7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome

Abstract

Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7^-/- mice, or in Dhcr7^-/- mice lacking one copy of Ret. Although kidneys from Dhcr7^-/- mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS.

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SmithLemliOpitz syndrome (SLOS; OMIM 270400, ORPHA818) is an autosomal recessive, multiple congenital malformation syndrome, with an incidence of approximately 01/50,000 live births. SLOS presents with a very heterogeneous phenotype, causing perinatal death in its most severe forms. In general, children aected by SLOS show growth retardation, developmental delay and intellectual disability. Craniofacial defects include micro-cephaly, micrognathia, and ptosis (dropping of the eyelid). Individuals aected by SLOS oen display postaxial polydactyly of the hands or feet, and syndactyly of the 2nd and 3rd toes. Internal malformations include renal hypoplasia, ambiguous genitalia in male, pyloric stenosis, colonic aganglionosis (Hirschsprung disease), holoprosencephaly and cardiac malformations. SLOS patients are hyperactive, display self-injurious behavior, and demonstrate autistic characteristics [reviewed in13].

SLOS is caused by inherited mutations of the 7-dehydrocholesterol reductase gene (DHCR7)4,5. DHCR7 gene

product catalyzes the last step of cholesterol biosynthesis, namely conversion of 7-dehydrocholesterol (7-DHC) into cholesterol. As a consequence, DHCR7 deciency impairs cholesterol production, resulting in elevated 7-DHC levels and typically decreased cholesterol levels. Cholesterol is a major structural component of plasma membranes, is the precursor of steroid hormones, bile acids and constitutes a critical component of plasma membrane microdomains known as lipid ras. Moreover, cholesterol is necessary for proper function of the sonic hedgehog (Shh) pathway6, and 7-DHC is the precursor of vitamin D. Given the multiple and important functions of cholesterol, it is not surprising that the molecular mechanisms by which such altered cholesterol/7-DHC ratio cause the variety of developmental abnormalities observed in SLOS patients are only partially understood.

Department of Experimental Medicine, Universitat de Lleida/Institut de Recerca Biomdica de Lleida (IRB Lleida), Spain.

Departments of Internal Medicine (Renal Present address: Correspondence and requests for materials should be addressed to M.G.-F. (email: [email protected]) or M.E. (email: [email protected])

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The GDNF family ligands (GFLs) constitute a group of neurotrophic factors (GDNF, NRTN, PSPN, ARTN) that regulate several aspects of the developing nervous and genito-urinary systems7. GFLs signal through a common tyrosine kinase receptor (Ret), and one of the four co-receptors known as GFR, which bind directly to GFLs providing ligand specicity. GFR co-receptors are attached to the outer leaet of the plasma membrane via a glycosyl-phosphatidyl inositol anchor, which targets them to lipid ras. Stimulation by GFLs prompts recruitment of Ret to lipid ras, where the GFL-GFR-Ret ternary complex interacts with Src family kinases to elicit maximal biological responses8. Interestingly, stimulation of Ret by soluble GFR1 also drives Ret to lipid ras where it interacts with FRS29. Moreover, partitioning of Ret to lipid ras not only promotes association with key signaling intermediates, but prevents its proteasomal-mediated degradation10. Finally, a very recent report shows that knockin mice expressing a GFR1 mutant localized outside lipid ras phenocopy several aspects of Ret knockout mice, conrming the importance of GDNF-driven translocation of Ret to lipid ras for its proper function11.

In vivo, Ret signaling is necessary for the proper development of the autonomic nervous system and the genito-urinary system. Mice bearing a variety of Ret mutations (as well as mutations in certain GFLs or their co-receptors) show dierent degrees of colonic aganglionosis, thus mimicking Hirschsprung disease in humans. On the other hand Ret signaling is crucial to kidney development, and mice bearing mutations in Ret or its co-receptor GFR1 develop several kidney defects ranging from complete agenesis to dierent degrees of kidney hypoplasia1218. Finally, mice lacking either ARTN or GFR3 develop ptosis owing to failure of sympathetic neurons form the cervical superior ganglion to innervate the tarsus muscle19,20.

