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1 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
2 PRESTO, and 3 CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
4 Laboratory for Cell Culture Development, and 5 Molecular Neuropathology Group, Brain Science Institute, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
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Introduction |
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(Barth et al. 2007). FKBP38 also functions as a co-chaperone with the heat shock protein HSP90. It promotes the folding and export of cystic fibrosis transmembrane conductance regulator (Wang et al. 2006b), and it regulates trafficking of HERG, a voltage-dependent delayed-rectifier K+ channel (Walker et al. 2007). In addition, the HSP90–FKBP38 complex interacts with NS5A and thereby plays an important role in the replication of hepatitis C virus RNA, suggesting that the co-chaperone function of FKBP38 regulates the activity of NS5A (Okamoto et al. 2006).
Although FKBP38 is expressed in all tissues examined, it is especially abundant in the central nervous system, both in neurons and in glial cells (Nielsen et al. 2004). The expression level of FKBP38 in cells is affected by various conditions. In a transgenic mouse model of neurocristopathy, the abundance of FKBP38 is increased in partially differentiated Schwannoma cells, which are regarded as neural crest derivatives, implicating FKBP38 in differentiation of neural crest-derived cells (Jensen et al. 1993; Pedersen et al. 1999). Over-expression of the tuberous sclerosis (TSC) proteins, which are tumor suppressors, triggers a reduction in cell size accompanied by up-regulation of FKBP38, suggesting that the regulation of cell size by TSC1 and TSC2 depends on FKBP38 (Rosner et al. 2003). FKBP38 was recently shown to be an endogenous inhibitor of mammalian target of rapamycin (mTOR), with this inhibitory activity being antagonized by Rheb in response to growth factor stimulation or nutrient availability (Bai et al. 2007). The TSC1–TSC2 complex thus appears to regulate mTOR signaling by two mechanisms: First, the TSC1–TSC2 complex stimulates the intrinsic GTPase activity of Rheb, resulting in Rheb inactivation and consequent facilitation of the inhibitory effect of FKBP38 on mTOR. Second, the TSC1–TSC2 complex also increases the level of FKBP38, resulting in suppression of mTOR function. These findings suggest that FKBP38 is a critical regulator of growth factor- or nutrient-induced cell growth.
To investigate the physiological roles of FKBP38, we have now generated mice nullizygous for the FKBP38 gene (Fkbp38). We found that the Fkbp38–/– mice die shortly after birth manifesting a defect in neural tube closure in the thoraco-lumbar-sacral region (spina bifida). An abnormal increase in the frequency of apoptosis and abnormal extension of nerve fibers were implicated in the development of this defect. We identified protrudin, a key regulator of Rab11-mediated membrane trafficking during neurite extension, as a protein that interacts with FKBP38 (Shirane & Nakayama 2006). Protrudin was found to be hyperphosphorylated in Fkbp38–/– mice, possibly accounting for the abnormal extension of nerve fibers. During our analysis of Fkbp38–/– mice, another group (Bulgakov et al. 2004) reported the generation of mice that lack only a portion of the protein and which appear to show a more severe phenotype (embryonic death at embryonic day 13.5) than our FKBP38 nullizygous mice.
