Human Molecular Genetics, 2000, Vol. 9, No. 7 1067-1074
© 2000 Oxford University Press
LMX1B transactivation and expression in nail–patella syndrome
1Children’s Hospital, University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany, 2Department of Molecular and Human Genetics and 8Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA, 3Istituto Nazionale per la Ricerca sul Cancro, Centro di Biotecnologie Avanzate, Genova, Italy, 4Hormone Research Unit, University of California at San Francisco, San Francisco, CA, USA, 5Institute of Human Genetics, University of Hamburg, Butenfeld 42, D-22529 Hamburg, Germany, 6Research Unit, Shriners Hospital for Children 3101 S.W. Sam Jackson Park Road, Portland, OR 97201, USA and 7Department of Pathology and Human Anatomy, Loma Linda University, Loma Linda, CA 92350, USA
Received 22 November 1999; Revised and Accepted 28 January 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail–patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix–loop–helix protein, E47/shPan1, can regulate LMX1B action. While co-transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nail–patella syndrome (NPS, MIM 161200) is an autosomal dominant, pleiotropic condition characterized by hypoplastic nails, hypoplastic or absent patellae, joint abnormalities with elbow contractures, iliac horns and renal insufficiency. We and others have demonstrated that NPS results from mutations within the LMX1B gene (1,2). Furthermore, Chen et al. (3) show that Lmx1b–/– mice exhibit renal and skeletal dysplasia similar to that observed in NPS patients. The analysis of the Lmx1b–/– mice and previous studies of ectopic expression of Lmx1b in chick limbs (4,5) show that LMX1B is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level during vertebrate development. The LMX1B gene encodes a LIM homeodomain (LIM-HD) protein belonging to a large family of transcription factors characterized by common cysteine-rich zinc-binding motifs that are important in mediating protein–protein interactions (6–8).
The group of LIM-HD proteins within the family of LIM proteins has been shown to be especially important in specifying various developmental programs (9–13). Despite the size of the LIM-HD gene family and its importance in development, LMX1B is the only known LIM protein to date in which subtle mutations have been found to be responsible for a human genetic disorder. An extensive mutation analysis of 41 NPS families by McIntosh and co-workers demonstrates that no correlation exists between disease severity and type or location of the mutation (14). While the biological importance of LMX1B is clear, comparatively little is known about its gene targets and the molecular mechanisms by which it functions. For example, until its correlation with the human phenotype, LMX1B function during renal development previously was unappreciated.
Regulation and targets of several other LIM-HD transcription factors have been the subject of intense study. The closely related LIM-HD protein Lmx1a/Lmx1.1 binds to and transactivates the rat insulin promoter (15). The target sequence element FAR1/FLAT contained within this promoter has been shown to also bind the hamster Lmx1b/Lmx1.2 ortholog (16). While the biological significance of this binding is unknown, it serves as a useful probe for homeodomain function of these two related LIM-HD proteins. Since the human and hamster Lmx1b homeobox sequences are 100% identical, we hypothesized and showed that the human LMX1B HD can also bind the rat FAR1/FLAT minienhancer element (1,14).
Given the function of LIM domains for mediating protein–protein interactions, a putative LIM domain-interacting protein (NLI/Ldb1/Clim2) has been described independently by several groups (17–19). Jurata and others (20–22) demonstrated direct interaction of Lmx1a with Ldb1. Moreover, Lmx1a and Lmx1b have both been shown to cooperate independently with the basic helix–loop–helix (bHLH) protein E47/shPan1 in activating an insulin gene promoter–reporter construct (15,16). Finally, Jurata and others (21) demonstrated direct interaction of all three components Lmx1a, Ldb1 and E47/Pan1 in transfection studies.