Given the overlapping spectra of congenital abnormalities found both in SLOS patients and Ret mutant mice and the necessity of intact lipid ras for optimal Ret function, in the present work we aimed to elucidate whether abnormal Ret signaling plays a role in the pathogenesis of SLOS. Analysis of the genito-urinary and enteric nervous systems of Dhcr7/ or Dhcr7/; Ret+/EGFP mice revealed no abnormalities consistent with compromised Ret signaling. Although kidneys from mutant mice branched at a slower pace than those from wild type littermates when explanted in the absence of exogenous cholesterol, sympathetic neurons from mutant mice showed normal responses to GDNF under the same conditions. Moreover, critical components of Ret signaling including GFR 1 or Src family kinases from knockout mice partitioned to the lipid ra fraction in otation gradients, suggesting that 7-DHC allows formation of lipid ras that efficiently support Ret signaling. In support with this notion, removal of cholesterol followed by replenishment with 7-DHC efficiently supported GDNF-mediated phosphorylation of Akt in MG87 broblasts cultured in the absence of cholesterol. Collectively, the data indicate that aberrant Ret signaling is unlikely to play a major role in the pathogenesis of SLOS.

Although no major defects in kidney and digestive system were described in Dhcr7 knockout mice, we sought to investigate whether subtle abnormalities reecting compromised Ret signaling could be identied in these mice. Macroscopic examination of the genito-urinary system of newborn mutant mice revealed no overt malformations consistent with aberrant Ret signaling such as kidney aplasia or hypoplasia, cystic or supernumerary kidneys, or ureter abnormalities (Fig.1A, le panel). Hematoxylin-eosin staining showed that mutant kidneys had normal architecture, with well-organized cortex and medulla, a prominent nephrogenic zone, and no cystic dilations or signs of hydronephrosis (Fig.1A, le panel). The number of glomeruli was also similar between Dhcr7 knockout mice and their wild type littermates (Fig.1B), consistent with proper branching of the ureteric bud. We next wanted to check the status of enteric nervous system. Immunostaining of colon from newborn mice with PGP9.5, which labels enteric neuronal precursors21 showed no signs of colonic aganglionosis, the hallmark of Hirschsprung disease (Fig.1B, le panel). Enteric plexus density was assessed throughout the entire gut by means of acetylcholinesterase staining, which labels cholinergic enteric neurons, revealing no dierences between genotypes (Fig.1B, right panel).

Ret The above results were not surprising in light of the relatively mild phenotype of Dhcr7 null mutants when compared to SLOS patients. Besides cle palate and immature lungs, Dhcr7 knockout mice show no overt developmental abnormalities reminiscent of the human disease22. It is usually assumed that such discrepancies are due to dierences in transplacental cholesterol transport between species, being it obligated in rodents23,24 but

not humans25. Thus, maternal cholesterol transfer to embryos, possibly via Abca1 cholesterol transporter26, would compensate cholesterol deciency in murine but not human embryos. To determine whether reduction in Ret gene dosage could unveil suboptimal Ret signaling in the context of Dhcr7 deciency, we crossed Dhcr7 mutant mice to heterozygous Ret knockin mice expressing one copy of EGFP from the Ret locus27, thus allowing easy tracking of Ret-expressing cells. As shown in Fig.2, removal of one copy of Ret did not aect ureteric bud branching or enteric plexus density (n 3 for each genotype), thus reinforcing the idea that no major abnormalities related to abnormal Ret signaling are found in Dhcr7 knockout mice. Taken together, the above observations are not consistent with abnormal Ret function in the developing urinary and nervous systems of Dhcr7 knockout mice.