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Results |
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The protein-coding region of Fkbp38 comprises exons 3–10 (Fig. 1A). Bulgakov et al. recently generated mice that lack exons 6–8 of Fkbp38 (Bulgakov et al. 2004), in which it remains possible that a partial FKBP38 protein is generated by in-frame splicing of exons 5–9. The protein encoded by the mutant allele (
Ex.6–8) would lack most of the three tetratricopeptide repeat (3 x TPR) domain but would still contain the NH2-terminal region including most of the FKBP domain as well as the COOH-terminal region including the calmodulin binding and transmembrane domains (Fig. 1B). This protein might thus be expected to act as a dominant negative mutant, and the phenotype of the corresponding mutant mice may be expected to differ from that of FKBP38 nullizygous mice. To generate a loss-of-function mutant of Fkbp38 in mice, we designed a targeting vector to delete the genomic region from exon 3 to exon 7. The resulting mutant allele (Ex.3–7) thus lacks the first ATG codon as well as the DNA sequence encoding the FKBP domain and most of the 3 x TPR domain (Fig. 1B). Furthermore, the fact that an ATG codon is present at a position corresponding to codon 357 of the wild-type allele suggests that a protein consisting of only 46 amino acids might be generated from the mutant allele if splicing of exon 2 to exon 8 occurs. To examine the stability of the proteins encoded by the two mutant alleles of Fkbp38, we transfected HEK293T cells with expression vectors for wild-type FKBP38 or for the proteins encoded by Ex.6–8 (246 amino acids) or by Ex.3–7 (46 amino acids) and then measured the amounts of the corresponding mRNAs and proteins (Fig. 1C). Although the amounts of the three mRNAs were similar, the translation product of the Ex.3–7 allele was not detected, probably as a result of its instability, whereas that of the Ex.6–8 allele was readily detected. These data thus indicate that deletion of exons 3–7 constitutes a null mutation of Fkbp38.
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Ex.3–7 allele of Fkbp38 by electroporation with the targeting vector. The transformed ES cells were injected into C57BL/6J mouse blastocysts, and chimeric males that transmitted the mutant allele in the germ-line were obtained. Heterozygotes were intercrossed to produce homozygous mutant mice (hereafter referred as FKBP38 nullizygous or Fkbp38–/– mice). We verified the homologous recombination event by PCR (Fig. 1D) and Southern blot (Fig. 1E) analyses of the mutant mice. Neither full-length nor truncated FKBP38 was detected in Fkbp38–/– mice by immunoblot analysis (Fig. 1F). Furthermore, immunofluorescence analysis of mouse embryonic fibroblasts prepared from Fkbp38–/– embryos did not yield a signal for FKBP38, which was localized predominantly to the mitochondrial membrane of wild-type MEFs (Fig. 1G). These results thus indicated that homozygous disruption of exons 3–7 of Fkbp38 indeed gives rise to mice nullizygous for FKBP38. Loss of FKBP38 results in neural tube and skeletal defects
Whereas mice with homozygous disruption of exons 6–8 of Fkbp38 die in utero at E13.5, those with homozygous disruption of exons 3–7 (Fkbp38–/– mice) were found to complete embryonic development but to die soon after birth (Table 1). The heterozygotes appeared normal and were healthy and fertile. Mating of Fkbp38+/– heterozygotes yielded Fkbp38+/+, Fkbp38+/– and Fkbp38–/– embryos, which were detected in the expected mendelian ratio from E10.5 to E18.5. The Fkbp38–/– neonates were abnormally small and exhibited cyanosis, dehydration, malformed limbs, and a lack of milk in the stomach (Fig. 2A,B). The most prominent characteristic of the Fkbp38–/– mice, however, was spina bifida, which manifested with a penetrance of 100% (Fig. 2C). Some homozygous mutant embryos also exhibited talipes varus (Fig. 2D) or curled tail (Fig. 2E) associated with the spina bifida (Juriloff & Harris 2000; Copp et al. 2003). Talipes varus is often observed in mice with spina bifida as well as in humans with this condition (Harris & Juriloff 2007). In addition, the spine of Fkbp38–/– mice manifested pronounced scoliosis, with the vertebral arches having failed to fuse at the dorsal midline between the forelimbs and hind limbs, whereas all other components of the vertebrae were present and showed normal ossification (Fig. 2F). Fkbp38–/– mice also exhibited malformation of the ribs, which were dilated and flattened and appeared to be responsible for the dyspnea observed in neonates. Analysis of transverse sections of the homozygous mutants at the lumbar level revealed the prominent malformation of the spinal cord (Fig. 2G). The vertebrae were open with a large vertebral body, short but stretched vertebral arch and crushed spinal cord. Notable abnormalities of internal organs were not detected (data not shown).
