analysis of the formation of head cartilage by Alcian Blue cartilage staining in swimming tadpoles at NF stage 45 (NF-St. 45). Meckel’s cartilage, ceratohyal cartilage, branchial cartilage and basihyal cartilage is shown in control standard- mo-injected (a) and rmnd5a-mo-injected (c,d) tadpoles (is, injected side; nis, non-injected side). A schematic of WT cartilage formation is shown in image b. (H) Quantitative representation of craniofacial changes in rmnd5a-mo-injected and standard-mo-injected tadpoles, with phenotypes displayed as a bar graph (embryos with a phenotype as a percentage of the total number scored; black, phenotype; gray, no phenotype). n=number of injected embryos analyzed. ***P≤0.001 (chi-square test).
	Fig. 7. Suppression of rmnd5a function interferes with neural patterning. (A–C) Embryos injected with rmnd5a-mo were used for in situ hybridization with (A) sox2 ( pan-neural marker), (B) shh (axial mesoderm and SHH target gene in the floor plate) and (C) nkx2.1 (marker of the ventral forebrain) at the indicated NF stages (st.; st. 14 in Aa, Ba; st. 15 in Bb; st. 17 in Ab, Bc, Bc′; st. 20 in Bd; st. 34 in Ac–Ac′′, Be–Be′′′′ and C). Injected sides are shown on the right of the images (red color) and in Ac′′, Ae′′, Ae′′′′ and Ca′′′, non-injected sides are shown on the left of the images and in Ac, Ae and Ca. Images show anterior views (Aa, Ab, Ac′, Ba-Bc, Be′, Be′′′, Cb), lateral views (Ac, Ac′′, Be, Be′′, Be′′′′, Ca, Ca′′′) or superior views (Bd). Expression of sox2 at NF stage 14 is shown in image Aa (anterior view; n=29/ 31); the yellow arrow indicates less patterned sox2 expression. The white arrows in images Ab, Bb and Bc indicate delayed development of the neural tube and smaller hemisphere. Yellow dashed lines in images Ab, Bb and Bd highlight sickle-shaped curvature towards the injected side. Green dashed line in image Ab indicates less patterned sox2 expression of the injected side within the presumptive brain region. Red arrow in image Ab indicates alleviated formation of the eye vesicle. Yellow arrow in image Ac′ marks the shifted olfactory anlage, and the oval indicates the remnant of the larval eye (visible in Ac–c′′). (B) Suppression of rmnd5a function results in almost normal shh expression domains. Yellow arrows indicate the prechordal plate; image Bc′ shows an inner view of an opened larva at NF stage 17. Yellow dashed line in image Bd indicates bending of the larva. Reduction of forebrain and eye development can be seen upon Rmnd5a- knockdown (yellow dashed oval) compared to the non-injected side (green dashed oval) in image Be′. Green arrow in Be′′′ shows primary expression of shh in the notochord; yellow arrow in Be′′′ and Be′′′′ shows secondary domain of shh expression in the floor plate of the neural tube. (C) Expression of nkx2.1 in NF stage 34 embryos (images a,a′,a′′′) and 20 μm vibratome sections (Ca1–a9, positions as indicated in image Ca; frontal plane shown in image Ca′′). In image Ca′′, the green arrow indicates the prospective lens on the non-injected side, the red arrow indicates a reduction of nkx2.1 expression on the injected side, the green oval indicates the prospective eye on the non-injected side, and the yellow oval indicates the prospective eye on the injected side. Asterisk indicates frontal section of the anterior head shown in Ca′′ and Ca6. Ca and Ca′′′ show lateral views of the non-injected side and injected side of the embryo, respectively. Scale bars: 300 μm. (D) Model of the primary cilium and a proposed function of the cilia-localized GID complex in the regulation of SHH protein homeostasis in the cilium. Left: primary cilium with a functional cilia-localized GID complex and functional SHH signaling response. Protein homeostasis of SHH components in the cilium is maintained (green). Right: primary cilium with aberrant protein homeostasis, reduction of SHH signaling components – namely GLI2 and PTCH1 – in the cilium and dysfunctional SHH signaling response without a functional GID complex (red).
	Fig. S1. Localization of Rmnd5a in the Xenopus laevis epidermis. Embryos were single or double injected with mRNAs (240pg~800pg per injection) at the 4-cell stage encoding (A) a) GFP-Rmnd5a (green), b) Centrin4-RFP (magenta), c) and Ac-α-tubulin (blue), d) merge; (B) (overview, left panel, GFP- Rmnd5a (green) and Centrin4-CFP (blue); middle panel, GFP-Rmnd5a and Ac-α-tubulin (magenta); right panel, Ac-α-tubulin (magenta) and Centrin4-CFP (blue)). In the yellow boxes (B), zoom sections of distinct areas of Xenopus laevis epidermis: 1) Centrioles, 2) MCC basal bodies, 3) midbody are represented. Immunofluorescence was performed on embryos fixed at embryonic stage 17 in 4% paraformaldehyde at 4°C overnight. Imaging was performed on a Zeiss LSM 700 confocal microscope. (Scale bar in A=10μm, B overview= 10μm, D. b-h=3μm).
