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Expression of SHOX in Human Fetal and Childhood Growth Plate

Endocrine Research Unit (C.J.F.M., M.T.H., J.A.B.), Royal Children’s Hospital Foundation Research Centre, and Department of Paediatrics and Child Health (C.J.F.M., H.R.H., L.M.C., M.T.H., J.A.B.), University of Queensland, Royal Children’s Hospital, Brisbane QLD 4029, Australia; Institute of Human Genetics (R.B., G.R.), Heidelberg University, 69120 Heidelberg, Germany; and Division of Genetics and Development (I.A.G.), Department of Pediatrics, University of Washington, Children’s Hospital and Regional Medical Center, Seattle, Washington 98112

Address all correspondence and requests for reprints to: Professor J. A. Batch, Endocrine Research Unit, Royal Children’s Hospital Foundation Research Centre, Royal Children’s Hospital, Brisbane QLD 4029, Australia. E-mail: j.batch{at}mailbox.uq.edu.au.

	Abstract

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Abnormalities in the growth plate may lead to short stature and skeletal deformity including Leri Weil syndrome, which has been shown to result from deletions or mutations in the SHOX gene, a homeobox gene located at the pseudoautosomal region of the X and Y chromosome. We studied the expression of SHOX protein, by immunohistochemistry, in human fetal and childhood growth plates and mRNA by in situ hybridization in childhood normal and Leri Weil growth plate. SHOX protein was found in reserve, proliferative, and hypertrophic zones of fetal growth plate from 12 wk to term and childhood control and Leri Weil growth plates. The pattern of immunostaining in the proliferative zone of childhood growth plate was patchy, with more intense uniform immunostaining in the hypertrophic zone. In situ hybridization studies of childhood growth plate demonstrated SHOX mRNA expression throughout the growth plate. No difference in the pattern of SHOX protein or mRNA expression was seen between the control and Leri Weil growth plate. These findings suggest that SHOX plays a role in chondrocyte function in the growth plate.

	Introduction

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LONGITUDINAL GROWTH TAKES place in the growth plate through a process called endochondral bone formation. In this process, chondrocytes in the reserve zone are recruited to start clonal proliferation (proliferative zone) and then undergo differentiation, which is followed by apoptosis (hypertrophic zone) and eventually mineralization. The balance between proliferation and differentiation is a crucial regulatory step controlled by a number of transcription factors, hormones, and growth factors.

Abnormalities in the growth plate may lead to short stature and skeletal deformity seen in many skeletal dysplasias including the Leri Weill dyschondrosteosis (LWD). We and others have shown that LWD results from deletions or mutations in the SHOX gene (1, 2, 3), a homeobox gene located at the pseudoautosomal region of the X and Y chromosome (4, 5). Langer mesomelic dysplasia, a very severe form of limb shortening and dwarfism, has recently been shown to be the homozygous form of LWD (6). Using histological techniques, we have also recently shown that the distal radial physis from SHOX haploinsufficient LWD subjects has a reduction in the size of the proliferative zone of chondrocytes, with a loss of the normal columnar stacking of chondrocytes within this zone (7).

Transcription of the SHOX gene may give rise to a variety of transcripts. At least two transcription initiation sites have been described (8), producing transcripts that differ in length at the 5'-untranslated region and in efficiency of translation. In addition, proteins of different lengths may arise as a result of alternate splicing (9).

RT-PCR and in situ hybridization studies on human tissues have identified SHOX gene expression in bone marrow fibroblasts, skeletal muscle, placenta, heart, fetal kidney, and the limbs of embryos (5, 10), but no SHOX protein expression studies have been performed on human tissues including growth plate.

Because we have demonstrated that the growth plate is abnormal in LWD individuals with SHOX gene deletions/mutations (7), we hypothesized that the SHOX gene is expressed in the growth plate and plays a role in growth plate function. In the present study, we therefore aimed to document the ontogeny of SHOX protein expression in human fetal growth plate and evaluate the expression pattern of SHOX mRNA and protein in the normal childhood growth plate and the growth plate of individuals who are haploinsufficient for the SHOX gene.