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Histopathologic examination of the brain of embryos at E18.5 revealed that Fkbp38–/– mice showed marked malformation of the cerebrum and cerebellum (Fig. 3A). The neuroepithelium of the cerebrum was disorganized and showed an increased density of neurons. The cerebellum appeared immature, with fewer grooves compared with wild-type embryos. Some of the mutant embryos developed exencephaly, characterized by an open cranial neural tube, in addition to the spinal neural tube defect (Fig. 3B,C). In addition, histopathologic examination revealed that all mutant embryos, even those that did not develop exencephaly, exhibited malformation of the brain neural tube (Fig. 3D,E). A closed neural tube with an enlarged lumen and aggregated neuroepithelium were conspicuous in most mutant mice. Marked malformation of the thalamus and cortex, accompanied by a shrunken medial cortex with overlap of the dorsal cortex, were apparent. These abnormalities appeared attributable to disorder and piling up of cells of the medial cortex and dorsal cortex. Abnormal aggregation of neurons was apparent in some mutant embryos (Fig. 3E). Together, these observations showed that the arrangement of cells in the brain was highly disorganized in Fkbp38–/– mice.
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We further characterized the abnormal aggregation of neurons in FKBP38 nullizygous mice by immunohistofluorescence analysis. To examine whether this abnormal aggregation was the result of overproliferation of the neuroepithelium, we stained sections with antibodies to phosphorylated histone H3 (Fig. 4A). No marked difference in the number of proliferating cells in the neuroepithelium was apparent between Fkbp38+/+ and Fkbp38–/– embryos, indicating that the hypertrophy in the FKBP38-deficient neuroepithelium is not likely the result of overproliferation. We then analyzed the distribution of neuronal progenitors in the neural tube (Fig. 4B). Staining with antibodies to MAP2 (marker of differentiated neurons) or with the antibody TUJ1 (specific for βIII-tubulin in post-mitotic neurons) revealed that the cell aggregates in the mutant embryos were composed of differentiated neurons. These MAP2+TUJ1+ cells partially overlapped with cells that stained with antibodies to calretinin, but the central region of the aggregates was not positive for this marker of interneurons. The radial fibers revealed by antibodies to nestin were also disorganized in the mutant embryos. Furthermore, the aggregated cells were positive for the homeodomain proteins Pax6, Mash1, or Islet1. Pax6, which is expressed in the lateral cortex of the normal brain, was ectopically expressed in the dorsal cortex and medial cortex of Fkbp38–/– mice (Fig. 4C). Many cells in the abnormal aggregates were positive for both Pax6 and Mash1 (Fig. 4D), which are expressed in a mutually exclusive manner in the normal brain. In addition, Islet1, which marks dorsal root ganglion cells and differentiated motor neurons, was abnormally coexpressed with Pax6 and Mash1 in the mutant brain (Fig. 4B). These results suggested that the abnormal cell aggregation in the mutant brain is attributable to a disordered distribution of neuronal progenitors that results from a defect in cell differentiation or migration rather than from overproliferation.
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The gross appearance of the spinal cord of Fkbp38–/– mice at E16.5 was characterized by marked kinks at the thoracic to sacral levels (Fig. 5A). Immunofluorescence staining with antibodies to nestin revealed that the arrangement of the radial fibers in the spinal cord was abnormal in the mutant embryos (Fig. 5B). In horizontal sections, the radial fibers of wild-type mice extend radially in all directions whereas those of Fkbp38–/– mice showed a dot-like (rather than a fiber-like) pattern, indicating that the axis of the fibers in the mutant mice was inclined. An unbalanced extension of the rostral–caudal axis in the mutant embryos may result in an incline of plates on which the radial fibers extend. In addition, histopathologic examination revealed that the neural tube of Fkbp38–/– mice was highly deformed with a thin epithelium and markedly dilated central canal at the cervical level, whereas it was reduced in size but maintained a normal overall structure at the sacral level (Fig. 5C). The formation of dorsal root ganglia was also markedly defective in the mutant embryos. The ganglia were rudimentary especially at the lumber-sacral levels of the spinal cord, probably as a result of an increased frequency of apoptosis during development (see below).
