Overlapping expression patterns of the multiligand endocytic receptors cubilin and megalin in the CNS, sensory organs and developing epithelia of the rodent embryo

Gene Expression Patterns 6 (2005) 69–78 www.elsevier.com/locate/modgep

Overlapping expression patterns of the multiligand endocytic receptors cubilin and megalin in the CNS, sensory organs and developing epithelia of the rodent embryo Emeline Asse´mata, Franc¸ois Chaˆteleta, Jacqueline Chandelliera, Fre´de´ric Commob, Olivier Casesc, Pierre Verrousta, Renata Kozyrakia,* a

Inserm UMR 538, Faculte´ de Me´decine Saint-Antoine, Universite´ Pierre et Marie Curie, 27 rue de Chaligny, 75012 Paris, France b Department of Anatomopathology, Hoˆpital Tenon, Assistance Publique des Hoˆpitaux de Paris, 75020 Paris, France c Inserm U 616, Hoˆpital de la Pitie´-Salpe´trie`re, 47 bd de l’Hoˆpital, 75013 Paris, France Received 7 March 2005; accepted 26 April 2005 Available online 18 July 2005

Abstract Cubilin and megalin are multiligand epithelial endocytic receptors well characterized in the adult kidney and ileum where they form a complex essential for protein, lipid and vitamin uptake. Although inactivation of the megalin gene leads to holoprosencephaly and administration of anti-cubilin antibodies induces fetal resorptions or cranio-facial malformations their function in the developing embryo remains unclear. We recently showed that both proteins are strongly expressed by the maternal–fetal interfaces and the neuroepithelium of the early rodent embryo where they co-localize and form a complex important for nutrient uptake. The aim of the present study was the further investigation of cubilin expression at later developmental stages of the rodent embryo and its correlation to that of megalin. Immunohistochemical and in situ hybridization analysis showed striking similarities in the spatial and temporal expression patterns of cubilin and megalin. The electrophoretic mobility of both proteins was identical to that of the adult as revealed by Western blot analysis. Cubilin and megalin were strongly expressed in the sensory organs, the central nervous system, the respiratory and urogenital tracts as well as in the thymus, parathyroids and thyroid. In each site, the expression mainly concerned epithelial structures and correlated with the onset of epithelial induction. Depending on the site, a decreased or restricted expression was observed by the end of the gestation for both proteins. q 2005 Elsevier B.V. All rights reserved. Keywords: Cubilin; Megalin; Multiligand endocytic receptors; Sensory organs; Epithelia

1. Results and discussion Endocytosis in the adult renal proximal convoluted tubule and ileum is largely mediated by two multiligand receptors cubilin and megalin (Verroust and Christensen, 2002). It is now well established that in these sites, cubilin and megalin form an oligomeric complex essential for protein, vitamin A, D, B12 and lipid uptake (Kozyraki, 2001). Cubilin is a 460 kDa membrane protein composed by highly interactive modules, EGF (Epidermal Growth Factor) and CUB (Complement Uegf Bmp1) domains, * Corresponding author. Tel.: C33 1 40 01 13 29; fax: C33 1 40 01 13 90. E-mail address: [email protected] (R. Kozyraki).

1567-133X/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2005.04.014

responsible for the multiligand properties of the receptor (Moestrup et al., 1998). Megalin is a member of the LDLR (Low Density Lipoprotein Receptor) gene family that associates with and drives endocytosis of cubilin and its ligands since cubilin lacks a transmembrane domain (Christensen and Verroust, 2002; Moestrup et al., 1998). Two lines of evidence suggest that cubilin and megalin are important for normal embryonic growth. Injection of anti-cubilin antibodies into the pregnant rat before gestational day 10 (E10) leads to embryonic resorptions or severe malformations of the rostral part of the embryo (Sahali et al., 1988). Megalin knock-out mice present holoprosencephaly, vitamin D-deficiency and die perinatally probably because of respiratory insufficiency (Nykjaer et al., 1999; Willnow et al., 1996). Recent studies suggest involvement of megalin in the sonic hedgehog (SHH) and the bone morphogenic protein 4 (BMP4)

