Abstract
Deletion of the Mesd gene region blocks gastrulation and mesoderm differentiation in mice. MESD is a chaperone for the Wnt co-receptors: low-density lipoprotein receptor-related protein (LRP) 5 and 6 (LRP5/6). We hypothesized that loss of Wnt signaling is responsible for the polarity defects observed in Mesd deficient embryos. However, because the Mesd deficient embryo is considerably smaller than Lrp5/6 or Wnt3 mutants, we predicted that MESD function extends more broadly to the LRP family of receptors. Consistent with this prediction, we demonstrated that MESD function in vitro was essential for maturation of the β-propeller/EGF domain common to LRPs. To begin to understand the role of MESD in LRP maturation in vivo, we generated a targeted Mesd knockout and verified that loss of Mesd blocks WNT signaling in vivo. Mesd mutants continue to express pluripotency markers, Oct4, Nanog, and Sox2, suggesting that Wnt signaling is essential for differentiation of the epiblast. Moreover, we demonstrated that MESD was essential for the apical localization of the related LRP2 (Megalin/MEG) in the visceral endoderm, resulting in impaired endocytic function. Combined, our results provide evidence that MESD functions as a general LRP chaperone, and suggest that the Mesd phenotype results from both signaling and endocytic defects resulting from mis-folding of multiple LRP receptors.
Keywords: mesd, lrp, low-density lipoprotein receptor related protein, visceral endoderm, megalin, lrp2, lysosome, chaperone, wnt
INTRODUCTION
The mesoderm development (Mesd) functional region was originally defined by complementation analysis of overlapping albino deletions (Holdener et al., 1994). Embryos homozygous for deletions that encompass Mesd lack mesoderm and do not form a posterior primitive streak (Holdener et al., 1994; Hsieh et al., 2003). Although loss of Mesd does not affect the localization of the anterior visceral endoderm (AVE), the anterior-posterior axis of the Mesd mutant embryo is oriented along the short axis of the embryo and contrasts markedly to the wild-type, embryo, which is oriented along the long axis (Hsieh et al., 2003; Rivera-Perez and Magnuson, 2005). The Mesd deficient primitive streak defects are similar to that observed in Wnt3 or Lrp5/6 knockouts (Liu et al., 1999; Kelly et al., 2004), suggesting that the Mesd interval is essential for Wnt signaling. Physical mapping and transgenic rescue experiments demonstrated that the polarity and mesoderm differentiation defects resulted from loss of a single gene, Mesd (previously named Mesdc2), from the deletion interval (Hsieh et al., 2003). Several observations suggest that Mesd encodes a molecular chaperone for the Wnt co-receptors LRP5 and LRP6: MESD promotes membrane localization of LRP5/6 in co-transfection assays, is localized within the ER, interacts with LRP5/6 in co-immunoprecipitation experiments, and prevents aggregation of over expressed LRP5/6 in COS1 cells (Hsieh et al., 2003).
In addition, Boca, the Drosophila MESD homologue, promotes membrane localization of Arrow and Yolkless in vivo (Culi and Mann, 2003). Arrow and Yolkless are structurally similar to LRP5/6 and are also members of the low-density lipoprotein receptor-related (LRP) family. Boca can also facilitate trafficking of other LRP members such as Drosophila LpR2 and human LDLR in cell culture, as well as isolated β-propeller/EGF domains in S2 cells (Culi et al., 2004). These results suggest that MESD function likely goes beyond maturation of LRP5/6. This prediction is consistent with the observation that embryos homozygous for deletions encompassing Mesd are considerably smaller, and have significantly expanded parietal endoderm compared to Wnt3 or Lrp5/6 mutant embryos. Combined, these data suggest that MESD function extends more broadly to the LRP family. Consistent with this prediction, Mesd appears ubiquitously expressed in adult and embryo tissues (Wines et al., 2001 and unpublished observations).
The 10 major LRPs share similar structural domains (Strickland et al., 2002). All are transmembrane proteins characterized by an intracellular domain that may contain up to three NPxY sequences, coupling the receptor to endocytic machinery and signaling cascades. The LRP extracellular domains consist of three major motifs: a cysteine-rich complement-like repeat unit also known as LDL-A repeats, an EGF-like domain, and a six-bladed β-propeller domain made up of YWTD peptide repeats (Strickland et al., 2002; May et al., 2007; Willnow et al., 2007). The interface between the β-propeller and C-terminal EGF domain largely consists of hydrophobic residues that extend from the EGF side chains and is positioned between the second and third propeller blades of the β-propeller domain. This encourages the EGF domain to pack tightly against N-terminal β-propeller, burying the β-propeller/EGF interface (Jeon et al., 2001). Individual LRP family members differ in both the number and orientation of these three different extracellular domains. These differences result in unique receptor functions in various tissues that include, but are not limited to, fatty acid clearance from the blood, neuronal organization, protein and vitamin reabsorption from the urine, signaling in a variety of tissues, and limb patterning (Willnow et al., 2007). Here, we provide new insight into the trafficking of LRP family members. Our studies provide evidence that MESD is a general LRP chaperone essential for WNT signaling, epiblast differentiation, and LRP mediated endocytosis in the visceral endoderm.