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FKBP38 is expressed in developing neurons associated with the neural tube, dorsal root ganglia, and notochord (Fig. 6A). Given that FKBP38 inhibits apoptosis through interaction with the anti-apoptotic proteins Bcl-2 and Bcl-xL (Shirane & Nakayama 2003), we examined whether the frequency of apoptosis might be increased in the regions of FKBP38 nullizygous embryos in which FKBP38 is normally expressed. Immunofluorescence analysis with antibodies to the cleaved (activated) form of caspase-3 revealed few apoptotic cells in the developing spinal cord of wild-type embryos at E10.5. In contrast, the number of apoptotic cells was markedly increased in the dorsal root ganglia and in the region surrounding the notochord of Fkbp38–/– embryos at this time (Fig. 6B,C). Apoptotic cells in the neural tube of the mutant embryos were not as frequent as those in dorsal root ganglia, but their number was still increased substantially compared with that in control embryos. We also examined signaling by Shh in the mutant embryos, given that FKBP38 has been implicated as a negative regulator of Shh signaling during development (Bulgakov et al. 2004). In the spinal cord of Fkbp38–/– embryos at E12.5, the area expressing the gene for Shh, a marker of ventral fate, as well as that for Nkx2.2, a downstream transcription factor, was expanded dorsally but did not express the gene for the dorsal fate marker Wnt1 (data not shown), consistent with previous observations with the FKBP38 mutant mice generated by Bulgakov et al. Given that secretion of Shh by the notochord establishes the ventral pole of the dorsal–ventral axis in developing embryos, the increased frequency of apoptosis in the notochord of the mutant embryos may result in their ventralization (Copp et al. 2003).
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Immunofluorescence analysis of differentiated neurons with the TUJ1 antibody revealed abnormal extension of nerve fibers in the spinal cord of Fkbp38–/– embryos at E14.5 (Fig. 7A). The neurons positive for βIII-tubulin in wild-type embryos extend their axons on the same side of the neural tube, whereas the axons of such neurons appeared to project transversely over the midline to the opposite side of the neural tube in Fkbp38–/– embryos. Immunofluorescence analysis of the peripheral nervous system with antibodies to periferin, a marker for intermediate filaments type III, also revealed the abnormal extension of nerve fibers in the spinal cord of Fkbp38–/– embryos (Fig. 7B).
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To identify the molecular mechanism responsible for the abnormal projection of neurites in Fkbp38–/– mice, we screened a human brain cDNA library with FKBP38 as the bait in the yeast two-hybrid assay. One of the positive clones that we obtained encoded a previously uncharacterized protein that was subsequently designated protrudin on the basis of its ability to promote neurite extension (Shirane & Nakayama 2006). To examine the specificity of the binding of protrudin to FKBP38, we tested whether it also interacts with FKBP12 or FKBP52, two proteins structurally related to FKBP38, both in yeast (Fig. 8A) and in mammalian cells (Fig. 8B). Protrudin interacted with FKBP38, but not with FKBP12 or FKBP52, in both systems, suggesting that the protrudin–FKBP38 interaction is specific. Given that protrudin regulates membrane recycling through interaction with the small GTPase Rab11, resulting in promotion of neurite formation, and that this latter effect of protrudin depends on its phosphorylation triggered by nerve growth factor and mediated by the extracellular signal-regulated kinase (ERK) signaling pathway, we examined the phosphorylation state of protrudin in the brain of wild-type and Fkbp38–/– mice. Two-dimensional (2D) PAGE analysis within the pH range of 4–7 followed by immunoblot analysis with antibodies to protrudin revealed that the spots corresponding to protrudin in the brain of the mutant mice were markedly shifted in the acidic direction compared with those in the brain of wild-type mice (Fig. 8C), suggesting that the level of protrudin phosphorylation is increased in the mutant brain. We confirmed these results with high-resolution (pH 3–5.6) 2D-PAGE (Fig. 8D). These observations thus suggest that FKBP38 regulates the neurite-extending activity of protrudin by inhibiting protrudin phosphorylation, and that the loss of FKBP38 results in hyperphosphorylation of protrudin and consequent dysregulation of neurite extension in the developing nervous system.