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signaling pathways and thus in embryonic patterning of the ventral forebrain (Spoelgen et al., 2005). To understand the function of cubilin during development we recently analyzed its expression in the peri-implantation embryo (Assemat et al., 2004). We showed that cubilin is co-expressed together with megalin by the rodent trophectoderm and after implantation by the visceral yolk sac. Our data suggest that the cubilin–megalin complex already forms at the blastocyst stage and that high density lipoproteins (HDL) binding to cubilin accounts for cholesterol delivery to the embryo during early gestation. We also showed that the forming rodent neuroepithelium at E10 is positive for both cubilin and megalin (Assemat et al., 2004). Although expression of cubilin in the developing kidney has previously been reported (Sahali et al., 1993) no data exist concerning its presence in other embryonic tissues. The purpose of this study was the analysis of the overall expression pattern of cubilin and its correlation to that of megalin in the rodent post-implantation embryo, from E11 and throughout gestation. Immunohistochemical, biochemical, and in situ hybridization analysis revealed

a very similar distribution of cubilin and megalin in the sensory organs, the central nervous system (CNS) and various developing epithelia. 1.1. Localisation in the brain and spinal cord Similar localization of cubilin and megalin was observed in the developing brain and spinal cord (Figs. 1 and 2). At E11 (Figs. 1A,E, 4A) both receptors were detected in the neuroepithelium of the neural tube, and the notochord. Beginning at E12 (Fig. 1B,C,F,G) the expression became progressively restricted in the ventricular zone of the ventral neural tube although some immunoreactivity could also be found dorsally. From E13, cubilin and megalin were mainly detected in the ventral regions (Figs. 1D,H, 2A–D) of the four ventricles, in the optic recesses and the ventral diencephalon, and in the ventral spinal cord (not shown). The infundibular recess at the floor of the forebrain (giving rise to neurohypophysis) and the future Rathke’s pouch (Fig. 1I–K) were similarly stained for both cubilin and megalin. The notochord became negative for both cubilin

Fig. 1. Immunolocalisation of cubilin (cub) and megalin (meg) in the developing neural tissues. (A) and (E) Expression of megalin and cubilin, respectively, on transverse sections of the neural tube at E11. Staining in the neuroepithelium (neur) and the notochord (nt) as well as in cells of the gut. (B), (C), (F) and (G) Transverse sections of the spinal cord and notochord (nt) at E12. Note megalin and cubilin staining in the floor plate (fp) and notochord. (D) and (H) Transverse sections through the mesencephalon at E13. Similar staining of cubilin and megalin in the floor plate (fp) of the third ventricle (3V). (I), (J) and (K) Similar expression of cubilin and megalin on sagittal sections of the developing pituitary at E12, (I) and (J), and E14, (K). Note staining in the Rathke’s pouch (rp) and in the infudibular recess (inf) of the diencephalon (dienc). Scale bars: 100 mm in A, B, E, F, I, J and K, 50 mm in C, D, G and H.

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Fig. 2. Distribution of cubilin (cub) and megalin (meg) in the CNS as revealed by immunostaining and ISH. (A) Sagittal section providing an overview of megalin expression in the forebrain (ventral lateral ventricle (lV) and choroid plexus (chp) at E14. (B) Higher magnification of the same region immunoreactive for cubilin. Note staining in the cortical hem. (C)–(E) The ventricular zones of the ventral third (arrow in C) and fourth ventricles (3V, 4V) and the choroid plexuses similarly expressed cubilin and megalin at E14. Some residual immunoreactivity could also be observed dorsally. (F), and (G) Weak mRNA (arrow in F) and protein expression of cubilin on transverse and sagittal sections, respectively, through a lateral ventricle at E17. (H)–(J) Sagittal sections through the third and fourth ventricles at E17. The ventricular zone of the ventral thalamus (th) and the fourth ventricle express both cubilin and megalin. (ge) ganglionic eminence, (hpc) hippocampus, (pg) pineal gland recess and (cb) cerebellum. Scale bars: 200 mm in A, C, D, G, H, I and J, 100 mm in B and F, 50 mm in E.