	Materials and Methods

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All investigations were performed at the Royal Children’s Hospital, Brisbane and associated facilities. The collection of blood samples for genetic analysis and materials for immunohistochemical analysis were undertaken after approval from The Royal Children’s Hospital Ethics Committee and informed consent from the subjects and/or guardian.

Growth plate samples

Growth plate sections from seven different fetuses between 8 and 40 wk gestation without dysmorphic features and absent/minimal postmortem maceration were studied. Gestational age was estimated from the results of an antenatal ultrasound scan or dates of the mother’s last menstrual period. The specimens had been fixed in 10% neutral buffered formalin saline for 24 h and then embedded in paraffin without decalcification. Paraffin blocks of costochondral junction were sectioned at 5-µm intervals and placed on Supafrost glass slides. One slide from each block was then stained with hematoxylin and eosin by standard methods. Distal radial growth plate was obtained at the time of physiolysis (Vickers’ procedure) (11) for correction of bilateral Madelung deformity from four subjects with LWD and SHOX haploinsufficiency (7). Proximal tibial growth plate was obtained from four control subjects at the time of surgical intervention (physiodesis) for leg length discrepancy. All growth plate specimens were fixed in 10% formalin, decalcified in 10% EDTA, mounted, and sectioned at 5-µm intervals on to Supafrost glass slides.

Genetic analysis

The molecular diagnosis of LWD was made according to previously described methods (3, 7).

Growth plate SHOX immunohistochemistry using diaminobenzidine (DAB)

Paraffin sections of growth plate were dewaxed, rehydrated to Tris-buffered saline (TBS), and treated with 0.1% trypsin at 37 C for 10 min. Exogenous peroxidase was quenched with 3% H₂O₂ in TBS, and the nonspecific binding sites were blocked by incubation with 10% normal goat serum. Rabbit-anti-SHOX antibody (kindly supplied by Professor G. Rappold (Institute of Human Genetics, Heidelberg University, Heidelberg, Germany) (5) (1:200 in PBS) was applied, and incubated at 4 C overnight. The biotin/streptavidin-horseradish peroxidase system (Zymed, San Francisco, CA) was used to detect antibody binding and DAB (Zymed, San Francisco, CA) was used to visualize the result. The cells were then counterstained with hematoxylin for 2 min before dehydration with xylene and mounting with Depex.

Antigen retrieval methods were tested, including autoclaving in 0.1% citric acid and treatment with of 0.1% trypsin or 0.1% proteinase K at 37 C for 10 min, without alteration of the pattern of staining. Internal negative controls were performed by replacing the primary antibody with TBS or rabbit isotype control antibody. Skeletal muscle was used as the internal positive control (5) for SHOXa protein expression in fetal growth plate sections. Sections were examined using an Eclipse E600 microscope (Nikon, Tokyo, Japan), and photos were taken using a SPOT RT digital camera with software version 3.0 (Diagnostic Instruments, Inc., Sterling Heights, MI).