RESULTS
Loss of Mesd blocks epiblast differentiation and WNT signaling
The Mesd deletion phenotype was originally characterized using albino deletions that remove approximately 39 genes from a nearly 4 Mb region of chromosome 7 (Holdener et al., 1994; Hsieh et al., 2003). A Mesd transgene, can restore mesoderm differentiation and gastrulation in deletion homozygotes (Hsieh et al., 2003), suggesting that these defects result from loss of a single gene from the deletion interval. To verify that other genes within the deletion do not contribute to aspects of the deletion phenotype not observed in Wnt3 or Lrp5/6 mutant embryos, we generated a Mesd knockout, Mesdtm1bch (referred to in this paper as Mesd-KO), that replaced most of Mesd exon 1 and all of exons 2 and 3 with a neomycin resistance gene (Figure 1A). The Mesd-KO allele was distinguished from the parental allele by Southern blot analysis or PCR (Figure 1B and Methods).
Whole-mount analysis of embryos obtained from intercrossing animals heterozygous for the Mesd-KO demonstrated that the knockout and deletion phenotypes were indistinguishable (Figure 1C) (Wines et al., 2000). At embryonic day (E) 7.5, wild-type littermates were undergoing gastrulation. The primitive streak was fully extended, and mesoderm differentiation was well underway (Figure 1C, left). In contrast, embryos homozygous for the Mesd-KO were characterized by expanded parietal endoderm surrounding an underdeveloped egg cylinder lacking a primitive streak and any mesodermal derivatives (Figure 1C, right), similar to that described for deletion homozygotes (Wines et al., 2000; Hsieh et al., 2003).
We also observed similarity between Mesd deletion homozygotes and the Mesd-KO using molecular markers. In wild-type embryos at E 8.5, T was expressed in the primitive streak as well as in the notochord (Figure 2A). As gastrulation progressed and the wild-type epiblast differentiated, the pluripotency markers Oct4, Sox2, and Nanog are characteristically down regulated (Figure 2C, E, and G) (Scholer et al., 1990; Avilion et al., 2003; Chambers et al., 2003; Hart et al., 2004). In contrast, T transcripts were expressed in Mesd-KO (n = 2) littermates in the extraembryonic ectoderm adjacent the epiblast (Figure 2B) and Oct4 expression persisted in the epiblast (Figure 2D). These patterns are similar to that observed in deletion homozygotes and in wild-type embryos prior to gastrulation (Hsieh et al., 2003; Scholer et al., 1990; Rivera-Perez and Magnuson, 2005). Similarly, we observed continued expression of Sox2 (n = 7) and Nanog (n = 3) throughout the Mesd-KO epiblast (Figure 2D, F, H) despite their down-regulation in wild-type littermates. These data suggested that MESD function was essential for repression of pluripotency markers and subsequent differentiation of the epiblast.
We hypothesized that the loss of primitive streak formation and mesoderm differentiation in Mesd mutant embryos results from a block in WNT signaling, despite the expression of Wnt3 in E 7.5 Mesd mutants (Hsieh et al., 2003). To determine if Wnt signaling was activated in E 7.5 Mesd mutants, we introduced a Wnt-reporter, BAT-gal, into the Mesd-KO background, and tested the ability of mutant embryos to activate the reporter (Figure 3). Embryo genotypes were confirmed by PCR (Figure 3 insets). This BAT-gal reporter encodes nuclear β-galactosidase under the control of a β-catenin-sensitive bipartite promoter containing 7 TCF-LEF binding sites upstream of a minimal Siamois promoter (Maretto et al., 2003). X-gal staining in E 7.5 embryos heterozygous for Mesd-KO identified active WNT signaling in the primitive streak and nascent mesoderm at E7.5 (Figure 3A). β-gal activity was not detected in E 7.5 Mesd-KO embryos that carry the BAT-gal reporter, confirming that loss of Mesd blocked WNT signaling.