	Fig. S2. Reduction of GID-complex function results in aberrant protein homeostasis in primary cilia. (A) Representative microscope images of PTCH1 localization. Cells were treated with cilia-inducing medium (high-glucose DMEM with 0.5% serum) for 24 h and fixed. Axonemes were stained with anti-acetylated TUBA4A (ac-tubulin) antibody (green); anti- PTCH1 antibody (red) was used to visualize PTCH1 protein and DAPI (blue) to stain DNA. Images of respective cells were merged (merge). Scale bars, 20μm. (B) Relative PTCH1 protein level per axoneme. PTCH1 fluorescence intensity (red) was measured in the area demarcating the axoneme (green) in WT and KO cells and normalized with the level of acetylated- tubulin, average WT was set to 1. Y-axes show relative ciliary PTCH1 level. Unpaired t-test n = 15 (WT), n = 15 (KO). ****, P < 0.0001. (C) Representative microscope images of GLI1 localization. Cells were treated with cilia-inducing medium (high-glucose DMEM with 0.5% serum) for 24 h and fixed. Axonemes were stained with anti-acetylated TUBA4A (ac-tubulin) antibody (green); anti-GLI1 antibody (red) was used to visualize GLI1 protein and DAPI (blue) to stain DNA. Images of respective cells were merged (merge). Scale bars, 20μm. (D) Relative GLI1 protein level per axoneme. GLI1 fluorescence intensity (red) was measured in the area demarcating the axoneme (green) in WT and KO cells and normalized with the level of acetylated-tubulin, average WT was set to 1. Y-axes show relative ciliary GLI1 level. Unpaired t-test n = 30 (WT), n = 30 (KO). ns, P > 0,05. (E) Relative nuclear GLI1. GLI1 fluorescence intensity (red) was measured in the area demarcating the nucleus (blue) in WT and KO cells and normalized with the signal intensity of DAPI, average WT was set to 1. Y-axes show relative nuclear GLI1 level. Unpaired t-test n = 30 (WT), n = 30 (KO). ns, P < 0,0001. (F) Representative microscope images of SUFU localization. Cells were treated with cilia-inducing medium (high-glucose DMEM with 0.5% serum) for 24 h and fixed. Axonemes were stained with anti- acetylated TUBA4A (ac-tubulin) antibody (green); anti-SUFU antibody (red) was used to visualize SUFU protein and DAPI (blue) to stain DNA. Images of respective cells were merged (merge). Scale bars, 20μm. (G) Relative SUFU level in cilia. SUFU fluorescence intensity (red) was measured in the area demarcating the nucleus (blue) in WT and KO cells and normalized with the signal intensity of acetylated-tubulin, average WT was set to 1. Y-axes show relative ciliary SUFU level. Unpaired t-test n = 15 (WT), n = 15 (KO). ns, P >0,05.
	Fig. S3. Suppression of RMND5A function is not associated with defects in the segmentation of pronephric kidney during development. (A) rmnd5a- morpholino (2.5 pmol/embryo) injected embryos (NF st.36) were subjected to in situ hybridization with indicated marker probes; slc12a3 (a, b) and slc5a9 (c, d), and slc5a2 (e, f). (B) rmnd5a-morpholinos injected embryos were used for in situ hybridizations (WMISH) (NF36) with indicated marker atp1a1. a’ and b’ show zoom sections of embryos kidneys. Abbreviations: is, injected side; nis, non-injected side.
	Fig. S4. Illustration of anti-RMND5A antibody specificity. Western blot analysis on NIH 3T3 cells. GAPDH serves as loading control. The anti-RMND5A antibody detects Rmnd5a in WT NIH 3T3 cells at its predicted molecular weight of 44 kDa. In contrast, the antibody does not provide a signal in NIH 3T3 cells lacking Rmnd5a.
	Fig.6.Ba GID genes are expressed in ciliated organs during X. laevis development. Spatial analysis of expression of GID complex subunits rmnd5a (A) and mkln1 (B) in X. laevis. WMISH of WT X. laevis embryos at developmental stage 34 (St. 34; lateral views in Aa and Ba) and the corresponding transverse (b,e,d) and sagittal (c) sections (ov, otic vesicle; pe, prosencephalon; ba, branchial arches; kd, pronephric kidney; gc, ganglion cells). Images are representative of three experiments. Images in Ab and Bb are the result of tiling multiple fields of view. Scale bars: 300 μm in Aa–c and Ba–c; 100 μm in Ad, Ae, Bd and Be.
	Fig.6.Aa GID genes are expressed in ciliated organs during X. laevis development. Spatial analysis of expression of GID complex subunits rmnd5a (A) and mkln1 (B) in X. laevis. WMISH of WT X. laevis embryos at developmental stage 34 (St. 34; lateral views in Aa and Ba) and the corresponding transverse (b,e,d) and sagittal (c) sections (ov, otic vesicle; pe, prosencephalon; ba, branchial arches; kd, pronephric kidney; gc, ganglion cells). Images are representative of three experiments. Images in Ab and Bb are the result of tiling multiple fields of view. Scale bars: 300 μm in Aa–c and Ba–c; 100 μm in Ad, Ae, Bd and Be.
	Embryos (NF-St. 32, NF stage 32; ... nis, non-injected side) were used for in situ hybridization with the pax6 marker.
	Fig.S3 Embryos (NF st.36) were subjected to in situ hybridization with indicated marker probes; slc12a3 (a). Abbreviations: nis, non-injected side.
	Embryos (NF st.36) were subjected to in situ hybridization with indicated marker probes; slc5a9 (c). Abbreviations: nis, non-injected side.
	Embryos (NF st.36) were subjected to in situ hybridization with indicated marker probes; slc5a2 (e). Abbreviations: nis, non-injected side.
	Embryos (NF st.36) were subjected to in situ hybridization with indicated marker probes; atp1a1 (a). Abbreviations: nis, non-injected side.

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