In situ hybridization

The plasmid pSHOX/SK containing the full-length coding region of SHOXa, cloned into the Hindlll/Smal site of pBluescript SK, was a gift from Professor G. Rappold (Institute of Human Genetics, Heidelberg University, Heidelberg, Germany) (5). Sense (and antisense) digoxigenin (DIG)-labeled RNA probes were generated by linearizing the vector with Xbal (or Clal) and transcribing with T7 (or T3) RNA polymerase in the presence of DIG-labeled nucleotides (Roche, Stockholm, Sweden). The probes were shortened to approximately 150 bp by exposure to carbonate buffer (pH 10.2), 60 C, 48 min. Rehydrated tissue sections were refixed in 4% paraformaldehyde, 4 C, 20 min, and treated sequentially with 0.3% Triton X-100 in PBS for 15 min, proteinase K (10 µg/ml) at 37 C for 30 min, 100 mM glycine in PBS for 5 min, 0.2 M HCl for 10 min, and then freshly prepared 0.1 M triethanolamine buffer (pH 8) with 0.25% acetic anhydride for 10 min. Sections were rinsed in diethylpyrocarbonate H₂O, dehydrated through an ethanol series, and allowed to air dry before being hybridized overnight at 55 C with hybridization buffer containing 50% formamide, 1x Denhardt’s, 10% dextran sulfate, 3 M NaCl, 10 mM Tris, 1 mg/ml yeast tRNA, 1 mg/ml herring sperm DNA, and DIG-labeled RNA probes at 250 ng/ml. Sections were washed in 2x saline sodium citrate (SSC) in 50% formamide (55 C, 30 min), digested with RNase A (20 µg/ml, 37 C, 30 min), and further washed in 2 x SSC (55 C,15 min) and 0.1x SSC (55 C, 15 min). Blocking with 10% normal sheep serum (1 h) was followed by overnight incubation with 1:500 anti-DIG-AP Fab fragments (Roche). Color development occurred over a 2- to 6-h period using 4-nitro blue tetrazolium chloride (315 µg/ml), 5-bromo-4-chloro-3-indoyl-phosphate (175 µg/ml), levamisole (5 mM) in 0.1 M Tris (pH 9.5), 0.1 M NaCl, and 0.05 M MgCl₂. Sections were lightly counterstained using nuclear fast red, 5 min, and mounted with Ultramount aqueous mounting medium (Dako, Carpinteria, CA).

	Results

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Fetal growth plate

The sections of fetal costochondral junction used for these studies were those that demonstrated the best preservation of growth plate architecture. Specifically, specimens representing the following time points were available: 12, 16, 22, 28, 31, 35, and 40 wk gestation. All fetal sections demonstrated retraction of the chondrocytic cytoplasm toward the center of the cells. This artifact may reflect postmortem changes or other fixation/sectioning effects. SHOX protein was demonstrated in fetal chondrocytes from 12 wk gestation to term. The growth plates were assessed for evidence of SHOX protein immunostaining by chondrocyte zone, i.e. reserve, proliferative, and hypertrophic. The intensity of stain among the different chondrocyte zones was evaluated by visual means. Microphotographs representing immunostaining results from the gestational ages (16 and 35 wk) are shown in Fig. 1, A–H, whereas a summary of all immunostaining results is presented in Table 1. At 12 wk gestation the plane of section did not demonstrate the full progression of chondrocytes from the reserve zone to the area of primary spongiosum; therefore, it was not possible to comment on differential expression within the zones of the growth plate. Despite this, cytoplasmic staining for SHOX protein was demonstrated in the chondrocytes at 12 wk. For 16 and 22 wk gestation, SHOX protein immunostaining of the chondrocytes was demonstrated within the reserve, proliferative, and hypertrophic zones of the growth plate with equal intensity. From 28 wk gestation, there was a decrease in the intensity of immunostaining for SHOX protein in the late hypertrophic, apoptotic zone of the growth plate. Decreased staining and staining of variable intensity throughout the proliferative and hypertrophic zones were features of later gestational age samples. Within the reserve zone, SHOX protein was consistently demonstrated in the cytoplasm of all cells.

View larger version (110K): [in this window] [in a new window]   FIG. 1. SHOX protein DAB immunostaining in fetal growth plate sections (x40). A, Fetal costochondral junction stained for SHOX protein demonstrating positive brown staining of chondrocytes in the reserve (R), proliferative (P), and hypertrophic (H) zones. Skeletal muscle (M) also stains for SHOX and was used as a positive control in subsequent sections. B, Negative control for SHOX immunostaining in which SHOX antibody was replaced with rabbit IgG isotype control. C–E, SHOX immunostaining at higher magnification in specific zones of 16-wk fetal growth plate (x400). C, Reserve. D, Proliferative. E, Hypertrophic. F–H, SHOX immunostaining in specific zones of 35-wk fetal growth plate (x400). F, Reserve. G, Proliferative. H, Hypertrophic.

Molecular genetics results. For growth plate tissue sections, the control subjects were C1 (male, 12.3 yr), C2 (male, 13.3 yr), C3 (male, 13.8 yr), and C4 (male, 15.2 yr). All were shown to have a normal karyotype with G banding, and fluorescence in situ hybridization (FISH) analysis performed from lymphocytes demonstrated two SHOX signals in all cases.