and megalin (not shown). Starting at E13 the newly formed choroid plexuses (Fig. 2A,B,D,E) expressed cubilin and megalin. Staining was also observed in the primordium of the pineal gland (not shown), the hippocampal ventricular zone and was particularly strong in the cortical hem (Fig. 2A,B). After E15 the expression of cubilin and megalin decreased progressively; the only sites that remained positive were the choroid plexuses, the ventricular zone of the ventral lateral ventricle and the cortical hem (Fig. 2G,I) as well as the ventricular regions of the ventral third and fourth ventricles (Fig. 2H–J) and the spinal cord (not shown). ISH analysis confirmed the immunostaining data for both cubilin and megalin although cubilin mRNA signal was weak (Fig. 2F). Some residual immunoreactivity

could still be detected in the pineal gland recess (Fig. 2H) and the neuroyhypophysis (not shown). 1.2. Localisation in the sensory organs At E11 cubilin and megalin were found in the optic vesicle, which, after invagination a day later gives rise to the optic cup. Both the outer layer of the cup, future pigment layer of the retina, and the inner, future nervous layer of the retina, strongly expressed cubilin and megalin (Fig. 3A,B). Similar staining was observed at E13 and 14; at these stages the optic stalk also expressed both receptors (Fig. 3C–F). Staining decreased progressively and at E18 only a faint immunoreactivity could be observed at the outer most layer

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Fig. 3. Immunolocalisation of cubilin (cub) and megalin (meg) in the developing eye. Immunolocalisation of cubilin and megalin in the developing eye at E12 (A, B), E13 (C, D), E14 (E, F) and E18 (G, H). Note the intense staining in the neural retina (nr) and the transient staining of the retinal pigmental epithelium (rpe). Lens (L) is negative. (os) optic stalk, (eyd) eyelid. Scale bars: 50 mm in A and B, 100 mm in C–F and H, 250 mm in G.

of the neural retina and the presumptive ciliary body region (Fig. 3G,H). The same distribution pattern was observed by ISH analysis (not shown). No expression was found in the lens vesicle, corneal ectoderm and the perioptic mesenchyme. Beginning at E11 the otic placode and the olfactory pit were stained for both receptors (Figs. 4A–C and 5A–C). At E12 both the dorsal and ventral halfs of the otic vesicle and the dorsally extending anlagen of the endolymphatic duct were stained (Fig. 4D). Two days later the thickened regions of the otic epithelium, the presumptive sensory epithelia, and the thin-walled nonsensory epithelium of the otocyst expressed cubilin and megalin (Fig. 4E,F). The endolymphatic duct was and remained negative

thereafter for both proteins. After E14 the sensory epithelia of the vestibular (utricle, saccule) and the cochlear components as well as epithelia of the semicircular ducts expressed cubilin and megalin and this pattern was maintened at E18 (Fig. 4G). At this stage cubilin and megalin were found in the vestibular and cochlear hair cells. ISH analysis further confirmed the overlapping expression of cubilin and megalin in the vestibular organs, the cochlear (Fig. 4H,I) and the semicircular ducts. At E14 the olfactory epithelium (Fig. 5D,E) and the forming vomeronasal organ (not shown) strongly expressed megalin; the distribution of cubilin in these sites was similar although the staining was fainter (Fig. 5F). Cubilin and megalin mRNA were detectable at this stage although