The β-propeller/EGF domain of LRPs depends on MESD for proper folding
The Mesd phenotype is characterized by failure to form a primitive streak or differentiate mesoderm. The phenotype is similar to knockout of Wnt3 or Lrp5/6. However, unlike Wnt3 and Lrp5/6, the Mesd epiblast is smaller and surrounded by an expanded parietal endoderm (Figure 1C). These phenotypic differences suggested that MESD function extends beyond LRP5/6 trafficking. To begin to identify other proteins that might require MESD function, we mapped LRP5/6 domains that require MESD for trafficking (Figure 4). To accomplish this, we utilized a soluble receptor secretion assay in which we assayed the ability of COS-1 cells to secrete soluble truncated LRP5/6 receptors (Figure 4A) in the presence or absence of MESD. Transfection of human IgG was used as a transfection and secretion control. In the absence of exogenous MESD, soluble receptors containing the entire extracellular domain of LRP6 (LRP6 ECD) were not secreted into the media unless Mesd was co-transfected, suggesting that the extracellular domain of LRP6 is highly dependent upon MESD (Figure 4B). We saw similar dependency on Mesd co-transfection for secretion of soluble LRP5 and LRP6 receptors containing two, three, and four β-propeller/EGF domains (LRP6 βP1-2, βP1-3, and βP1-4) as well the first LRP5 β-propeller/EGF domain (LRP5 βP1). In contrast, the first LRP6 β-propeller/EGF domain (LRP6 βP1) is secreted independently of exogenous MESD, though LRP6 constructs containing multiple β-propeller/EGF domains that include LRP6 βP1 do require MESD for maturation. Together, these observations demonstrated that MESD facilitates trafficking of the β-propeller/EGF domains of LRP5 and 6, and are consistent with previous data suggesting a similar role for Boca (Culi et al., 2004). In addition, these data suggested that individual β-propeller/EGF domains might have distinct requirements for MESD (Figure 4B). This raised the possibility that MESD in vivo may facilitate trafficking of LRPs other than LRP5/6, and suggested that differences between the Mesd-KO and Wnt3 or LRP5/6 mutant embryos could result from defects in trafficking of these receptors. Consistent with this prediction, we determined that the LRP1 second β-propeller/EGF (LRP1 βP2) also requires exogenous MESD in the soluble receptor secretion assay (Figure 4B).
LRP2 requires MESD for apical membrane localization
To determine which LRPs have the potential to contribute to the Mesd mutant phenotype, we characterized the expression of LRP receptors that contain β-propeller/EGF domains at E 6.5 and E7.5 using RT-PCR (Figure 5). We detected transcripts from all the LRPs with the exception of LRP1b, which is not expressed until after E 11.0 (Li et al., 2005). Among these receptors, Lrp1 and Lrp2 are strongly expressed and are two of the largest members of the LRP family, containing seven and eight β-propeller/EGF domains, respectively. Given the results from our in vitro secretion assays, we predicted that the maturation of these receptors would be highly sensitive to the presence or absence of MESD.
LRP1 and 2 are strongly expressed in the VE. LRP2 is present on the apical membrane in a complex with Amnionless (AMN) and Cubilin (CUBN), similar to what is reported for other absorptive epithelia (Figure 6A, A′, B, C) (Sahali et al., 1988; Kalantry et al., 2001; Kozyraki and Gofflot, 2007). We also observed LRP2 on the apical surface of the epiblast, although the distribution appeared more punctate than that observed on the VE membrane (Supplemental Figure 1B). In the absence of Mesd, LRP2 was detected in a diffuse intracellular pattern in the VE and epiblast (Figure 6D, D′ and Supplemental Figure 1 D). In contrast, loss of MESD did not interfere with the apical localization of AMN and CUBN (Figure 6E, F), suggesting that MESD function is not generally required for trafficking of apically localized proteins or for localization of proteins that contain only the EGF motif (CUBN) or cysteine-rich domains (AMN) (Moestrup et al., 1998; Tanner et al., 2003). Moreover, the aberrant localization of LRP2 in Mesd mutants provided in vivo evidence that MESD function was not limited to LRP5/6 but extended more broadly to the LRP family of receptors.
To determine if loss of MESD simultaneously disrupted localization and function of multiple LRP family members, we performed an endocytosis assay using receptor associated protein (RAP), which specifically binds to the complement-like repeats in the extracellular domain of LRPs (Herz et al., 1991; Bu and Schwartz, 1998). This approach has been used to measure expression of membrane-localized, functional LRP1 in cortical neurons (Bu et al., 1994). Using a similar approach, we compared uptake of fluorescently labeled RAP (488-RAP) in wild-type and Mesd mutant embryos, with or without unlabeled RAP competitor (Figure 6 and Supplemental Figure 2). The VE of wild-type embryos labeled extensively (Figure 6G and G′); whereas in the presence of 200-fold excess unlabeled RAP, labeling of the VE was significantly reduced (Supplemental Figure 2). In contrast, the Mesd mutant VE cells showed a significant decrease in fluorescence, at levels comparable to that observed when wild-type embryos were incubated with unlabeled RAP (Figure 6H and H′). These results suggested that loss of Mesd disrupts general LRP function in the VE.
Loss of MESD results in smaller lysosome size in visceral endoderm
In the VE, LRP2 likely functions as a scavenger receptor, similar to its role in the kidney (Willnow et al., 1996; Verroust et al., 2002). Kidney proximal tubule cells from LRP2-deficient mice fail to endocytose transferrin, lysozyme, vitamin-D binding protein, or retinol-binding protein. Instead, these proteins are improperly excreted into the urine (Leheste et al., 1999; Kozyraki et al., 2001). For this reason, we predicted that loss of a prominent scavenger receptor such as LRP2, and more likely loss of both LRP1 and LRP2, from the apical cell surface of the VE will impair endocytosis in the VE.