in vitro To examine Ret signaling avoiding the confounding eect of maternal cholesterol transfer, we decided to place embryonic metanephroi from Dhcr7 mutant embryos in culture, in the absence of any exogenous source of cholesterol. As stated in the introduction, ureteric bud branching is critically dependent on GDNF-Ret signaling, both in vivo and in explant culture. We explanted metanephroi from E12.5 wild type and null embryos in dened medium containing retinoic acid, and counted ureteric bud tips aer two and ve days in vitro. We pooled data from wild type and Dhcr7 knockout mice irrespectively of the Ret allele expressed as no dierences on ureteric bud branching were found

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Figure 1. Genito-urinary and enteric nervous systems do not show developmental alterations in a SLOS mouse model. (A) Macroscopic and microscopic appearance of kidney and lower urinary tracts (le panel), and glomeruli number (right panel) of newborn wild type and Dhcr7 knockout mice. (B) Representative images of acetyl cholinesterase (AChE) and PGP9.5 staining (le panel) and acetyl cholinesterase-positive ber density (right panel) of newborn wild type and Dhcr7 knockout mice.

between Ret+/EGFP and Ret+/+ embryos within a given Dhcr7 genotype (data not shown). We then stained Ret+/+ metanephric explants with an antibody against CalbindinD-28k, a marker of the ureteric bud28, or directly photographed Ret+/EGFP metanephroi under a uorescence microscope. We used both techiniques at both 2days in vitro and 5days in vitro . As shown in Fig.3A, small but signicant dierences between wild type and Dhcr7 knockout metanephroi were found aer two days in culture, which increased with time in culture (Fig.3B). Thus, ureteric bud branching is reduced in Dhcr7 null embryonic kidneys when cultured in dened medium and are therefore devoid of exogenous cholesterol.

Even though signaling by the GDNF-GFR1-Ret axis is the major player in ureteric bud branching, the above observations do not directly prove suboptimal Ret signaling in this system. To directly assess biological responses downstream of Ret, we chose sympathetic neurons from the superior cervical ganglion as a model system. The importance of lipid ras in Ret signaling has been extensively characterized in sympathetic neurons from the superior cervical ganglion (SCG)10. We rst used immunoblots to phosphorylated ERK as a readout of Ret signaling in sympathetic neurons cultured for ve days in the presence of lipid-depleted serum, and stimulated with GDNF in the presence soluble GFR1. As shown in Fig.4A, no major dierences on GDNF-mediated ERK phosphorylation were found between wild type and mutant neurons. We next assessed survival of sympathetic neurons in the presence of increasing concentrations of GDNF and a xed concentration of sGFR1. As shown in Fig.4B, no dierences between genotypes were observed at any of the concentrations assayed. Neurite outgrowth of SCG explants in response to GDNF was also similar between wild type and Dhcr7 null mice (Fig.4C). Thus, at least for sympathetic neurons, mutation of Dhcr7 does not appear to have any consequence on GDNF-induced Ret signaling.

As stated in the introduction, GFR1, which is located to lipid ras by virtue of its glycosylphosphatydilinositol anchor, recruits Ret to these membrane domains upon ligand stimulation. We next investigated whether lack of eect of Dhcr7 mutation on Ret bioactivity in neurons was due to normal partition of GFR1 to lipid ras. To this end, we generated otation gradients from neonatal brain extracts of both control and Dhcr7 null mice. Importantly, cholesterol does not cross the blood-brain barrier and thus the contribution of maternal cholesterol to central nervous system drops from E10-11 onwards, being 90% of neonatal brain cholesterol of embryonic origin29. Therefore, 7-DHC but

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Figure 2. Reducing Ret dosage does not unveil developmental defects of the genito-urinary and enteric nervous systems of Dhcr7 knockout mice. Representative uorescence images metanephroi (top and middle panel) and colon (bottom panel) from Dhcr7+/+; Ret+/EGFP and Dhcr7/; Ret+/EGFP embryos of the indicated

ages. No overt dierences in ureteric bud branches or plexus density are observed.