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Ex.6–8 mice) die in utero at E13.5 manifesting defects in neural tube formation and eye development (Bulgakov et al. 2004). The posterior neural tube of the mutant mice is excessively ventralized as a result of expansion of Shh signaling, suggesting that FKBP38 is a negative regulator of such signaling. Given that neural tube defects alone do not usually result in the death of mouse embryos in utero, the cause of the embryonic mortality of the mutant animals remains unknown. In contrast, we have now shown that mice lacking exons 3–7 of Fkbp38 (Ex.3–7 mice) survive embryonic development but die soon after birth, likely as a result of pronounced dyspnea. The Ex.3–7 mice exhibit a variety of abnormalities including neural tube defects, an abnormal body axis, aberrant skeletal formation, disorganization of the neuroepithelium, and abnormal extension of nerve fibers, all of which are likely attributable to an increased frequency of apoptosis or dysregulation of protrudin in addition to increased Shh signaling.
The phenotypic differences between the two types of mice are likely attributable to differences in the deleted region of the Fkbp38 locus, although a contribution of differences in genetic background or environmental factors cannot be excluded. In Ex.6–8 mice, the splicing of exon 5 to exon 9 in frame potentially results in the expression of a truncated mutant protein containing the first methionine residue, most of the FKBP domain, the calmodulin binding motif, and the transmembrane domain. Such a partial product may influence the function of proteins that associate with FKBP38, including calmodulin (Edlich et al. 2005), calcineurin, Bcl-2 and Bcl-xL (Shirane & Nakayama 2003), the proteasome (Nakagawa et al. 2007), and mTOR (Bai et al. 2007), through gain-of function or dominant negative effects, especially given that the PPIase activity of the FKBP domain is thought to be the most important activity of FKBP38. In contrast, the mutant allele generated by the deletion of exons 3–7 potentially encodes a protein of only 46 amino acids, which is highly unstable if produced at all. We therefore conclude that Ex.3–7 mice are nullizygous for FKBP38 function.
Defects in neural tube closure are relatively common in humans, with a frequency of approximately 1 in 1000 births (Juriloff & Harris 2000). Given that the etiology of such defects is genetically complex, establishment of a mouse model would be expected to provide insight into the human condition (Copp et al. 2003; Harris & Juriloff 2007). The FKBP38 nullizygous mice examined in the present study exhibit neural tube defects with 100% penetrance, suggesting that FKBP38 plays an important role in neural tube formation. Abnormal cell aggregates were detected in the brain of Fkbp38–/– mice. In addition, the spinal cord of the mutant animals exhibited abnormal extension of nerve fibers. The abnormal structure of the neuroepithelium and altered differentiation of neuronal precursors in the neural tube apparent in the FKBP38 nullizygous mice indicate that the primary defect is associated with development of the neuroepithelium.
Given the presence of ventral neural progenitors in the dorsal neural tube in Fkbp38–/– mice, we examined Shh signaling in these animals. Shh is a major organizer of dorsoventral patterning and is required for the induction of motor neurons and adjacent interneuron progenitors in the ventral neural tube (Copp et al. 2003). FKBP38 has been implicated as a negative regulator of Shh signaling during development, and we confirmed that expression of the Shh gene, a marker of ventral fate, was expanded dorsally in the FKBP38 nullizygous embryos, consistent with previous observations with Ex.6–8 mice (Bulgakov et al. 2004). The phenotype of FKBP38 nullizygous embryos resembles that of embryos that lack protein kinase A (PKA), suggesting that FKBP38 and PKA may perform similar functions (Huang et al. 2002). PKA inhibits the Hedgehog signaling pathway by phosphorylating and thereby affecting the conformation of Smoothened, a transducer of Hedgehog signaling (Jia & Jiang 2006; Zhao et al. 2007). These observations thus suggest that FKBP38 may also contribute to the regulation of Smoothened.
