LRP2/Megalin Is Required for Patterning of the Ventral Telencephalon Robert Spoelgen*, Annette Hammes*, Uwe Anzenberger*, Dietmar Zechner, Olav M
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Research article Development and disease 405 LRP2/megalin is required for patterning of the ventral telencephalon Robert Spoelgen*, Annette Hammes*, Uwe Anzenberger*, Dietmar Zechner, Olav M. Andersen, Boris Jerchow and Thomas E. Willnow† Max-Delbrueck-Center for Molecular Medicine, Berlin, 13092, Germany *Authors contributed equally †Author for correspondence (e-mail: [email protected]) Accepted 10 November 2004 Development 132, 405-414 Published by The Company of Biologists 2005 doi:10.1242/dev.01580 Summary Megalin is a low-density lipoprotein receptor-related ventral forebrain. As a consequence of absent SHH activity, protein (LRP2) expressed in the neuroepithelium and the ventrally derived oligodendroglial and interneuronal cell yolk sac of the early embryo. Absence of megalin expression populations are lost in the forebrain of megalin–/– embryos. in knockout mice results in holoprosencephaly, indicating Similar defects are seen in models with enhanced signaling an essential yet unidentified function in forebrain through BMPs, central regulators of neural tube development. We used mice with complete or conditional patterning. Because megalin mediates endocytic uptake megalin gene inactivation in the embryo to demonstrate and degradation of BMP4, these findings indicate a role for that expression of megalin in the neuroepithelium but not megalin in neural tube specification, possibly by acting as in the yolk sac is crucial for brain development. During BMP4 clearance receptor in the neuroepithelium. early forebrain development, megalin deficiency leads to an increase in bone morphogenic protein (Bmp) 4 expression Key words: LDL receptor-related proteins, Bone morphogenic and signaling in the rostral dorsal neuroepithelium, and a proteins, Sonic hedgehog, Yolk sac, Holoprosencephaly, Mouse, subsequent loss of sonic hedgehog (Shh) expression in the LRP2 Introduction axis into discrete hemispheres. This defect is likely to be the Development Low-density lipoprotein (LDL) receptor-related proteins result of defective patterning during development involving (LRPs) are multifunctional cell surface receptors structurally improper specification of the rostral portion of the neural tube. related to the LDL receptor (Nykjaer and Willnow, 2002). Many genetic pathways involved in neural tube patterning have been implicated in the etiology of HPE (Wallis and Muenke, Initially considered endocytic receptors involved in lipoprotein 1999). In particular, alterations in pathways that specify the metabolism, recent studies revealed important roles for LRPs dorsoventral axis of the rostral neural tube may cause this in the regulation of many signaling processes (Nykjaer and syndrome. For example, increased dorsal signaling through Willnow, 2002; Herz and Bock, 2002). For example, LRP5 and bone morphogenic proteins (BMPs), members of the LRP6 bind WNT proteins and WNT inhibitors, and act as co- transforming growth factor β superfamily, and through WNT receptors for Frizzled in determining pattern formation in the proteins, results in HPE (Golden et al., 1999; Wallis et al., early embryo (Pinson et al., 2000; Wehrli et al., 2000; Mao et 1999; Anderson et al., 2002; Lagutin et al., 2003). Also, loss al., 2001a; Mao et al., 2001b), while apolipoprotein E receptor- of sonic hedgehog (SHH) expression, a factor that specifies the 2 and very low-density lipoprotein receptor function as ventral neural tube, leads to holoprosencephalic syndrome in receptors for Reelin, a signaling factor that controls neuronal humans and in mice (Chiang et al., 1996; Roessler et al., 1996). migration in the embryonic brain (Trommsdorf et al., 1999; Concerning a role for megalin in forebrain development, Hiesberger et al., 1999; D’Arcangelo et al., 1999). some evidence suggests involvement of the receptor in the LRP2, or megalin (herein referred to as megalin), is another SHH pathway (McCarthy et al., 2002; Farese and Herz, 1998). member of the LDL receptor gene family essential for brain Sonic hedgehog is a secreted protein expressed in the development (Saito et al., 1994; Willnow et al., 1996). At mid- notochord and the floorplate that undergoes autocatalytic gestation, the receptor is expressed in the neuroepithelium of the cleavage into a 19 kDa amino terminal (SHH-N) and a 25 kDa embryo as well as in the visceral endoderm of the yolk sac carboxyl terminal (SHH-C) domain. During cleavage, a (Willnow et al., 1996; Zheng et al., 1994). Targeted disruption cholesterol moiety becomes covalently attached to SHH-N, the of the respective gene in the mouse results in holoprosencephalic active fragment that binds to the receptor Patched (Ptch1) and syndrome and in