not cholesterol is the major sterol in brains from Dhcr7 knockout mice. As shown in Fig.5, GFR1 was mainly found in the high-buoyant, lipid ra fractions, in both wild type and Dhcr7 mull mice. Partition of other critical components of Ret signaling such as Src family kinases was also similar in wild type and Dhcr7 knockout mice (Fig.5). Distribution of lipid ra (GM1 ganglioside, caveolin-1 and otillin-1) and non-ra (transferrin receptor) markers indicated correct fractionation of the samples. Thus, loss of Dhcr7 does not aect distribution of critical components of Ret signaling to lipid ras in brain, suggesting that 7-DHC can efficiently support Ret signaling. To directly test this hypothesis, we conducted cholesterol depletion followed by 7-DHC replenishment experiments. We rst conrmed that depletion of membrane cholesterol by means of methyl--cyclodextrin (MCD) aected signaling downstream of Ret in the mouse broblast cell line MG87 stably expressing Ret and GFR1, a widely used model to study Ret signaling. As shown in Supplemental Fig. 1, GDNF-mediated phosphorylation of both ERKs and Akt were aected by increasing concentrations of MCD to dierent extents, being the PI3-K/ Akt pathway more sensitive to cholesterol depletion. Since MCD was toxic at high concentrations even for short exposure times, we used the lowest eective concentration of this compound (4 mM) and focused on p-Akt phosphorylation for replenishment experiments. As shown in Fig.6A, replenishment of cellular membranes with 7-DHC supported GDNF-mediated Akt phosphorylation to levels comparable to those obtained in parallel cultures not subjected to depletion/replenishment treatments, or to cells replenished with cholesterol. Interestingly, replenishment with 7-DHC in the presence of BM15766, a novel Dhcr7 inhibitor, did not substantially changed Akt phosphorylation, indicating that conversion of 7-DHC to cholesterol was not responsible for rescuing the pathway. To assess the efficacy of these stripping/replenishment experiments, we measured both 7-DHC and cholesterol in cell extracts by means of liquid chromatography quadrupole time-of-ight mass spectrometry (LC-QTOF-MS/MS), as described in methods. MCD depleted approximately half of the cellular cholesterol,

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Figure 3. Ureteric bud in vitro branching is modestly reduced in Dhcr7 knockout mice. (A) Calbindin immunostaining of E12.5 kidney explants of the indicated genotypes cultured in the absence of exogenous source of cholesterol for 2days (le panel). Quantication of ureteric bud tip number aer 2days in vitro (2DIV). (B) EGFP immunouorescence of E12.5 kidney explants of the indicated genotypes cultured in the absence of exogenous source of cholesterol for 5days (le panel). Quantication of ureteric bud tip number aer 5days in vitro (5DIV) (right panel).

which was efficiently replenished by MCD:Cholesterol. Importantly, MCD:7-DHC complexes did not increase cholesterol above levels found in cells treated with MCD alone, indicating that 7-DHC was not being converted to cholesterol in cells exposed to MCD:7-DHC. Finally, we only detected 7DHC in cells treated with MCD:7-DHC complexes but not in the rest of conditions (Fig.6B,C). Taken together, these experiments indicate that 7-DHC efficiently supports Ret signaling.

In the present work we examined the hypothesis of whether suboptimal Ret signaling could explain some of the developmental abnormalities found in SLOS patients. Such hypothesis was formulated based on the overlapping phenotypes of SLOS patients and Ret mutant mice, and the observation that lipid ras are critical for proper Ret signaling.

Even though the genetic defect causing SLOS is well-established, the molecular mechanisms leading to the plethora of developmental malformations found in SLOS patients are only partially understood. Besides being a fundamental component of plasma membranes, cholesterol serves as precursor for a number of important bio-molecules such as bile acids, steroid hormones and signaling molecules. Such multiplicity of functions makes it unlikely that a single molecular mechanism underlies the pathogenesis of SLOS. Moreover, not only cholesterol deciency but the toxic eects of 7-DHC (or its derived oxysterols) buildup may account for some of the complications found in SLOS patients3,30. A number of observations suggest that membrane structural and functional properties may be aected in SLOS patients. 7-DHC has been shown to induce changes in the physico-chemical properties of both articial and cellular membranes, including uidity, electric potential, and curvature3134.