Ultrastructural comparison of wild-type and Mesd-KO embryos provided indirect evidence that endocytosis was impaired in the Mesd-KO (Figure 7A and B). In the wild-type embryo (Figure 7A), the visceral endoderm cells were columnar in shape with a basally located nucleus. The apical membrane of the VE is populated by numerous microvilli that maximize the surface area available for membrane receptor localization and absorption of proteins and complexes, similar to that observed in the cells in the kidney proximal convoluted tubule (PCT) (Moestrup and Verroust, 2001). In addition, the VE contained numerous membrane-enclosed organelles in the cytoplasm, thought to be endocytic components including endosomes and lysosomes (Nagy, 2003). Although the VE cells from Mesd-KO embryos (Figure 7B) appeared shorter in height, they maintained apical-basal polarity and contained numerous apical microvilli and a basally-localized nucleus. In addition, we observed the fine network of ER, as well as a number of mitochondria throughout the cytoplasm indicating a basic level of normal cell function. In contrast, the size of the membrane-enclosed organelles was reduced significantly in Mesd-KO. Given the role of LRP2 and LRP1 in endocytosis of ligands/nutrients, we hypothesized that the ultrastructural defects reflected a reduction in either endosomes or lysosomes resulting from loss of LRP-mediated endocytosis.
Lysosomes are collectively identified as membrane-enclosed organelles that contain acid hydrolases and integral lysosomal membrane proteins (Luzio et al., 2007; Saftig and Klumperman, 2009). Lysosomes can form by fusion of late endosomes and serve to breakdown material delivered through late endosomes, phagosomes, and autophagosomes (Saftig and Klumperman, 2009). We observed similar apically localized EEA1-positive early endosomes in both wild-type and Mesd mutant embryos (Figure 7C and 7D, magenta) suggesting that although loss of Mesd may affect endocytosis of LRP-specific ligands it does not generally impair non-LRP mediated endocytosis. In contrast, in wild-type embryos, LAMP antibodies identified large vacuole-like lysosomes throughout the apical cytoplasm (Figure 7C and C′, green). These structures were concentrated basally to the early endosomes. Despite the normal distribution of early endosomes in Mesd-KO embryos, lysosome size was reduced in mutant embryos (Figure 7D, D′, green). The reduction in lysosome size was confirmed with LysoTracker Red staining (Figure 7E and F). In wild-type embryos, LysoTracker Red labeled round lysosomes that averaged 2.5μm in diameter with a standard deviation of .38μm (Figure 7E) (Koike et al., 2009). In contrast, lysosomes in Mesd-KO embryos were less abundant, more granular, and considerably smaller in diameter, averaging 1μm in diameter with a standard deviation of .36μm (Figure 7F). Quantification of wild-type LysoTracker Red staining using ImageJ resulted in an average pixel intensity of 63, where quantification of Mesd LysoTracker Red staining resulted in an average pixel intensity of 27, or 43% of wild-type LysoTracker Red staining. These observations are consistent with the absence of large membrane-bound organelles in TEM images of mutant VE (Figure 7A, B). In LRP2-deficient kidney proximal tubule cells, the size of the lysosomes are similarly reduced, suggesting that lysosome size can be regulated by the amount of ligand uptake (Christensen et al., 2003; Nielsen et al., 2007). Taken together, these studies provide evidence that that MESD-dependent LRP localization regulates endocytosis and lysosome formation in the VE.
DISCUSSION
Our studies demonstrated that MESD function in vitro is essential for maturation of the LRP5/6 and LRP1 β-propeller/EGF domains. Furthermore, soluble receptors that contained two or more β-propeller/EGF domains increasingly depended on exogenous MESD for maturation. Most LRPs contain at least one β-propeller/EGF domain, and LRP1 and LRP2 contain seven and eight domains, respectively (Strickland et al., 2002). For this reason, we hypothesized that MESD function extends to the LRP family of receptors, and predicted that phenotypic differences between Mesd deletion homozygotes and Lrp5/6 or Wnt3 knockouts resulted from defects in trafficking multiple LRPs. By characterizing a Mesd knockout, we provided in vivo evidence that MESD was essential for WNT signaling and visceral endoderm function. In addition, the continued expression of pluripotency markers, Oct4, Sox2, and Nanog, in Mesd mutant embryos suggests that MESD function is required for epiblast differentiation. We cannot exclude the possibility that defects in the mutant visceral endoderm contribute to the failure of the epiblast to differentiate. However, given the role of MESD in promoting maturation of the WNT co-receptors LRP5/6, and the observation that Wnt3 mutants continue to express Oct4 and neither Wnt3 nor Lrp5/6 mutants differentiate mesoderm (Liu et al., 1999; Kelly et al., 2004), we hypothesize that WNT signaling is essential for epiblast differentiation. The continued expression of pluripotency markers in Mesd and Wnt3 mutant embryos is striking considering these mutants do not maintain expression of Nodal, and Nodal mutant epiblast differentiates into neural precursors (Camus et al., 2006).