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Fig. 4. Distribution of cubilin (cub) and megalin (meg) in the developing inner ear as revealed by immunostaining and ISH. (A) Overview of megalin expression on a transverse section of an E11 embryo, and (B) and (C) higher magnifications of the otic placode (Ot pl) at the same stage stained for megalin and cubilin. (D)–(F) Sagittal sections of the otic vesicle (Ot ves) at E12 (D) and its components at E14 (E) and (F). Staining in the otic epithelium of the presumptive utricule (u) and saccule (s). No labelling in the forming endolymphatic duct (ed). (G) Megalin expression on transverse sections through the vestibular (utricule, saccule) and cochlear (coch) components of the inner ear at E18. (H) and (I) mRNA expression of megalin (MEG) and cubilin (CUB) on transverse sections of the cochlear components at E18. (fb) forebrain, (hb) hindbrain, (ac) amniotic cavity. Scale bars: 200 mm in A, G and H, 100 mm in B, D, E, F and I, 50 mm in C.

the signal for cubilin was weaker (not shown). After E15 the vomeronasal organ is completely formed and the olfactory epithelium becomes pseudostratified. It is composed by primarily three cell types: the apical supporting cell layer, acting as glia, and the underlying sensory neurons which co-express cubilin and megalin at E17-18 (Fig. 5G–J) and throughout gestation. The basal cells of the olfactory epithelium remain negative for both receptors. No staining was detected in the developing snout and whiskers. 1.3. Localisation in the gastrointestinal tract Cells lining the lumen of the foregut were similarly stained at E11 for cubilin and megalin (Fig. 1A,E). At E12 some cells of the intestinal tube and the epithelial cells lining the stomach were positive (Fig. 6A,B). The newly formed dorsal pancreas was faintly labeled; the signal became stronger a day later in some, presumably epithelial, cells of the pancreatic bud (Fig. 6C,D) and progressively disappeared after E14 for both cubilin and megalin. Transient staining was also detected in the developing oesophagus between E14 and E17 (Fig. 6F,G). Some expression was observed and maintained at the epithelia of the small intestine (not shown).

1.4. Localisation in the respiratory tract Cubilin and megalin were detected in the epithelium of the tracheal diverticulum at E11 (not shown). During the early pseudoglandular stage of the lung development, at E13 and 14, they were localized at the bronchial lumen of the developing lung buds (Fig. 6E) and at the apical regions of the epithelial cells lining the trachea (Fig. 6F). Beginning at E17, late pseudoglandular stage, the signal became fainter for both proteins (Fig. 6G,H) and decreased steadily through the canalicular stage and until the end of the gestation where only some bronchi remained positive (not shown). 1.5. Localisation in the urogenital system At E11 the newly formed mesonephric cord and Wolffian duct co-expressed cubilin and megalin. The mesonephric tubules, the ureteric bud, and the Mu¨llerian duct (E12-17) expressed both proteins at a high level (Fig. 7A,C,D,G). ISH analysis at E12 also suggested strong mRNA expression for both cubilin (Fig. 7B) and megalin (not shown). At E14 the rapidly differentiating conjunctive cells of the metanephric mesenchyme strongly expressed cubilin and megalin (Fig. 7C). From E15 abundant staining was observed in

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Fig. 5. Immunolocalisation of cubilin (cub) and megalin (meg) in the developing nose. (A) Megalin expression on a sagittal section of the forebrain at E12. In (B) and (C) higher magnifications of the invaginating olfactory placode (op) stained for megalin and cubilin, respectively. Note staining in the neuroepithelial cells of the telencephalic vesicle (tv) in the lateral ventricle (lV) and in the optic stalk (os). (D) Sagittal section through the olfactory epithelium at E14 labelled for megalin and higher magnifications in (E, megalin) and (F, cubilin). Staining in the medial and lateral olfactory pit (mop, lop) of the olfactory epithelium (oe). (G) Megalin expression on a transverse section through the olfactory epithelium and the vomeronasal organ (vno) at E17. (H) At E18 the supporting cell layer (scl) and the sensory olfactory nerve layer (sonl) of the olfactory epithelium co-expressed cubilin and megalin. In higher magnifications megalin (I) and cubilin (J) in the apical (ase) and basal vomeronasal sensory epithelium (bse). No staining in the nasal septum (ns). Scale bars: 250 mm in A, D, G and H, 100 mm in B, C, E, F, I and J.