These changes likely account for functional alterations of membrane proteins such as the NMDA and serotonin receptors35,36. Of particular interest are the abnormalities found in lipid ras, as cholesterol is one important component of these membrane microdomains. Thus, 7-DHC has been shown to induce changes in both the lipid and protein composition of ras37,38. Likewise, changes in lipid ra stability may account for impaired degranulation responses of mastocytes in response to IgE39, or for alterations in caveolae and the potassium channel BKCa in skin broblasts40. Despite these observations, our data show that GFR1 (and Src-family kinases) equally partition to lipid ra fractions in brain extracts from both wild type and Dhcr7 null mice, suggesting that at least in central neurons Ret would be correctly recruited to lipid ras upon GDNF stimulation. It is important to stress that brain cholesterol is synthesized by the embryo from E11.5 onwards, and therefore maternal cholesterol is likely to be residual in brains from newborn Dhcr7 null mice. Even though GFR1-mediated recruitment is not the only mechanism by which Ret moves to lipid ras upon ligand stimulation9, recent in vivo data show that it constitutes the major mechanism involved in such translocation11.

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Figure 4. Dhcr7/ sympathetic neurons properly respond to GDNF stimulation. (A) Superior cervical ganglion neurons from wild type and Dhcr7 knockout were cultured and stimulated with GDNF as described in methods. Total lysates were resolved by SDS-PAGE and probed with the indicated antibodies (le panel). The graph on the right panel shows the relative intensity of phospho-ERK signal normalized by tubulin. (B) GDNF-mediated survival of SCG neurons was evaluated aer 48hours in the presence of growing concentrations of GDNF. (C) Representative images of SCG explants aer ve days in the presence of 50ng/ml GDNF.

We have found that embryonic kidney explants from knockout mice branch at a slightly slower pace than their wild type counterparts. Even though GDNF-Ret signaling is the master regulator of kidney branching, we do not know whether these defects are caused by impaired Ret signaling in mutant kidneys. In light of our results with sympathetic neurons we nd such possibility unlikely, although we cannot exclude the possibility that sensitivity of Ret signaling to 7-DHC varies depending on the cellular context. Thus, mice expressing a non-ra version of GFR1 (GFR1TM/TM mice) show defects on urogenital, enteric and motor but not sympathetic nervous system11. However, the renal phenotype of GFR1TM/TM mice is a fully penetrant, complete kidney agenesis, whereas we observe a modest branching defect that at the most suggests that 7-DHC partially supports Ret signaling in the ureteric bud. Alternatively, other signaling molecules that play a secondary role in kidney branching might be aected by lack of cholesterol/increased levels of 7-DHC. Thus, signaling by FGF10 is able to induce kidney morphogenesis in animals lacking Ret and Sprouty141. Similarly, perturbation of Shh signaling causes kidney defects in mice ranging from kidney hypoplasia to kidney agenesis42,43. Interestingly, altered SHH signaling is consistent with some SLOS features such as holoprosencephaly and polydactyly, and previous cell culture work demonstrates diminished responses to Shh in cholesterol-depleted cells44. However, the eect of Dhcr7 on the activity of the Shh pathway is controversial. Studies in Xenopus show that Dhcr7 negatively regulates the pathway by inhibiting Smo, in a reductase-independent manner45. In contrast, Bijlsma and colleagues46 show that secretion of pro-vitamin D3 (whose precursor is 7-DHC) inhibits Smo activity, suggesting that Dhcr7 should activate the pathway by keeping the levels of 7-DHC low. In contrast, a more recent work provides compelling genetic and pharmacological evidence that reduced cholesterol rather than increased 7-DHC or oxysterol levels block Shh pathway by impairing Smo activity47. All these observations point to a complex regulation of the Shh pathway by sterols beyond their role as covalent modications of the Shh molecule. Another intriguing mechanism by which 7-DHC might regulate kidney morphogenesis is through modulation of the Wnt/-catenin pathway. Thus, a very recent report establishes that elevated 7-DHC but not diminished cholesterol levels are responsible for aberrant neuronal dierentiation of induced pluripotent stem cells derived from SLOS patients, via inhibition of the Wnt/-catenin pathway48. Interestingly, genetic inactivation of -catenin in the ureteric bud in mice leads to kidney agenesis/dysgenesis owing to reduced ureteric bud branching49.