The following LWD subjects were studied for SHOX expression in growth plate sections: LW1 (female, 13.6 yr) and LW2 (female, 14.3 yr) both had a large-scale SHOX deletion detected from the SHOX-cosmid FISH analysis performed on peripheral lymphocyte metaphases; LW3 (female, 10.5 yr old) and LW4 (male, 14.9 yr) did not have large scale SHOX deletions detected by FISH. However, for LW3, DNA sequencing of SHOX coding exons and exon-intronic boundaries identified a hemizygous point mutation at the boundary between intron 2 and exon 3 (IVS2-1 G->A). LW4 was found to be hemizygous for the X chromosome SHOX flanking marker, DXYS233, due to inheritance of a null allele at this locus (3). SHOX expression studies in bone marrow fibroblasts in these two latter cases indicated an absence of mRNA expression from both the mutation in the donor splice site allele (IVS2-1 G->A) and the SHOX null allele (LW4), confirming SHOX haploinsufficiency in both cases (3). The results and details of these molecular genetic analyses as well as the growth plate histology for LW1 and LW3 have been reported previously (7).

Immunohistochemistry for SHOX

SHOX protein immunohistochemistry was performed on proximal tibial growth plate from control subjects C1–4. The growth plates from C1 and C2 showed columns of chondrocytes, with progression through the reserve, proliferative, and hypertrophic zones. Representative microphotographs of the SHOX immunostaining pattern observed for C2 are shown in Fig. 2, A–C. The growth plates from C3 and C4 were from older, more skeletally mature children and demonstrated growth plates near fusion. In these sections the columns of chondrocytes had been replaced with chondrocytes seen singularly or in clusters.

View larger version (106K): [in this window] [in a new window]   FIG. 2. SHOX protein DAB immunostaining (A–C) and SHOX mRNA in situ hybridization (D–F) in childhood growth plate sections. A–C, SHOX immunostaining in reserve (R), proliferative (P), and hypertrophic (H) zones, respectively of growth plate (x400). Regions of positive staining are indicated by arrows. Note the strong staining for SHOX protein in the hypertrophic region (C) and the almost complete absence of staining for SHOX protein in the proliferative zone (B). D–F, In situ hybridization staining observed using DIG-labeled SHOX antisense probe in reserve, proliferative, and hypertrophic zones, respectively, of growth plate (x400). Note the presence of staining for SHOX mRNA in reserve, proliferating, and hypertrophic chondrocytes, indicated by arrows. G–I, Negative control for in situ hybridization using DIG-labeled SHOX sense probe (x400).

The pattern of SHOX immunostaining observed in control growth plates was compared with the pattern observed in growth plates from LWD individuals. Although the linear zone of progression of the chondrocytes was disrupted as described previously (7), the pattern of SHOX immunostaining was unchanged. The results from all subjects, LWD and control, are summarized in Table 1. There was no difference in SHOX protein immunohistochemical staining between the relatively normal growth plate and that demonstrating frank dysplasia.

In situ hybridization for SHOX

To confirm the distribution of SHOX protein in growth plate sections observed by immunostaining, we performed in situ hybridization for SHOX mRNA on childhood growth plate sections from both control (n = 4) and LWD (n = 4) individuals. Results obtained for C2 are shown in Fig. 2, D–I. Using DIG-labeled antisense probe to SHOX, mRNA-positive staining was observed throughout the reserve zone, proliferative zone, and early hypertrophic zone chondrocytes of childhood growth plates (Fig. 2, D–F). Duplicate slides probed with a DIG-labeled sense probe gave no significant levels of staining (Fig. 2, G–I). Strong in situ hybridization signal for SHOX mRNA was seen in reserve, late proliferative, and hypertrophic regions of the control growth plates. Staining in the early proliferative zone was often weaker (Table 2). The pattern of staining in LWD was unchanged in that SHOX mRNA was observed in the reserve, proliferative, and early hypertrophic regions.

	Discussion

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The work presented provides the first evidence that SHOX protein is expressed in the human growth plate from 12 wk gestation up to the time of growth plate fusion in late childhood and supports the notion that SHOX is an important gene in skeletal development and growth.