perinatal lethality (Willnow et al., 1996). activates downstream SHH target genes through Smoothened However, the exact mechanism by which megalin contributes to (Smo). Cholesterol modification of SHH-N is crucial for forebrain formation, and why holoprosencephaly (HPE) ensues proper activity, perhaps by spatially restricting SHH activity in megalin-deficient mice, is still unclear. (Porter et al., 1996). Expression of megalin in the yolk sac and Holoprosencephaly is defined as a failure of the its ability to take up lipoproteins suggests a possible function prosencephalon (forebrain) to separate along the mid-sagittal for the receptor in delivery of cholesterol-rich lipoproteins 406 Development 132 (2) Research article from the maternal circulation to the embryo for SHH activation Apoptosis and proliferation assays (Willnow et al., 1992; Farese and Herz, 1998). Alternatively, Embryonic day 10.5 embryos were dissected, fixed, paraffin- expression of megalin in the neuroepithelium and its ability to embedded and sectioned as described above. After de-waxing and bind SHH indicates a more direct effect as a co-receptor in rehydration, the sections were incubated with 20 µg/ml proteinase K SHH signaling (McCarthy et al., 2002). in 10 mmol/l Tris/HCl, pH 7.4 for 20 minutes at 37°C. Detection of In the present study, we used mice in which the megalin gene apoptotic cells was performed using the TUNEL reaction kit was ubiquitously inactivated (including yolk sac and (www.Roche.com) following manufacturer’s instructions. For neuroepithelium) or conditional mutants with loss of megalin detection of proliferation, paraffin sections were incubated with rabbit anti-phosphohistone H3 antibody (1:200; Upstate, Lake Placid) only in the neuroepithelium to examine the contribution of the followed by secondary Cy3-conjugated sheep anti-rabbit IgG (Sigma, receptor in these tissues to forebrain formation. Sustained www.sigma.com), and detection by immunofluorescence microscopy. expression of megalin in the yolk sac did not rescue mice from HPE, indicating that a receptor activity in the neuroepithelium BIAcore analysis and cell culture experiments is essential for normal forebrain development. In the Carrier-free preparations of recombinant human BMP4 and neuroepithelium, megalin activity affects dorsal and ventral BMP5 were purchased from Research and Development patterning of the forebrain, and promotes ventral cell fate (www.RnDSystems.com). Recombinant His-tagged RAP was 2+ specification. produced and purified from bacteria by standard Ni affinity chromatography. As a source for WNT1 we used conditioned medium from Rat-2 cells infected with a retroviral Wnt1 expression construct Materials and methods that excrete large amounts of the protein (Rat-2/WNT1) and compared it with medium from cells infected with the empty expression vector Animals (Rat-2/MV7) (cell lines kindly provided by A. M. C. Brown, Cornell The generation of mice with targeted disruption of the megalin gene University, New York) (Giarre et al., 1998). Interaction of megalin has been described (Willnow et al., 1996). The megalin gene defect with BMP4, BMP5 and RAP (0.5-1 µmol/l) or WNT1 (20-fold was analyzed in receptor-deficient and in somite-matched wild-type concentrated medium) was tested by surface plasmon resonance littermates on a hybrid (129SvEmcTer × C57BL/6N) and on an inbred (SPR) as described before (Hilpert et al., 1999). For cell uptake CD1 genetic background. Both lines gave identical results. For studies, BMP4 was iodinated according to the IODOGEN protocol conditional inactivation of the megalin gene in the epiblast, animals (specific activity 3.1×108 cpm/µg). Replicate monolayers of rat homozygous for a loxP-modified allele of the megalin gene choriocarcinoma (BN16) cells were incubated for 2 hours with 1.6 lox/lox (megalin ) (Leheste et al., 2003) were crossed with mice carrying ng/ml of 125I-BMP4 in the presence of buffer, 200 µmol/l chloroquine, a knock-in of the Cre transgene into the mesenchyme homeobox 2 or 100 µg/ml RAP. Degradation of ligand was determined as the total tm1(cre)Sor gene locus (Meox2 ) obtained from Jackson Laboratories amount of 125I-labeled trichloroacetic acid-soluble material released (www.JAX.org) (Tallquist and Soriano, 2000). Embryos from into the culture medium and expressed as picogram ligand per mg of lox/wt tm1(cre)Sor lox/lox breeding of megalin /Meox2 mice with megalin total cell protein (Hilpert et al., 1999). animals were used for phenotypic analysis following genotyping of Development embryo- or yolk sac-derived tissues. Results Histological and immunohistological analysis Megalin deficiency impairs forebrain development Embryos or yolk sac tissues were dissected from pregnant mice at around mid-gestation designated time points, fixed overnight in 4% paraformaldehyde in PBS at 4°C, and embedded in paraffin. Routine