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Figure 5. The Ret co-receptor GFR1 is properly located to lipid ra fractions in Dhcr7/ mice.

(A) Representative immunoblots of consecutive fractions from wild type and Dhcr7 knockout brain homogenates subjected to sucrose density centrifugation. Six equal fractions were taken from the top of the tubes and labeled from fraction 1 (non-ras) to fraction 6 (ras). Protein in fractions was resolved by SDS-PAGE and probed with the indicated antibodies. (B) Relative intensity of GFR1 bands (mean SEM) from three independent experiments.

In conclusion, our results show that 7-DHC has the ability to support Ret signaling in several model systems, indicating that impaired Ret function is unlikely to contribute to the pathogenesis of SLOS.

All experimental procedures involving mice were in accordance with national and regional guidelines and approved by the experimental Animal Ethic Committee of the University of Lleida. Dhcr7 null mice22 were purchased from Jackson Laboratories (Bar Harbor, Maine). Mice bearing a oxed Ret allele followed by EGFP (RetoxEGFP) have been previously described27. These mice were crossed to transgenic mice expressing Cre recombinase under the control of phosphoglycerate kinase 1 promoter [Jackson Laboratories50] leading to widespread excision of the oxed Ret allele and subsequent expression of EGFP from the Ret locus.

Newborn mouse gut was dissected and xed in 4% paraformaldehyde for 1 to 2h at 4C, transferred to saturated sodium sulfate, and stored overnight at 4C. Acetylcholinesterase staining was then performed exactly as described51. Quantication of enteric plexus ber density was determined from randomly selected elds per mouse, by counting the number of upper and right side crossing bers points of a dened grid as described by52.

Kidneys from wild type and Dhcr7 knockout newborn littermates were dissected, xed in 4% paraformaldehyde, dehydrated and embedded in paraffin blocs. Histological analysis was performed on serial 5m sections stained with Hematoxylin and Eosin. Kidneys were sectioned and glomeruli were counted as previously described53.

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Figure 6. 7-DHC supports Ret signaling. (A) MG87 cells were treated or not with MCD for 30min to deplete cholesterol (stripping), followed by 30min replenishment step with MCD alone, loaded with 7-DHC or with cholesterol, or with 7-DHC in the presence of the Dhcr7 inhibitor BM15766. Cells were then stimulated with GDNF for 10minutes. Total lysates were resolved by SDS-PAGE and probed with the indicated antibodies.(B) Representative extracted ion chromatograms and MS spectrum for 7-DHC and Cholesterol in samples from cells replenished with MCD:7-DHC complexes. Chromatograms shown retention times of the compounds and the MS peak used for quantication. The ions selected for quantication was m/z (M +H)+(H2O): 367.3360 for 7-DHC and m/z (M+H)+(H2O): 369.3519 for cholesterol. (C) Relative abundances of 7-DHC and cholesterol measured by means of LC-QTOF-MS in two independent experiments. Numbers indicate the ratio of the indicated metabolite (7-DHC or cholesterol) with respect to an internal standard (13C-cholesterol).

N.d.: not detected.