It is uncertain whether apoptosis and mis-oriented neurite extension are attributable to the deregulation of Shh signaling. A previous report showed that apoptosis of neurons is inhibited Shh signaling (Thibert et al. 2003). Given that apoptosis is actually increased in FKBP38-nullizygous mice, it is unlikely that the increased apoptosis is the result of Shh activation in FKBP38-nullizygous mice. Also, Shh signaling was shown to inhibit neurite outgrowth (Trousse et al. 2001) or to play a repulsive role in axon guidance (Bourikas et al. 2005). These data are also inconsistent with our observation that the loss of FKBP38 results in the abnormally activated neurite extension, suggesting that Shh activation in FKBP38-nullizygous mice is not the cause of abnormal extension of the neurons. On the other hand, some studies demonstrated that Shh serves as a chemoattractant in axon guidance at least in some condition (Charron et al. 2003; Kolpak et al. 2005). Given that the abnormal extension of axons in FKBP38-nullizygous mice was not oriented to the notochord where Shh concentration is high, however, it is also unlikely that the mis-orientation of neuronal axons is the result of Shh hyperactivation in FKBP38-nullizygous mice.
Apoptosis is a highly regulated cellular process that is essential for embryonic development. FKBP38 interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL, thereby promoting their localization to mitochondria, as well as with calcineurin. These interactions result in the suppression of apoptosis by FKBP38 in cultured cells. The number of apoptotic cells was greatly increased in the posterior neural tube of FKBP38 nullizygous mice at E10.5. However, the number of apoptotic cells in the neural tube, including the brain and lumbar spinal cord, was similar in wild-type and mutant embryos after E12.5 (data not shown), suggesting that the time window of FKBP38 action during neural development is relatively small. These results show that endogenous FKBP38 exhibits anti-apoptotic function specifically in the posterior neural tube. Mice that lack Bcl-xL exhibit a diffuse pattern of apoptosis in the neural tube (Motoyama et al. 1995), whereas those deficient in Bcl-2 do not show a neuronal phenotype (Nakayama et al. 1993; Veis et al. 1993; Nakayama et al. 1994). These observations suggest that antagonism of Bcl-xL function by FKBP38 is intrinsic to normal development of the neural tube.
Abnormal extension of nerve fibers in the neural tube of Fkbp38–/– mice was found to be accompanied by hyperphosphorylation of protrudin, an FKBP38-binding protein. Protrudin contains an atypical Rab11 binding domain and GDI consensus sequence, and it promotes neurite formation through interaction with the guanosine diphosphate (GDP)–bound form of Rab11 (Shirane & Nakayama 2006). The association of protrudin with Rab11 is promoted by phosphorylation of the former protein, which is mediated by ERK signaling triggered by nerve growth factor. Forced expression of protrudin in cultured cells induced localized membrane extension and the consequent formation of long protrusions, whereas down-regulation of protrudin by RNA interference induced membrane extension in all directions, resulting in inhibition of neurite formation. Hyperphosphorylation of protrudin is therefore likely responsible for the abnormal extension of nerve fibers in FKBP38 nullizygous mice. FKBP38 may inhibit the phosphorylation of protrudin by sequestering it from ERK or by inducing a conformational change of protrudin that reduces its accessibility to ERK. Future studies with cultured cells may shed light on the precise mechanism by which FKBP38 regulates the function of protrudin. The diverse effects of FKBP38 ablation are thus likely explained by alteration of the function of proteins that bind to FKBP38, such as Bcl-2 and Bcl-xL as well as protrudin.