some renal vesicles that eventually transform into convoluted tubules; beginning at this stage the primitive glomeruli strongly expressed cubilin and megalin (Fig. 7I). However, by the end of gestation, mature glomeruli only maintained expression of megalin in the podocytes. Cubilin and megalin were expressed in the developing ureteric bud by E13 and thereafter (Fig. 7C,H). From E14 the epidydimic structures were positive for both proteins; no staining was observed in the gonads, ovary or testis (Fig. 7E,F). 1.6. Localisation in the thyroid, parathyroids and thymus Both proteins were detected at the apical pole of the first branchial arch epithelium at E11. At E12 (Fig. 8A,B)

the pharyngeal pouches, I, III, IV and the transiently formed pouch II were positive for cubilin and megalin. At E13 pouch III (Fig. 8D) develops two diverticula strongly stained for cubilin and megalin: the ventral diverticulum that turns into the thymus and the rostral diverticulum that will give rise to the parathyroid. At this stage the newly formed thyroid gland (Fig. 8C,F) and the ultimobranchial body which develops from the caudal surface of the fourth pouch (Fig. 8E) were and remained positive for both cubilin and megalin (Fig. 8I,J). At E14-15 the parathyroid gland separates from the thymus; from this stage and later on a high immunoreactivity for both receptors was observed (Fig. 8G,H,L). During the same period both the endodermal part (from the ventral diverticulum of the third pouch) and

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Fig. 6. Immunolocalisation of cubilin (cub) and megalin (meg) in the gastrointestinal and respiratory tracts. (A) Overview of megalin expression on a sagittal section of an E12 embryo, and (B) higher magnification showing cubilin expression in the developing stomach (st) and gut. Note staining in the mesonephros (mesoneph). (C) and (D) Expression of megalin and cubilin on sagittal sections of the developing stomach (st) and pancreas (panc) at E14. (E) and (F) Sagittal sections through the developing lung, trachea (trach) and oesophagus (oesoph) at E14. Cubilin and megalin (not shown) staining marks the epithelia lining these structures. (G) Transverse section showing a fainter staining in the trachea (trach) and the oesophagus (oesoph) for both cubilin (G) and megalin (not shown) at E17. (H), Transverse section through the lung at E17. Some bronchi were stained for cubilin (H) and megalin (not shown). Scale bars: 250 mm in A and C, 50 mm in B and H, 100 mm in D, E, F and G.

the corresponding ectoderm (from the branchial clefts) of the developing thymus (Fig. 8G,H) expressed cubilin and megalin. After E18 only some, presumably, epithelial cells retained the staining (Fig. 8K). No labeling was observed in the spleen, the liver, or the musculo-skeletal system. In the developing heart both the myocardium and the endocardium were negative whereas the mesothelial cells of the pericardium expressed both

receptors. The mesothelial cells of the pleural and peritoneal cavities were also stained. Western blot analysis of the neural tube, the eye and the lung at various developmental stages showed that in these structures cubilin and megalin exhibited the same electrophoretic mobility as in adult kidney (Fig. 9). The antibodies used here were previously described (Assemat et al., 2004; Sahali et al., 1993). Control probing

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Fig. 7. Distribution of cubilin (cub) and megalin (meg) in the developing urogenital system as revealed by immunostaining and ISH. (A) and (B) Sagittal sections through the mesonephros at E12 (mesoneph). Protein and mRNA (CUB) expression of cubilin in the epithelium of the mesonephric ducts (mesoneph). (C) Sagittal section through the ureteral bud (ub) that reaches the metanephric mesenchyme (mm) at E14. The Wolffian (mesonephric) duct (Wod) is also stained. Note staining in the mesothelium (mesoth). Cubilin staining was similar (not shown). (D) Sagittal sections of the Wolffian (Wod) and Mu¨llerian (Mud) ducts in a female at E15. (E) and (F) Sagittal sections through the megalin and cubilin expressing mesonephric tubules (mt), future epidydime, at E16. The adjacent developing testis (Te) is negative. (G) and (H), Sagittal sections through the Wolffian (Wod) and the regressive Mu¨llerian (Mud) ducts of a male at E17. The ureter (ur) and the deferent duct (dd) are also labelled for both proteins. (I) Transverse section through the metanephros at E17; note staining for cubilin in the primitive glomeruli (arrow in I). Scale bars: 50 mm in A, B, D and G, 100 mm in C, E, F, H and I.