Neurons from the sympathetic superior cervical ganglion of newborn Dhcr7 mice cultured as described54 with minor modications. Briey, ganglia were enzymatically digested, dissociated and plated on rat tail collagen-coated dishes in medium containing 50 ng/ml NGF (Alomone labs, Jerusalem, Israel) and 10% lipid-depleted fetal bovine serum (Life Technologies, Carlsbad, CA) for 57days. For survival experiments cells were incubated with the indicated concentrations of GDNF plus 100 ng/ml Fc-GFR1 (R & D Systems, Minneapolis, MN). Neuronal survival was assessed by counting neurons of designated elds before and aer

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treatments at the indicated time points. For immunoblots cells were treated in complete medium as above, deprived of NGF overnight, and stimulated for 10 minutes with 50ng/ml GDNF plus 100ng/ml Fc-GFR1. For SCG explants, whole superior cervical ganglion from newborn Dhcr7 mice were dissected and plated in collagen matrices in the presence of NGF and charcoal stripped serum (Life Technologies, Carlsbad, CA). Twenty four hours later, each well was washed three times with PBS and switched to medium supplemented with GDNF (50 ng/ml) and charcoal stripped serum for ve days. MG87 broblasts stably expressing Ret and GFR1 were maintained in DMEM plus 10% lipid-depleted FBS.

Metanephroi were dissected from E12.5 embryos, placed on 0.4 m pore PTFE inserts (Millipore), and cultured on air-uid interphase with dened media in the bottom chamber [DMEM, 1x ITS supplement (Life Technologies) and 200 nM all-trans retinoic acid (Sigma, St. Louis, MO)]. Microscopy images were obtained at the indicated time points, and the number of ureteric buds was counted and expressed as percent of wild type. Culture media was changed every 48h.

A slight modication of the method used by Tansey and collaborators8 was used. Brains from newborn mice were homogenized in 0.5 ml of 0.1% Triton lysis buer [50 mM Tris-HCl (pH 7.4), 150mM NaCl, 5mM EDTA, 0.1% Triton X-100, 10mM NaF, 1mM Na2VO4, 50mM -glycerophosphate, 2mM Benzamidine, 1g/ml leupeptin and 1g/ml aprotinin] using a dounce tissue grinder, followed by four passages through a 22G needle. Homogenates were centrifuged at 3000 rpm at 4 C for 10 min, and supernatants were adjusted to 35% sucrose, placed in a SW55Ti ultracentrifuge tube (Beckman), and overlayed with 3.5ml of 30% Sucrose in 0.1% Triton lysis buer and 0.5 ml of 0.1% Triton lysis buer. Aer centrifugation (4 h, 200,000 g, 4C), six fractions were collected from the top, TCA-acetone precipitated, air dried, and resuspended in 50 l of Laemli sample buer.

Western blot was conducted as previously described54 using primary antibodies listed in Supplementary Table 1. Immunoblots were developed using a VersaDoc device (BioRad, Hercules, CA), and densitometry performed using the soware package ImageLab (BioRad). Ganglioside GM1 was visualized by directly probing membranes with HRP-conjugated cholera toxin B subunit (Millipore).

Tissues were xed in 4% paraformaldehyde and incubated in blocking buer (PBS containing 0.2% Triton X-100 and 2% BSA) overnight at 4C. Tissues were then incubated with the indicated primary antibodies (Supplementary Table 1) in blocking buer overnight at 4 C, washed several times in blocking buer at room temperature, and incubated with the appropriate DyLight-labeled secondary antibodies (Jackson ImmunoResearch Europe, Newmarket, Suolk, UK) for 1 h at room temperature (metanephric explants) or overnight at 4C (guts).

Membrane cholesterol was depleted by adding 4 mM MCD (Sigma) dissolved in serum-free medium to cells for 30min at 37C. For replenishment experiments, cholesterol depletion with 4mM MCD was followed by a 30 min incubation with 4 mM MCD alone, or complexed with 0.4 mM 7-DHC or 0.4 mM cholesterol in serum free medium. 7-DHC was complexed to MCD following the method of Christian and collaborators55. Briey, 7-DHC was dissolved in chloroform:methanol 1:1 (v:v) to a concentration of 50mg/ml. An aliquot of this solution was added to a glass tube and evaporated. Then the appropriate volume of MCD dissolved in serum-free medium was added, sonicated and mixed overnight at 37C. Solutions were ltered through a 0.45M lter before use. Cholesterol: MCD complexes were purchased from Sigma and adjusted to 1:10 molar ratio with MCD. When indicated, BM15766 (Sigma) was included at a nal concentration of 5M. Cells were then washed three times and stimulated with or without GDNF (50ng/ml) for ten minutes.