While Lmx1b is a unique mesenchymal determinant of dorsal patterning during vertebrate limb development, the mechanism by which mutations generate the NPS phenotype has not been addressed previously at the transcriptional level or correlated with its spatial pattern of gene expression. Studies in Drosophila show that the homolog to LMX1B, apterous, interacts with the Ldb ortholog, Chip, in a strict stoichiometric fashion to determine dorsal–ventral polarity in the wing (23). While heterozygous mutations in LMX1B cause NPS, the renal and skeletal dysplasia observed in Lmx1b–/– mice is of greater severity and only observed in homozygotes. While this may be due to differences in dosage sensitivities of the respective orthologous developmental programs to loss of Lmx1b function, the potential for a dominant-negative effect in humans cannot be ruled out, especially since deletions encompassing the entire gene have not yet been reported in NPS patients. To help address this question and to correlate the pathogenesis of NPS with Lmx1b function, we determined: (i) the pattern of Lmx1b expression in wild-type murine embryo sections and in cells expressing LMX1B; (ii) effect of human NPS mutations on LMX1B transactivation; and (iii) the potential for a dominant-negative effect of mutant LMX1B on wild-type LMX1B transactivation.
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RESULTS |
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Lmx1b expression during embryogenesis correlates with NPS phenotype
Previous whole-mount in situ hybridization showed that expression in the limb bud begins at E8.5 (24). To study directly the expression of Lmx1b in tissues affected in NPS, we performed in situ hybridization using murine Lmx1b on dorsal–ventral and anterior–posterior sections of developing murine limbs at E14.5 (Fig. 1A). Sections of forelimbs show strong expression in dorsal mesenchymal tissue destined to form tendons, ligaments and musculature. This expression exhibited a proximal–distal gradient and correlates with the degree of differentiation (i.e. greatest intensity in the distal less differentiated tissues). This was observed in both earlier and later sections at E12.5 and E16.5 (data not shown). Maximal expression was observed at E14.5 and was significantly diminished by E16.5, remaining in only the most distal dorsal tissues (nail mesenchymal precursor), suggesting a role for Lmx1b in initializing the specification of dorsal limb cell differentiation. Lmx1b expression in the limb was effectively abolished by E18.5.
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As expected, expression was found in patellar mesenchyme which ultimately gives rise to the patellar tendon and patella in hindlimbs. Interestingly, Lmx1b is also present in high concentration in dorsal mesenchyme within and surrounding developing joints. Furthermore, with increased expression anteriorly, another gradient of expression was found in the anterior–posterior axis of developing digits in both forelimbs and hindlimbs. The localization of Lmx1b to dorsal mesenchyme correlates well with the dorsal tissues (nail and patella) which are affected in NPS. Moreover, the prolonged expression in the tendinous patella precursor and concentration in anterior distal regions of digital mesenchyme correlate with more severe dysplasia of thumb nails and patellar phenotype. Gradation of nail dysplasia is observed from the anterior to posterior axis of the hand in NPS patients, paralleling the anterior–posterior gradient of Lmx1b expression (Fig. 1B). These data show that the tissues affected in NPS correlate with the dorsalizing function of LMX1B and with its graded expression in the anterior–posterior and proximal–distal axes during limb development. The NPS phenotype and pattern of Lmx1b expression is another example of interaction among signals which specify both the anterior–posterior and dorsal–ventral axes of the limb (4).
Functional consequences of mutations affecting the LIM B domain and homeodomain
Previous transfection studies demonstrated interaction of murine Ldb-1 and hamster Lmx1a (20–22). In our studies, we isolated human LDB1 by RT–PCR amplification of human chondrosarcoma (ATCC HTB94) RNA and confirmed its identity by sequence homology to murine and Xenopus Ldb1. Transfection of an LDB1 expression construct into 293 cells showed negligible transactivation of a reporter construct containing five copies of the rat insulin FAR1/FLAT enhancer element, a prolactin minimal promoter, and the luciferase reporter gene when compared with the reporter construct transfected with the empty vector (Fig. 2A). In contrast, co-transfection of LMX1B alone with the reporter resulted in 32-fold activation, while co-transfection of LDB-1 and LMX1B expression constructs showed dose-dependent inhibition of transactivation. LDB-1 appears to inhibit LMX1B-mediated transactivation of this reporter by 50%.
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Remarkable synergy between the LIM-HD proteins Lmx1a or Lmx1b and the bHLH protein E47/shPan1 on the rat insulin promoter has been reported previously (15,16). In the current study, co-transfection of human LMX1B and E47/shPan1 resulted in a >18-fold increase in transactivation when compared with LMX1B alone (Fig. 2B). The addition of LDB1 proportionately decreased the effect of LMX1B/E47 transactivation by 50%, similar to its effect on LMX1B alone.