with non-immune rabbit sera or secondary antibody only confirmed the specificity of anti-cubilin and anti-megalin antibodies. Moreover ISH analysis further confirmed these results. In summary, cubilin and megalin are expressed in several tissues of the rodent embryo, not only in the developing kidney and gut but also in the CNS, sensory organs, and various developing epithelia. In all these sites, with the only exception being the glomeruli, overlapping expression was observed even if cubilin staining was fainter in some sites. It is possible that this corresponded to a lower level of cubilin synthesis, as also suggested by ISH analysis. The observed pattern may also suggest that during development cubilin and megalin interact and form oligomeric complexes like in the materno–fœtal interfaces and the adult. The progressively restricted expression in the CNS, the eye, or the thymus as well as the transient expression coinciding with epithelial differentiation, like for instance in the pancreas, may suggest a role for cubilin and megalin in specific morphogenetic events during development.

2. Experimental procedures 2.1. Animals Pregnant Wistar rats were from Harlan (France). Rat embryos were analyzed from embryonic day 10 (E10) to E21. All animal care and handling were performed according to institutional guidelines (Bureau for Experimental Animal research (BEA) of Inserm and Department of Sciences de la Vie of CNRS) and to the French regulations on animal care during scientific experiments (de´cret no. 2001–464). 2.2. Immunohistochemistry Embryos were fixed in 75% ethanol, 2% formol and 5% acetic acid (AFA) and embedded in paraffin. Fourmicrometer paraffin sections were placed on SuperFrost Plus glass slides (CML, Villemaur sur Vanne, France) and kept overnight (ON) at 50 8C. The sections were dewaxed

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Fig. 8. Immunolocalisation of cubilin (cub) and megalin (meg) in the developing thyroid and the branchial pouches. (A)–(C), Sagittal sections through the branchial pouches 2 (P2), 3 (P3) and 4 (P4) and the thyroid diverticulum (Thyr) at E12. Note staining at the floor of the mouth cavity (mc). (D)–(F), Transverse sections through the branchial pouches P3 and P4, the ultimobranchial body (UB), and the thyroid (Thyr) at E13. (G) and (H), Sagittal sections through the developing parathyroid and the adjacent thymus stained for megalin at E14 and cubilin at E15. (I) and (J), Transverse sections at E16 through the parathyroid (Parath) the thyroid (Thyr), and the invading ultimobranchial body (UB). (K) Tansverse section through the thymus (Thym) at E18. Staining for megalin and cubilin (not shown) remains in some, probably epithelial, cells. (L) Transverse section through the thyroid at E21. Similar staining for cubilin and megalin (not shown) in the follicular epithelial cells of the thyroid (Thyr) and intense staining in the parathyroid cells (Parath). (PeC) pericardial cavity. Scale bars: 100 mm in A, G, K and L, 50 mm in B–F, H–J.

with toluene and rehydrated with distilled water through a series of alcohol solutions. After proteinase K treatment (10 mg/ml, 10 min at 37 8C) the sections were rinsed in TBS (0.15 M NaCl, 0.05 M Tris pH7.6) and incubated in 0.5% blocking reagent/TBS (TSA Biotin System, Perkin Elmer, Courtaboeuf, Fr.) for 10 min at room temperature (RT). Incubation with the primary antibodies, sheep anti-megalin (1:40,000) and rabbit anti-cubilin (1:15,000) (Sahali et al., 1988) was performed in a moist chamber at 4 8C ON as previously reported (Assemat et al., 2004). For cubilin only an additional incubation with a goat anti-rabbit antibody (20 min, RT) was performed. After rinsing, relevant biotinylated secondary antibodies (Jackson Immuno Research, Baltimore, USA) were added. TSA Biotin system