In the early post-implantation mouse embryo, the VE functions as an absorptive epithelium, as well as a signaling tissue (Beddington and Robertson, 1999; Bielinska et al., 1999; Baron, 2005). As an absorptive epithelium, the VE is responsible for filtering nutrients provided by the maternal bloodstream for early embryonic development to the developing embryo (Brent et al., 1990; Jollie, 1990; Cross et al., 1994; Bielinska et al., 1999). The absence of VE abnormalities in the Lrp2 knockout opens the possibility that structurally similar LRPs compensate for loss of LRP2 in the VE. Lrp1 and Lrp2 (Megalin) are structurally very similar and are both expressed in the VE (Chatelet et al., 1986; Herz et al., 1992), suggesting that these receptors could functionally substitute for one another. However, their role in VE endocytosis was unknown since these defects were not noted in either Lrp1 or Lrp2 knockouts. Lrp1 mutants were recovered at E 13.5, with defects in neural tube patterning (Herz et al., 1992), and Lrp2 mutants were recovered at birth, with 2% surviving to adulthood (Willnow et al., 1996; Nykjaer et al., 1999). Defects in apical endocytic components of the kidney proximal convoluted tubule in Lrp2 knockouts and proteinuria of LRP2-ligands, vitamin-D binding protein and retinol-binding protein, are consistent with our prediction that LRP2 is important for absorption in the VE (Christensen and Willnow, 1999; Leheste et al., 1999). However, VE absorption defects were not described in LRP2 knockouts, suggesting that related LRPs could compensate for lack of LRP2. This hypothesis is supported by the decreased endocytosis of 488-RAP in the visceral endoderm of Mesd mutant embryos. LRP1 is similar in structure and is also expressed in the VE (Herz et al., 1992). Since the LRP1 β-propeller/EGFs also required exogenous MESD for trafficking in cell culture, the VE endocytosis defects observed in Mesd mutants likely resulted from simultaneous misfolding of LRP1 and LRP2.
Other mutations that disrupt endocytosis or trafficking, including Dab2, Amn, Cubn, and Enpp2, share some similarity to the Mesd mutant phenotype. Endocytosis of LRP2 depends on the disabled homologue-2 (Dab2) adaptor protein that binds the intracellular NPxY motif of LRP2 as well as clathrin and AP2 (Morris and Cooper, 2001; Mishra et al., 2002). Dab2 mutants also fail to gastrulate and display a loss of apical vacuoles (Morris et al., 2002; Maurer and Cooper, 2005). However, Dab2 mutants fail to pattern the anterior VE (AVE), suggesting that loss of Dab2 has a wider range of effects than Mesd. On the apical surface in the VE, LRP2 forms a complex with the membrane receptors Amnionless (AMN) and Cubilin (CUBN) (Strope et al., 2004). Mutations in either AMN or CUBN decrease the volume of the apical VE, suggesting that these mutations could also impair absorption (Strope et al., 2004; Smith et al., 2006). Unlike the more severe Mesd or Dab2 phenotypes, disruption of Amn only blocks paraxial and lateral plate mesoderm differentiation resulting in embryonic lethality before E 11.5. Disruption of Cubn generates an almost identical phenotype to Amn and mutant embryos are not recovered at E 13.5. However, Cubn mutants exhibit a morphological defect in the definitive endoderm not reported in the Amn mutant (Tomihara-Newberger et al., 1998; Smith et al., 2006). Finally, Enpp2 mutant embryos show similar fragmentation of the characteristically large VE lysosomes (Koike et al., 2009). Enpp2 encodes a lysophospholipase enzyme that regulates lysosome size by stimulating the Rho-ROCK-Lim pathway (Koike et al., 2009). Although mutant embryos retain the ability to differentiate mesoderm and begin neurulation, the growth of the mutant embryos is significantly impaired. Combined, these mutant phenotypes suggest that growth defects in Mesd mutants could result from impaired endocytosis, storage, and breakdown of nutrients. However, because of the role of LRP1 and 2 in PDGF, TGFβ, and BMP4 signaling (reviewed in May et al., 2005; Lillis et al., 2008) we cannot exclude the possibility that defects in LRP-mediated VE or epiblast signaling also contribute to the Mesd mutant phenotype. This is underscored by the observation that high contribution chimeras comprised of Mesd mutant extraembryonic tissue and wild-type epiblast remain small and do not gastrulate (Hsieh et al., 2003), and the recent observation that PDGF signaling is important for regulation of primitive endoderm and epiblast size in the pre-implantation embryo (Artus et al., 2010). For this reason, it is likely that the Mesd phenotype is complex, resulting from defects in visceral endoderm or epiblast cell signaling, embryo nutrition, as well as possible secondary alteration in the interactions between the VE and epiblast. If primitive streak and mesoderm differentiation defects are solely related to MESD function in the epiblast, then we predict that epiblast-specific deletion of Mesd will closely resembles the Wnt3 or Lrp5/6 knockout phenotypes. Alternatively, we predict that loss of Mesd in the visceral endoderm could disrupt embryo nutrition as well as VE-epiblast interactions. In this case, we predict that conditional deletion of Mesd in the VE will result in defects more extensive than delayed embryo growth.