Aer depletion/replenishment, cells were lysed in 1 ml of ice-cold methanol, scraped o the dishes, and sonicated. Two ml of chloroform (containing 13C cholesterol as internal standard) were added to 1 ml of cell lysate and vortexed for 10 s. Then, 0.75 ml of KCl 0.7% were added and vortexed for 10s. The samples were centrifuged at 1000g during 15min at 4C and chloroform phase (lower) was separated in a glass tube, evaporated using a Speed Vac (Thermo Fisher Scientic, Barcelona, Spain) and resuspended in 200 l of methanol/chloroform (3:1)56. For LC-Q-TOF-based lipid molecular species analyses, lipid extracts were subjected to liquid chromatography-mass-spectrometry using a HPLC 1290 series coupled to ESI-Q-TOF MS/MS 6520 (Agilent Technologies, Barcelona, Spain). Two microliters of lipid extract were injected onto an XBridge BEH C18 shield column (100mmL2.1mm ID 1.7m; Waters, Milford, MA, USA) kept at 80 C. The mobile phases, delivered at 0.5 ml/min, consisted of ammonium formate (20 mM at pH 5) (A) and methanol (B). The gradient started at 50% B and reached 70% B in 14min and was followed by a slow gradient of 7090% B over 50min and an isocratic separation at 90% B for 15min. The mobile phase B subsequently reached 100% over 5 min and was maintained for an additional 5 min57. Data were collected in positive electrospray ionization-TOF operated in full-scan mode at 1003000m/z in an extended dynamic range (2GHz) (MassHunter Data Adquisition Sofware, Agilent Technologies, Barcelona, Spain), using N2 as nebulizer gas (5 L/min, 300C). The capillary voltage was 3500V with a scan rate of 1scan/s.

For targeted search of cholesterol, 7-DHC and 13C-cholesterol (internal standard) MassHunter Qualitative Analysis Soware (Agilent Technologies, Barcelona, Spain) was used. Specically, Find by formula algorithm was applied to select the main ion for each compound. These ions ((M + H) + (H2O) = 367.3360 for 7-DHC, (M + H) + (H2O) = 369.3519 for cholesterol and (M + H) + (H2O) = 370.3519 for 13C cholesterol) were used for metabolite quantication56.

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The ShapiroWilk test was used to determine whether variables followed a normal distribution. A Students t-test was used to determine dierences between normally distributed variables, otherwise a ManWhitneys U-test was applied. Dierences were considered statistically signicant when p <0.05.

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We thank Lidia Parra for technical support, and the Metabolomics Facility of IRB Lleida for lipidomic analyses. This work was supported by grants from Fundaci La Marat de TV3 (101810), and from Ministerio de Economa y Competitividad (BFU2010-17628 and BFU2013-47175), and funding from Suport als Grups de Recerca (2014 SGR 138) from Generalitat de Catalunya to ME. MG-F was supported by a grant from Fundaci La Marat de TV3. AM was supported by a predoctoral fellowship from Universitat de Lleida. C A is supported by a predoctoral contract from Universitat de Lleida. MV was supported by a predoctoral fellowship from AGAUR (Generalitat de Catalunya).

M.G.-F. conducted research, analyzed results, and wrote the paper. A.M. conducted research and analyzed results. C.A. conducted research and analyzed results. M.V. conducted research and analyzed results. S.J. contributed reagents and analyzed results. M.J. conducted research and analyzed results. J.R. conducted research and analyzed results. M.E. conceived research, analyzed results, and wrote the paper.

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Gou-Fbregas, M. et al. 7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome. Sci. Rep. 6, 28534; doi: 10.1038/srep28534 (2016).

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