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Genomic DNA corresponding to the Fkbp38 locus was isolated from a 129/Sv mouse genomic DNA library. The targeting vector was constructed by replacing a 5.2-kb fragment containing exons 3–7 of Fkbp38 with a cassette containing the mouse phosphoglycerate kinase (PGK) gene promoter, the neomycin phosphotransferase gene (neo), and a poly(A) sequence (Nakayama et al. 1996, 2000). The targeting vector thus contained 1.2- and 8.0-kb regions of homology 5' and 3' of the neomycin resistance marker, respectively. A cassette containing the PGK gene promoter, the thymidine kinase gene (tk) of herpes simplex virus, and a poly(A) sequence was ligated at the 3' end of the insert. 129/Sv mouse embryonic stem (ES) cells were subjected to electroporation with the linearized targeting vector (Fig. 1A) followed by positive (G418) and negative (ganciclovir) selection. Eleven positive ES cell clones were identified by polymerase chain reaction (PCR) analysis of 101 selected clones. To confirm the homologous recombination event, DNA prepared from PCR-positive clones was digested with EcoRI, transferred to a nylon membrane, and then subjected to Southern blot analysis with the probes as shown in Fig. 1A. ES cells heterozygous for the targeted mutation were microinjected into C57BL/6J mouse blastocysts, which were then implanted into pseudopregnant ICR female mice. The resulting male chimeras were mated with C57BL/6J females, and germ-line transmission of the mutant allele was confirmed by Southern blot analysis. Heterozygous offspring were intercrossed to produce homozygous mutant animals. For generation of Fkbp38–/– embryos, Fkbp38+/– mice were intercrossed and the time at which the vaginal plug was detected was considered embryonic day (E) 0.5. Animals were genotyped by PCR with three primers (5'-ACTGTTCCGCAAGGGCAAGGTG-3', 5'-AGACAACCAGGACTGAGTAGACGG-3', and 5'-TCCA GACTGCCTTGGGAAAAGCGC-3'). FKBP38 mutants were analyzed on a mixed 129/C57 genetic background. All mouse experiments were approved by the animal ethics committee of Kyushu University.
Histology
For histological analysis, embryos were dissected from the uterus at the desired stage of gestation, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated, and embedded in paraffin wax. Sections (thickness, 5 µm) were mounted on slides for staining with hematoxylin and eosin (H&E).
Immunohistofluorescence analysis
Fixed tissue was exposed to 20% (w/v) sucrose in PBS, embedded in Tissue-Tek O.C.T. compound (Sakura, Tokyo, Japan), and cryosectioned at a thickness of 50 µm. Sections were rehydrated in PBS, permeabilized with 0.01% Triton X-100 in PBS containing 0.1% bovine serum albumin, and exposed either to primary antibodies for immunohistofluorescence analysis or to toluidine blue for Nissl staining. Immunohistofluorescence analysis was performed with antibodies to FKBP38 (Shirane & Nakayama 2003), to microtubule-associated protein (MAP) 2 (Sigma, St. Louis, MO), to phosphorylated histone H3 (Cell Signaling, Danvers, MA), to neuron-specific βIII-tubulin, TUJ1 (Covance, Princeton, NJ), to calretinin (Zymed, San Francisco, CA), to nestin (Rat401, Developmental Studies Hybridoma Bank), to Pax6 (Convac, Brimsdown, UK), to Mash1 (R&D Systems Inc., Minneapolis, MN), to Islet1 (40.2D6; DSHB, Iowa City, Iowa), or to peripherin (Chemicon, Temecula, CA). Apoptotic cells were detected by immunohistofluorescence analysis with antibodies to activated caspase-3 (Cell Signaling) or with the use of a TUNEL staining kit (Boehringer, Ridgefield, CT). For staining of mitochondria, cells were incubated for 20 min with 100 nM MitoTracker (Molecular Probes, Carlsbad, CA) before fixation.
Skeletal staining
E16.5 embryos were placed in a water bath at 65 °C for 20–30 s to facilitate skin removal, and the deskinned embryos were eviscerated, fixed in 95% ethanol for 3 days at room temperature, and stained overnight at room temperature with 15% Alcian blue in 95% ethanol and 20% glacial acetic acid. The skeletons were washed with and then maintained overnight at room temperature in 95% ethanol. After exposure to 1% KOH for 4 h, they were stained for 2 h at room temperature with 5% alizarin red in 2% KOH. They were then incubated for 2 days at room temperature in 2% KOH followed by overnight incubations in mixtures of 2% KOH and glycerol (80 : 20, 60 : 40 and 40 : 60, v/v, sequentially). The skeletal preparations were stored in 2% KOH–glycerol (20 : 80, v/v).