Our studies provide convincing evidence that MESD facilitates trafficking of the LRP YWTD β-propeller/EGF domain and can function as a general LRP chaperone. The receptor-associated-protein (RAP) similarly facilitates trafficking LRPs through interaction with the LDL-A cysteine rich complement region (Andersen et al., 2000; Fisher et al., 2006). In contrast to Mesd mutants, Rap−/− mice are viable and fertile (Willnow et al., 1995; Birn et al., 2000). Since LRPs contain both β-propeller/EGF as well as LDL-A domains, we predict that MESD likely plays a more central role in LRP maturation and function than RAP. This is consistent with the recent proposal that the YWTD containing β-propeller repeat could facilitate release of RAP from the LRP LDL-A domain (Jensen et al., 2009). This would suggest that RAP function is dependent upon MESD mediated folding of the β-propeller/EGF domain. Given the diverse and overlapping roles of LRPs in endocytosis and cell signaling (Willnow et al., 2007), we predict conditional Mesd mutations will facilitate the analysis of functional redundancy among LRP members as well as the contribution of LRP misfolding to the development of diseases as diverse as atherosclerosis, alzheimers, diabetes, osteoporosis, and kidney disease.
METHODS
Mouse strains and generation of Mesd-KO
Mice heterozygous for the Mesd deletion (Del(7)Tyrc-3YPSD/Tyrc-ch) were maintained in a closed colony by crossing to (TyrC/Tyrc-ch) (Holdener et al., 1994; Wines et al., 2000). The Mesdtm1bch (Mesd-KO) was generated in 129 ES cells purchased from Cell & Molecular Technologies (Phillipsburg, NJ; currently part of Invitrogen, Inc.). Three hundred and eighty four colonies were screened by PCR and Southern blotting and three correctly targeted clones isolated. Chimeras were generated by injecting the ES-cells into C57BL/6J blastocysts. Chimeric males were tested for germline transmission of ES-cell descendants by mating to C57BL/6JxDBA/2J F1 females. ES-cell electroporation, screening, and injection into blastocysts were done with the assistance of the Stony Brook University Transgenic Facility. Heterozygous mice were backcrossed to C57BL/6J mice for over 8 generations and are currently maintained by backcross to C57BL/6J.
Southern genotyping
Tail DNA was digested with either HpaI or KpnI. The 5′ probe was generated by PCR amplification using primers: NotI-5′-Mesd 5′-GCGGCCGCACCAGTTTAATTGACAGTGATATTGAAAG-3′ and XhoI-5′-Mesd 5′-CTCGAGGAGCAACAGAAGGTCCGAGGCACACAG-3′. The amplified fragment detects a 15kb wild-type fragment and a 9.4kb Mesd targeted Hpa I fragment. The 3′ probe was generated by PCR amplification using primers: NotI-3′A-Mesd 5′-GCGGCCGCACATGCAGGGTGTCTGTTTTGGCAGTC-3′ and XhoI-3′A-Mesd 5′-CTCGAGCTGAAGTCTCAAACTGGCTTTGATGAG-3′ or NotI-3′B-Mesd 5′-GCGGCCGCACATAAGACATAGATGGAAATGACATTTC-3′ and XhoI-3′B-Mesd 5′-CTCGAGGCCACCTGCTAAAGGTCTTCTCTTCTG-3′. The amplified fragment detects a 14kb wild-type fragment and a 9kb Mesd targeted Kpn I fragment.
PCR genotyping
DNA isolated from animal tail biopsies was genotyped by PCR using a common reverse primer (5′-CAAAAGGATGAGTGCCCTGT-3′) located at the 3′ end of the third exon, and either a Mesdc2-ko forward primer (5′-GGGAGGATTGGGAAGACAAT-3′) in the neomycin resistance gene (262 bp) or a wild-type forward primer (5′-TCCAGTTGGTTTCCGTTCAT-3′) also located 3′ of the third exon (202 bp) (Figure 1A).
BAT-Gal reporter assay
Mice homozygous for the BAT-Gal reporter transgene were crossed to mice heterozygous for the Mesd targeted allele to generate double-heterozygous animals. Male and female mice heterozygous for the Mesd targeted allele and carrying at least one copy of the BAT-Gal reporter transgene were mated. Embryos were collected at E 7.5 and stained for the presence of β-galactosidase as previously described (Maretto et al., 2003). Embryos were observed and photographed (Zeiss stereo Discovery.V8 dissecting scope, Zeiss AxioCam MRc camera), and then lysed in 20μl of lysis buffer (1xPCR buffer, 0.2mg/ml proteinase K, 10mM DTT, 1.5mM MgCl2) at 50°C overnight. Lysates were boiled for 5 minutes then 2μl used for PCR genotyping using the following primers: lacZ forward (5′-CGGTGATGGTGCTGCGTTGGA-3′), lacZ reverse (5′-ACCACCGCACGATAGAGATTC-3′) and Mesd-KO primers described above).