The finding of SHOX protein in fetal growth plate from 12 wk gestation is in keeping with the in situ hybridization studies of Clement-Jones et al. (10), who reported SHOX mRNA in the primordial bone of human fetuses at 8 wk gestation. Together these data suggest a role for SHOX in early chondrogenesis.

Our data demonstrated that the immunostaining of SHOX protein within the growth plate varied through fetal development and childhood. This differential expression pattern suggests that the actions of the SHOX protein on the growth plate may differ throughout development to regulate growth. The pattern of SHOX mRNA, by in situ hybridization, in general coincided with the pattern of SHOX protein expression throughout the childhood growth plate with the exception of differential expression in the proliferative zone of the growth plate. This finding suggests that translational control of SHOX expression (8) may be a feature of SHOX regulation in this region of the growth plate.

SHOX expression in the LWD radial growth plate looks very similar to the expression pattern of SHOX found in the control tibia growth plate. On the basis of genetic and molecular findings, haploinsufficiency for SHOX mRNA is predicted for LWD individuals (3). In this study we did not attempt to quantify either mRNA or protein levels of SHOX in the LWD growth plate due to the lack of age and site-matched controls.

Because there is no mouse ortholog for the SHOX gene (12), traditional approaches to gene function studies are precluded. To unravel the function of this gene within the growth plate and its interactions with other growth-regulating proteins and molecules, the approach we and others have followed is to use observations of experiments of nature and in particular studies of SHOX haploinsufficiency in humans (7, 13, 14, 15, 16). Zinn et al. (6) demonstrated that Langer syndrome is the homozygous form of LWD. The histological analysis of growth plate in a fetus with Langer syndrome has shown disorganization of the proliferative zone with the chondrocytes lying in relatively small clusters without the parallel arrangement normally seen in this zone (17). The histological appearance of growth plate in both the homozygous (17) and haploinsufficient (7) forms of SHOX gene abnormality lead us to speculate that SHOX may play a role in chondrocyte stacking within the proliferative zone and differentiation of chondrocytes into the hypertrophic phenotype. These findings suggest that SHOX haploinsufficiency may result in premature terminal differentiation of proliferative chondrocytes with progression to the hypertrophic phenotype and accelerated growth plate fusion. It is also possible to speculate that within the reserve zone of chondrocytes, SHOX protein may function as a repressor of chondrocyte differentiation, retarding the progression of chondrocytes from the reserve phenotype to the proliferative phenotype. Clinical observations suggest that SHOX might interact with estrogen and fibroblast growth factor receptor 3 to regulate chondrocyte differentiation within the growth plate, although the precise role of SHOX within chondrocyte development requires further clarification (13, 16).

In summary, we have shown that SHOX protein is expressed in fetal and childhood growth plate in a developmentally specific pattern. An improved understanding of the function of the SHOX gene, its regulators, downstream targets, and subsequent interactions could help to explain the multiple phenotypes associated with SHOX haploinsufficiency. It may also provide opportunities for novel therapies to be developed for subjects with SHOX mutations and associated short stature and skeletal abnormalities.

	Acknowledgments

 
The contributions of Dr. Val Hyland, Mr. Simon Flanagan, and Ms, Monica Berry are acknowledged for the SHOX molecular analysis. Dr. Gail Phillips assisted with the fetal growth plate studies. The surgical contributions of Professor David Vickers and Dr. Robert LeBrom are gratefully acknowledged.

	Footnotes

 
This work was supported by grants from the Royal Children’s Hospital Foundation, the Australasian Paediatric Endocrine Group, and Novo Nordisk Pharmaceuticals. C.J.F.M. is the Royal Children’s Hospital Foundation/Woolworths Scholar.

Abbreviations: DAB, Diaminobenzidine; DIG, digoxigenin; FISH, fluorescence in situ hybridization; LWD, Leri Weill dyschondrosteosis; SSC, saline sodium citrate; TBS, Tris-buffered saline.

Received December 30, 2003.

Accepted April 25, 2004.

	References

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