To determine the effect of specific NPS mutations on this model of LMX1B transactivation, we generated LMX1B expression constructs containing representative NPS mutations (1,14). Since the majority of human mutations found are missense mutations affecting the HD, LMX1B cDNAs containing previously described human mutations affecting the third helix of the HD (N246K), the second helix of the HD (R226P), the first helix of the HD (A213P) and the N-terminal arm of the HD (R200Q) were generated. These mutations were all associated with classic NPS phenotypes. Interestingly, the R200Q and R226P HD mutations were shown to only partially decrease HD binding to the FAR1/FLAT element on electrophoretic mobility shift assay (EMSA; 14). Since premature termination mutations disrupting the HD and LIM domains theoretically may act in a dominant-negative manner, two additional LMX1B mutants were generated. The first contained a truncation of the C-terminal domain (amino acids 255 to the termination codon) while the second contained a missense mutation affecting a highly conserved cysteine in the LIM B domain (C142W). The C142W mutation was described previously in a patient with classic NPS (14).
Transfection of LMX1B containing each of the homeodomain missense mutations abolished transactivation by >90% (Fig. 2C). While mutations that were previously demonstrated to partially abolish DNA binding on EMSA (R200Q and R226P) did exhibit greater residual transactivation than those that had no detectable DNA-binding activity (A213P and N246K), the difference was not statistically significant. The truncation mutant and the C142W LIM B mutation showed significantly greater transactivation than the homeodomain mutants, but much less than wild-type LMX1B. The truncation mutant containing the remaining LIM A and B domains and the HD exhibited a 73% decrease in transactivation compared with wild-type controls, demonstrating that an activation domain resides within the C-terminus of LMX1B. The C142W mutation also retained significant residual transactivation, showing an ~70% decrease in activity of the reporter. Since the LIM B domain is required for LMX1B function (16), probably via interaction with the transcription apparatus including E47/shPan1, mutations in the LIM B domain potentially may result in a hypomorphic effect on transactivation of target genes.
Dominant-negative action versus haploinsufficiency of LMX1B mutations
The nature of the LMX1B mutations published to date (1,2,14,25) strongly implies haploinsufficiency in the pathogenesis of NPS. However, microdeletion of LMX1B has yet to be described in an NPS patient. Since renal and skeletal abnormalities are observed only in Lmx1b null mice and they are significantly more severe than those observed in NPS patients, a contributory dominant-negative effect in the human phenotype cannot be ruled out. To gain additional insight into the mechanism by which mutations in LMX1B might affect transactivation, we determined the effect of equimolar amounts of mutant LMX1B on wild-type LMX1B transactivation (Fig. 3A). In these studies, expression of LMX1B mutants containing missense mutations in the HD failed to transactivate the reporter but did not adversely affect normal transactivation by wild-type LMX1B. Co-transfection of mutant and wild-type LMX1B showed an ~50% dose-related decrease in transactivation when compared with wild-type LMX1B alone (Fig. 3A). Interestingly, in this assay, the R200Q and R226P mutants showed a slight but statistically significant greater contribution to total LMX1B transactivation compared with the A213P and N246K mutants. This paralleled the residual transactivation of reporter exhibited by each mutant when they were transfected alone previously (Fig. 2C). The residual transactivation observed in the transfection of LMX1B truncation and LIM B mutants alone was again evident in the mixing experiment where they contributed to wild-type LMX1B transactivation. These data show that depending on the nature of the mutation, LMX1B mutant protein either did not or only partially contributed to wild-type LMX1B transactivation in this assay. Importantly, it did not exert a significant dominant-negative effect which would have decreased transactivation by wild-type LMX1B.