In situ hybridization
Embryos from a heterozygous intercross of Mesd-KO animals were dissected at E 7.5 (data not shown) and E 8.5. In situ hybridization was performed as previously described (Hsieh et al., 2003). DNA constructs used to generate probes were provided by Drs. Ian Chambers (Nanog), Hans Scholer (Oct4), Bernard Herrman (T), and Sox2 (Du, 2010).
LRP secretion assay
Expression constructs
Construction of FLAG-Mesd is previously described (Hsieh et al., 2003). Soluble LRP receptors were tagged with a C-terminal C-myc epitope (KLGGGMEQKLISEEDLNGGGLE) and cloned into pRK5. Mouse LRP5 and LRP6 soluble receptors are comprised of the following amino acid sequences: LRP6 ECD, 1-1363; LRP6 βP1-4,1-1245; LRP6 βP1-3, 1-931; LRP6 βP1-2, 1-630; LRP6 βP1, 1-328; and LRP5 βP1, 1-337. EGFP-rho (EGFP/pRK5-SK) was a gift from Jen-Chih Hsieh. Human IgG heavy chain plasmid (hIgG-pRK5) was previously described (Hsieh et al., 1999; Hsieh et al., 2003).
Transfection of COS1 Cells
COS1 cells were seeded at 50% confluency in 12-well plates and transfected 24 hours later with a total of 1 μg plasmid DNA using Fugene 6 (Roche) following manufacturer’s directions. Transfections contained 0.3 μg of Mesd, 0.4 μg of LRP6, 0.1 μg of hIgG plasmid, 0.1μg of EGFP, and pCS2+ plasmid to bring total DNA to 1 μg. The cells were detached from the plate in 1ml of 5 mM EDTA/phosphate buffered saline (PBS), collected by centrifugation in a microfuge at 3000 rpm for 3 minutes, and lysed in 55 μl cold lysis buffer (1% Triton X-100/PBS containing final concentrations of 17mg/ml aprotinin, 10mg/ml benzamidine, 1mg/ml leupeptin, 3mg/ml antipain, 1M PMSF). 20μl of loading dye (Coligan, 1995–2002) were added to each sample and boiled for 5 minutes. The lysates (10 μl each) were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of β-mercaptoethanol followed by Western blot analysis. All transfections were repeated a minimum of three times. Representative Western blots are shown.
Western blotting, antibodies, image acquisition and quantitation
After transferring the proteins from SDS-polyacrylamide gels onto nitrocellulose membrane (.45μm PROTRAN, VWR) membranes were blocked overnight in 1% casein/Tris buffered saline (TBS) at 4°C, incubated with primary antibodies for 1 hour at room temperature, followed by fluorescently-labeled secondary antibody for 1 hour at room temperature. Membranes were washed with 1xTBST (0.05% Tween-20/TBS) 3×15 minutes before and after secondary antibody incubation. All antibodies were diluted in 1% casein/TBS. Rho-tagged LRP6 was detected using mouse monoclonal anti-rhodopsin (clone 1D4) at 1:1000. Flag-tagged MESD was detected using mouse monoclonal anti-FLAG (clone M2, Sigma) at 1:5000. Secondary antibody Alexa 680-labeled anti-mouse (Invitrogen) was used at 1:4000, and human IgG heavy chain was directly detected using IRDye800-labeled anti-human IgG (Rockland) at 1:10,000. Membranes were scanned using the Odyssey-Infrared Imaging System (LI-COR Biosciences), and the intensities of the bands of interest were determined from the captured images using the Odyssey imaging software.
RT-PCR
Total RNA from embryos or brain and spleen tissue lysates were prepared. First strand cDNA was synthesized with SuperScript III First-Strand Synthesis System (Invitrogen). Primer sequences used to amplify LRP family gene products are listed in Supplementary Table 1.
Immunohistochemical analysis of embryos
Mouse embryos were dissected at E 7.5 and fixed in 4% paraformaldehyde for 1 hour at 4°C. Embryos were embedded in paraffin (Leica EG1160) and sectioned (RMC MT910) at 7μm and mounted on glass slides (Fisherbrand Colorfrost/Plus). Embryo sections were dewaxed, rehydrated, and antigen retrieved by microwaving the slides for 5 min at 40% power in 0.1M sodium citrate pH 9/1xTBST. Slides were washed in 1xTBST(0.05% Tween 20), and blocked overnight in 5% bovine serum albumin (BSA)/1xTBST at 4°C. Primary antibodies used were sheep anti-gp330, 1:1000 (Dr. Pierre Verroust); rabbit anti-amnionless, 1:4000 (Dr. Elizabeth Lacy); and goat anti-cubilin, A-16/Y-20/T-16, 1:1000 (Santa Cruz Biotechnologies). Secondary antibodies used were goat anti-rabbit 1:1000, goat anti-sheep 1:1000, rabbit anti-goat 1:1000 (Vector Labs).