(Perkin Elmer, Courtaboeuf, Fr.) was applied according to the manufacturer’s instructions. The sections were then exposed to a working solution containing the DAB chromogen (Sigma fast 3,3 0 -diaminobenzidine tablet sets)

Fig. 9. Western blotting analysis of cubilin (A) and megalin (B) in tissues of E16 rat embryos. Western blotting analysis of cubilin (460 kDa) and megalin (600 kDa) on kidney (K), 5 mg of protein, or 20 mg of protein on brain (Br), lung (Lu) and eye (Ey) extracts of E16 embryos.

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for 10 min at RT according to the manufacturer’s instructions. The slides were counterstained with hematoxylin (Sigma, Lille-Lezennes, Fr.), dehydrated and mounted in Eukitt (Labonord, Villeneuve d’Ascq, France). 2.3. In situ hybridization For in situ hybridization digoxigenin-labeled probes were used. Briefly cDNA fragments of cubilin and megalin were subcloned in pDrive vectors (Qiagen, Courtaboeuf, Fr.). Probes corresponding to the CUB2 or 3 0 UTR domains of cubilin were previously described (Assemat et al., 2004). Plasmids for megalin were kindly provided by Pr. Emil de Heer, Leiden University Medical Center, The Netherlands (Luca et al., 1996). To generate sense or anti-sense cRNA probes, 1 mg of plasmid was linearized with BamHI and XhoI (Roche Diagnostics, Neuilly s/Seine, Fr.). In vitro transcription was performed using the Boehringer kit (Roche Diagnostics, Neuilly s/Seine, Fr.) and T7 or SP6 RNA polymerase in the presence of Dig-UTP. In situ hybridization was performed on fresh frozen rat embryo sections (10-mm-thick) as previously described (Vitalis et al., 2003). Tissue sections were postfixed for 10 min in 4% paraformaldehyde, washed in PBS treated with proteinase K (5–10 mg/ml), acetylated, washed in PBS, dehydrated, and air dried. After prehybridization sections were covered with hybridization buffer containing the appropriate probe and incubated at 65 8C ON. After several washes hybridization signal was detected by using alkaline-phosphatase conjugated antibodies against digoxigenin according to the manufacturer’s instructions (Roche Diagnostics, Neuilly s/Seine, Fr.). 2.4. Western blotting For Western blotting brain, eye, lung and kidney extracts of E16 rat embryos were used. Briefly collected organs were rinsed in PBS and homogenized in ice-cold buffer containing 250 mM sucrose, 5 mM EDTA, 10 mM HEPES, pH 7.8 and protease inhibitors (Complete, Roche Diagnostics, Neuilly s/Seine, Fr.). After centrifugation at 4 8C (15 min 13,000!g) the pellets were extracted in ice-cold buffer (2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 140 mM NaCl, pH7.8) containing protease inhibitors (Complete, Diagnostics, Neuilly s/Seine, Fr.) and 1% Triton X-100. The samples were centrifuged (4 8C, 13,000!g, 30 min) and the supernatants electrophoresed on 4–16% SDS-PAGE polyacrylamide gradient gels as previously described (Assemat et al., 2004). As positive controls, adult kidney extracts were similarly prepared.

Acknowledgements This work was supported by grants from the ‘Fondation pour la Recherche Me´dicale’ (FRM) to R. Kozyraki, from ‘Association pour la Recherche sur le Cancer’ (N83443), from the ‘ACI Biologie du De´veloppement et Physiologie Inte´grative’ (1A068G) and from the EEC (QLG1-CT-200201215 and Eurogene 2004-005085) to P. Verroust. E. Assemat was a recipient of a fellowship from the Ministe`re Franc¸ais de la Recherche. We would like to thank Pr. Patrice Callard for his help and Mrs Franc¸oise Illien for technical assistance.

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