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Cellular localization
Since LIM domains have been reported to mediate protein–protein interaction in the cytoplasmic compartment and regulation of transcription factor action may include sequestration outside of the nucleus, we determined the cellular localization of LMX1B expression in 293 cells permanently expressing LMX1B. The 293-LMX1B cell line was generated by transfection with the pcDNA-LMX1B expression construct (Fig. 3B). Anti-LMX1B polyclonal antibody stained 293 nuclei at low levels, but strongly localized expression to nuclei of transfected cells, suggesting that cytoplasmic sequestration does not play a role in LMX1B action.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To date, four genes are known to be expressed differentially along the dorsal–ventral axis during limb development. En-1 is expressed in the ventral ectoderm, r-Fng and the secreted factor Wnt 7a show expression in the dorsal ectoderm, while Lmx1b is expressed in the dorsal mesenchyme (for a review, see ref. 26). While r-Fng ultimately becomes restricted to the apical ectodermal ridge and is critical in distal limb outgrowth, the other genes have been shown by misexpression studies in chick and gene targeting studies in mice to be critical for establishing the dorsal–ventral cell fates of the developing limb. Consequently, mutations in Lmx1b result in a skeletal dysplasia affecting dorsal tissues, especially nail and patella. The human phenotype also exhibits abnormalities in the anterior–posterior axes as evidenced by the more severe nail dysplasia in the first and second digits, hypoplasia of the radial head, disproportionate prominence of the medial condyle of the femur and asymmetric development of joints. Our data show that this is probably due to a loss of the usual gradient of Lmx1b expression in NPS during limb development in both the proximal–distal and anterior–posterior axes. Our studies show that there is persistent and elevated expression in patellar mesenchyme and distal dorsal mesenchyme of first and second digits during development. Moreover, there is significant expression in the dorsal mesenchyme of developing joints. The presence of an anterior–posterior gradient in joint mesenchyme would correlate with the finding of asymmetric joint development: medial (anterior) versus lateral (posterior) dysplasia in the upper and lower skeletal elements. In fact, this effect is observed in the skeletal manifestations of Lmx1b null mice which have disproportionate growth of anterior versus posterior skeletal segments, i.e. ulnar hypoplasia.
LMX1B function requires not only DNA binding to target elements but also interaction with a complex transcriptional machinery which may include LDB1 and E47/shPan1. Our transfection studies show LDB1 down-regulation of LMX1B-mediated transactivation in both the presence and absence of E47/shPan1 in human cells. Studies in Drosophila suggest that a stoichiometric relationship is critical for normal function, and too much or too little of one particular component might have a hypomorphic effect on transactivation. This raises the possibility of dominant-negative action of subtle mutations in LMX1B on the transcription apparatus in the pathogenesis of NPS. Our data show that missense mutations in each of the helices of the HD can affect DNA binding and, ultimately, transactivation. Interestingly, human mutations that exhibit partial binding to target DNA sequences also exhibited a slightly greater transactivation of the reporter gene compared with those mutations which completely abolished DNA binding. This effect was observed to be significant in mixed transfection studies with wild-type LMX1B. At the same time, LIM B and truncation mutations which leave the HD intact result in a hypomorphic effect on transactivation, but were associated with classic NPS without correlation with severity or phenotypic manifestations in patients.
To address whether a dominant-negative effect might explain these observations, we modeled the in vivo situation by mixed transfection studies with equimolar amounts of wild-type and mutant LMX1B in the presence of E47/shPan1. In all cases, the human mutant LMX1B protein did not alter wild-type function, but instead contributed to transactivation of the reporter at a level directly correlated with its own endogenous transacting potential. While this situation cannot duplicate the more complex interactions in vivo, it does lend insight into the potential for competitive interactions with the E47/shPan1 coactivator. A higher order interaction requiring more than one component for a stable complex might be less susceptible to dominant-negative effects of one imbalanced protein member.