Immunofluorescence of embryo sections
Mouse embryos were dissected at E7.5 and fixed in 4% paraformaldehyde for 1 hour at 4°C. Embryos were washed with 1xPBS and infused with 30% sucrose/1xPBS overnight at 4°C. Embryos were then infused with OCT embedding media and frozen solid in cryomolds. Embryos were sectioned on a Microm HM-505E at 7–9μm and stored at −20°C. Cryosections were hydrated in 1xPBS for 15 minutes at room temperature, then sequentially treated with 0.5% saponin and 0.1%saponin/0.1%sodium borohydride/PBS for 10 minutes each. Sections were washed in 0.1%saponin/PBS 3×15 minutes and blocked in 0.1%saponin/PBS/5%NGS for 3 hours at room temperature, then probed with goat anti-LAMP1 or goat anti-LAMP2 (1D4B, ABL-93, Santa Cruz Biotechnology), and rabbit anti-EEA1 (324610, Calbiochem; sc-33585, Santa Cruz Biotechnology) at 1:50 overnight at 4°C. After incubation, slides were washed and probed with Alexa 488 anti-goat or 488 anti-rabbit. After washing, slides were treated with DAPI at 50ng/ml for 10 minutes at room temperature, mounted in Gel Mount, and sealed with nail polish.
Preparation of embryos for transmission electron microscopy
Samples used for transmission electron microscopy were processed using standard techniques by Susan van Horn of the electron microscopy facility at Stony Brook University. Briefly, samples were fixed with 2% paraformaldehyde and 2.5% EM grade glutaraldehyde in 0.1M phosphate buffer saline (PBS), pH7.4, overnight. Samples were then placed in 2% osmium tetroxide in 0.1M PBS pH 7.4, dehydrated in a graded series of ethyl alcohol and embedded in Durcupan resin. Ultrathin sections of 80nm were cut with a Reichert-Jung UltracutE ultramicrotome and placed on formvar coated slot copper grids. Sections were then counterstained with uranyl acetate and lead citrate and viewed with a FEI Tecnai12 BioTwinG2 electron microscope. Digital images were acquired with an AMT XR-60 CCD Digital Camera system and compiled using Adobe Photoshop.
LysoTracker Red staining
Embryos were dissected at E 7.5 in DMEM at 37°C and freed from the parietal endoderm and Reichert’s membrane. Embryos were allowed to recover for 15 minutes at 37°C in DMEM/10%FBS in a tissue culture incubator. Staining was performed as described (Koike et al., 2009) and visualized on a Leica DMIRE2. Lysosome pixel intensity and diameter measurements were done using ImageJ. To measure pixel intensity, ten regions of interest (ROI) corresponding to ten individual cells were measured. The mean gray value for each ROI was multiplied by the area of the ROI to obtain the “integrated density”. The integrated density values were averaged, and a standard deviation was calculated. To measure lysosome diameters, eleven measurements were taken, ten corresponding to individual lysosomes, and one corresponding to the 8μm scale bar. The measurements were converted to μm using the 8μm scale bar measurement, averaged, and a standard deviation value was calculated.
488-RAP endocytosis assay
Embryos were dissected at E 7.5 in DMEM at 37°C and freed from the parietal endoderm and Reichert’s membrane. Embryos were washed in 1mL of PBSc at 4°C (1xPBS/1mM CaCl2/0.5mM MgCl2) and incubated with either 50mM 488-RAP in 100μL PBSc at 4°C, or 50mM 488-RAP/10uM RAP in 50μL cold PBSc for 1.5 hours at 4°C. Embryos were washed on ice with 2 × 1mL PBSc, then placed in 1mL of pre-warmed 37°C PBSc and incubated for 10 minute at 37°C. After incubation, embryos were fixed for 1 hour at 4°C in 4% paraformaldehyde and permeabilized for 5 minutes in 0.5% saponin/PBS. Embryos were then stained for 20 minutes at room temperature with DAPI (200ng/mL) and rhodamine-phalloidin (1:50, Invitrogen), mounted in Gel Mount, sealed with nail polish and visualized on a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Confocal Microscope.
Supplementary Material
Acknowledgments
The authors thank Drs. Deborah Brown, Ian Chambers, Howard Crawford, Jianguang Du, Michael Frohman, Kat Hadjantonakis, Fred Hess, Elizabeth Lacy, Masayuki Masu, Hans Scholer, Dudley Strickland, and Pierre Verroust for valuable reagents, protocols, and/or advice; Susan van Horn, Juei-Suei Chen, and Guo-wei Tian for technical assistance. The authors also thank Drs. Howard Sirotkin and Gerald Thomsen for their critical reading of the manuscript. These studies were supported by NIH grants to BCH (GM53964) and to Drs. Robert Haltiwanger and BCH (CA123071).
Grant support: NIH: GM053964 and CA095407
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