In situ hybridization
In situ hybridization was performed as previously described (Motoyama et al. 2003). Mouse cDNAs for Sonic hedgehog (Shh), Nkx2.2, or Wnt1 were used as templates to generate digoxygenin-labeled RNA probes.
Yeast two-hybrid system and cDNA isolation
The human FKBP38 cDNA was fused to the DNA sequence for the DNA binding domain of LexA in the plasmid pBTM116 (kindly provided by S. Fields, Howard Hughes Medical Institute), which also contains the TRP1 nutritional gene. For screening, the resulting vector was introduced by transformation into the yeast L40 reporter strain together with a human brain cDNA library in the pACT2 vector (Clontech, Mountain View, CA), which contains the DNA sequence for the activation domain of GAL4 as well as the LEU2 nutritional gene. Transformants were selected by plating the yeast cells on DOB medium lacking tryptophan and leucine, and they were assayed for β-galactosidase activity. One positive clone was recovered in Escherichia coli and sequenced.
Immunoprecipitation and immunoblot analysis
Cells or tissues were homogenized in a solution (Triton lysis buffer) containing 40 mM Hepes–NaOH (pH 7.6), 150 mM NaCl, 0.5% Triton X-100, 10 mM MgCl2, 1 mM Na3VO4, 1 mM EDTA, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 µg/mL), leupeptin (10 µg/mL), 10 µM MG132 and 10% glycerol. The homogenate was incubated for 10 min on ice and then centrifuged at 10 000 g for 10 min at 4 °C. After determination of its protein concentration with the Bradford assay (Bio-Rad, Hercules, CA), the resulting supernatant was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and the separated proteins were transferred to an Immobilon-P membrane (Millipore, Billerica, MA) and probed with primary antibodies. Immune complexes were detected with Super Signal reagents (Pierce, Rockford, IL). For immunoprecipitation, cell or tissue extracts were incubated with both primary antibodies (or control immunoglobulin G) and protein G–Sepharose 4 Fast Flow (GE Healthcare Limited). Antibodies used for immunoblot analysis or immunoprecipitation included those to FKBP38 (Shirane & Nakayama 2003), to the HA (HA11, Roche, Tokyo), to calnexin (StressGen Biotechnologies, Victoria, CANADA), to HSP70 (BD Transduction Laboratories, San Jose, CA), or to the FLAG (Sigma).
Two-dimensional PAGE and immunoblot analysis
Fkbp38+/+ or Fkbp38–/– embryos were homogenized in Triton lysis buffer. The resulting extracts were applied to an Immobiline Dry Strip pH 3–5.6 or 4–7 (Amersham Biosciences, Uppsala, Sweden) for isoelectric focusing with an Ettan IPG phore Isoelectric Focusing System (GE Healthcare Limited, Buckinghamshire, UK) followed by SDS-PAGE. The separated proteins were then subjected to immunoblot analysis with antibodies to protrudin (Shirane & Nakayama 2006).
Cell culture, transfection and mRNA analysis
HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% FBS and transfected with the use of FuGENE 6 (Roche). mRNA were prepared from the cells with ISOGEN kit (NipponGene, Toyama, Japan), followed by reverse-transcriptase reaction with ReverTraAce kit (Toyobo, Osaka, Japan), and the cDNA was analysed by PCR with primers for exon 10 of Fkbp38 (5'-TCCATC CCGTGGAAATGGCTG-3') and (5'-TCAGTTCCT GCCAGCAATGAC-3').
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* Correspondence: Email: nakayak1{at}bioreg.kyushu-u.ac.jp
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Received: 9 February 2008
Accepted: 17 March 2008
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