Finally, the immunolocalization of LMX1B in the nucleus suggests that sequestration in the cytoplasm such as is the case for NF-
B action is not a mechanism for regulating LMX1B function and, hence, not a target of potential antimorphic or neomorphic action (27). Our data, together with the nature of mutations described to date in NPS patients, suggest that haploinsufficiency of LMX1B function results in classic NPS. While our studies show that hypomorphic mutations may exist as quantitated by in vitro assays, they do not exert sufficient activity in vivo to alter the phenotypic expression of skeletal or renal abnormalities. Ultimately, an understanding of LMX1B action during development will benefit from identification of downstream effectors in development, description of microdeletion patients with NPS and novel phenotypic associations with subtle mutations, e.g. missense substitutions in the activation domain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cloning the human LDB1 gene
The coding region of LDB1 was isolated by PCR amplification of reverse-transcribed human chondrosarcoma (ATCC HTB94) RNA (28). Primers were designed from regions of identity/homology between mouse (accession no. U69270) and Xenopus laevis (accession no. U74360). The primers used were as follows:
LDB1afor, 5'-GGATCCGCCAAGATGTTGG-3';
LDB1brev, 5'-ACACAGAGTCGGAGGTAGTT-3';
LDB1cfor, 5'-GATAAAGACGTGGCACTTC-3';
LDB1drev, 5'-TCACTGGGAAGCCTGTGA-3'.
Plasmid constructs
The full-length cDNAs of LMX1B and LDB1 were subcloned in pBluescript-SK(–) and used as templates for PCR-mediated construction of expression plasmids. For expression plasmids containing wild-type and mutant LMX1B, forward primer (LMX1B-for) in the 5'-untranslated region and reverse primer (LMX1B-rev) in the 3'-untranslated region, with respective BamHI and EcoRV linkers, were used to PCR amplify full-length cDNAs. The PCR products were then subcloned into the BamHI–EcoRV site of the pcDNA3.1(+) vector (Invitrogen) under the control of a cytomegalovirus (CMV) promoter. The constructs which contain point mutations in the LIM B domain (C142W) or the homeodomain (R200Q, A213P, R226P and N246K) were generated by a two-step mutagenesis protocol previously described (1). PCR amplification was first performed using LMX1B-for and Mut-rev primer and LMX1B-rev and Mut-for primer (see list below for specific Mut primers). The PCR products were subject to a second round of amplification with LMX1B-for and LMX1B-rev. For the LMX1B truncation mutant, the wild-type LMX1B cDNA was amplified with LMX1B-for and a reverse primer with an XhoI linker (see list below).
For the LDB1 expression plasmid, pcDNA-LDB1, the LDB1 cDNA was amplified using forward and reverse primers from the respective 5'-untranslated (LDB1-for) and 3'-untranslated (LDB1-rev) domains. The LDB1-for primer contains a 5' BamHI linker and the Kozak consensus sequence. LDB1-rev contains a 5' XbaI linker. The cDNA was cloned into the pcDNA3.1(+) expression vector and confirmed by DNA sequencing. All constructs were confirmed by DNA sequencing on a Perkin Elmer 377 automated sequencer.
The plasmid constructs containing E47/shPan1 (15,29) and the reporter plasmid with five copies of the proinsulin minienhancer element FAR1/FLAT, a rat minimal promoter and a luciferase reporter are described elsewhere (15).
List of primers: the mutated bases are underlined
LMX1B-for, 5'-GGATCCGCCAAGATGTTGG-3';
LMX1B-rev, 5'-CTGTAGTCTGTGCGGATATCAGGG-3';
LMX1B-trunc.rev, 5'-CTGCTGCTAGCGCCGTCACGCCAG-3';
R200Q-for, 5'-CGGAGGCCCAAGCGACCCCAGACCATCCTCACCAC-3';
R200Q-rev, 5'-GTGGTGAGGATGGTCTGGGGTCGCTTGGGCCTCCG-3';
A213P-for, 5'-CAGCAGCGAAGAGCCTTCAAGCCCTCCTTCGAGGTC-3';
A213-rev, 5'-GACCTCGAAGGAGGGCTTGAAGGCTCTTCGCTGCTG-3';
R226P-for, 5'-GCCTTGCCGAAAGGTCCCAGAGACACTGGCAGCTG-3';
R226P-rev, 5'-CAGCTGCCAGTGTCTCTGGGACCTTTCGGCAAGGC-3';
N246K-for, 5'-GTCCAGGTCTGGTTTCAGAAACAAAGAGCAAAGATG-3';
N246K-rev, 5'-CATCTTTGCTCTTTGTTTCTGAAACCAGACCTGGAC-3';
C142W-for, 5'-CCAGCTGCTGTGGAAGGGTGACTACGAGAAG-3';
C142W-rev, 5'-CTTCTCGTAGTCACCCTTCCACAGCAGCTGG-3';
LDB1-for, 5'-GGATCCGCCGCCATGCTGGATCGGGATGTGG-3'; and
LDB1-rev, 5'-CTAGTCTAGACTAGTGGGAAGCC-3'.
Cell culture
Human 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4.500 mg/l D-glucose) supplemented with 0.1 mM non-essential amino acids and 10% fetal bovine serum (FBS). Cells were cultured in humidified 95% air and 5% CO2 at 37°C. 293 cells permanently expressing LMX1B were isolated by transfections of 293 cells with pcDNA-LMX1B followed by selection with media supplemented with G418 (750 µg/ml). After 15 days of G418 selection, clones of resistant cells were isolated and expanded. LMX1B expression was confirmed by RT–PCR amplification of 293-LMX1B RNA, and copy number was determined by Southern hybridization to the LMX1B cDNA.
Transfections
Cells were plated at 2.5–3 x 105 cells/well in 6-well plates 15–20 h before transfection. Transfection of each set of plasmid DNAs into 293 cells was performed five times by using DNA–lipid complexes (Lipofectamine Plus, Gibco BRL, Gaithersburg, MD) according to the manufacturer’s protocol. A 0.2 µg aliquot of each plasmid and the pSVß-galactosidase vector were used in each transfection. After exposure to the DNA–Lipofectamine complex for 5 h in serum-free medium, the cells were washed with fresh medium supplemented with 10% FBS and then cultured in the same medium. At 24 h after transfection, cells were washed twice with phosphate-buffered saline (PBS), lysed, and luciferase and ß-galactosidase assays were performed using the ß-gal enzyme assay and luciferase assay systems according to the manufacturer’s protocol (Promega, Madison, WI). Luciferase activities were normalized to ß-galactosidase activities. Transfections were performed five times, and average values and standard deviations were calculated. Transfections were performed at two doses to ensure linear dose-related reporter activity.
In situ hybridization on tissue sections
The in situ hybridization with 35S-labeled riboprobes was performed as previously described (30). The Lmx1b cDNA probe contains part of the 3'-untranslated region and was PCR amplified from reverse-transcribed mouse kidney mRNA. The cDNA was inserted into the EcoRV site of pBSIISK(–) (Stratagene, La Jolla, CA). The expression was visualized under dark fluorescence and digital images were captured via a Sony DC-5000. Morphological composition and colorization of the expression pattern (red) was done in Photoshop (5.0).
Immunohistochemistry and antibody production
Rabbit anti-human LMX1B antibody was generated against keyhole limpet hemocyanin (KLH)-conjugated peptides specific for the LIM B domain (NH2-D-M-K-P-A-K-G-Q-G-S-Q-S-K-G-S-G-D-C-COOH). Sera were isolated at 10 weeks after the second booster injection and tested by western analysis to the KLH-conjugated peptide (data not shown).
LMX1B-stably transfected cells were washed in 1x PBS and then fixed in 4% paraformaldehyde at 4°C for 30 min. For the detection of LMX1B protein, the Vectastain Elite ABC-Kit (anti-rabbit) as well as the DAB substrate kit for peroxidase (Vector Laboratories, Burlingame, CA) were used according to the manufacturer’s recommendations. The anti-LMX1B antibody was used at a 1:500 dilution. The stained cells were washed with deionized water, dehydrated with ethanol, incubated in xylol and preserved in mounting medium (Cytoseal, Stephens Scientific, USA).
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ACKNOWLEDGEMENTS |
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We are grateful to Rossi Dawn for editorial assistance. The work was supported by the Deutsche Forschungsgemeinschaft (S.D.D., A.W. and B.Z.), the National Institutes of Health (AR44738, B.L.), (HD01204, K.C.O.), March of Dimes Birth Defects Foundation (B.L.), the National Arthritis Foundation (B.L.), the Baylor College of Medicine Child Health Research Center (B.L.), Baylor College of Medicine Mental Retardation Research Center (B.L.) and the Shriners Research Program (#8540, W.A.H.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 713 798 8835; Fax: +1 713 798 5073; Email: blee@bcm.tmc.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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