Identification of novel SHOX target genes in the developing limb using a transgenic mouse model

Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development

DSA-Based 2D Perfusion Measurements in Delayed Cerebral Ischemia to Estimate the Clinical Outcome in Patients with Aneurysmal Subarachnoid Hemorrhage: A Technical Feasibility Study

(1) Background: To predict clinical outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH) and delayed cerebral ischemia (DCI) by assessment of the cerebral perfusion using a 2D perfusion angiography (2DPA) time–contrast agent (CA) concentration model. (2) Methods: Digital subtraction angiography (DSA) data sets of n = 26 subjects were acquired and post-processed focusing on changes in contrast density using a time–concentration model at three time points: (i) initial presentation with SAH (T0); (ii) vasospasm-associated acute clinical impairment (T1); and (iii) directly after endovascular treatment (T2) of SAH-associated large vessel vasospasm (LVV), which resulted in n = 78 data sets. Maximum slope (MS in SI/ms), time-to-peak (TTP in ms), and maximum amplitude of a CA bolus (dSI) were measured in brain parenchyma using regions of interest (ROIs). First, acquired parameters were standardized to the arterial input function (AIF) and then statistically analyzed as mean values. Additionally, data were clustered into two subsets consisting of patients with regredient or with stable/progredient symptoms (or Doppler signals) after endovascular treatment (n = 10 vs. n = 16). (3) Results: Perfusion parameters (MS, TTP, and dSI) differed significantly between T0 and T1 (p = 0.003 each). Significant changes between T1 and T2 were only detectable for MS (0.041 ± 0.016 vs. 0.059 ± 0.026; p = 0.011) in patients with regredient symptoms at T2 (0.04 ± 0.012 vs. 0.066 ± 0.031; p = 0.004). For dSI, there were significant differences between T0 and T2 (5095.8 ± 2541.9 vs. 3012.3 ± 968.3; p = 0.001), especially for those with stable symptoms at T2 (5685.4 ± 2967.2 vs. 3102.8 ± 1033.2; p = 0.02). Multiple linear regression analysis revealed that a) the difference in MS between T1 and T2 and b) patient’s age (R = 0.6; R2 = 0.34; p = 0.009) strongly predict the modified Rankin Scale (mRS) at discharge. (4) Conclusions: 2DPA allows the direct measurement of treatment effects in SAH associated DCI and may be used to predict outcomes in these critically ill patients

Regulated genes in transgenic mice and validation of <i>Ctgf</i> as a target.

<p>(A): qRT-PCR using limb RNA (E12.5-E14.5) from wildtype (Wt) and transgenic littermates (Tg) (N = 8–10 for each stage). Measurements were carried out individually, in duplicates, and normalized to <i>Adam9</i> and <i>Sdha</i>. Relative normalized values are presented on the y-axis. Significances are indicated in each diagram by asterisks (*: <i>p</i>≤0.05, **: <i>p</i>≤0.01, ***: <i>p</i>≤0.001). Variations are indicated by the standard deviation (SD). In 7/8 candidates an upregulation was confirmed as significant in at least one embryonic stage. (B): nCounter analysis of <i>CTGF</i> and <i>SHOX</i> expression in NHDF and U2OS cells after transient transfections of <i>SHOX</i> and <i>p.Y141D</i>. <i>CTGF</i> is significantly downregulated in NHDF cells, whereas it is significantly upregulated in U2OS cells. Values on y-axis represent absolute counts of mRNA, normalized to <i>ADAM9</i>, <i>HPRT1</i> and <i>SDHA</i>. Significancies are indicated by asterisks. (C): <i>In situ</i> hybridization using a <i>Ctgf</i> antisense riboprobe on embryonic limbs from wildtype and <i>SHOX</i>-transgenic littermates (N = 8) at stage E12.5. In transgenic embryos, enhanced and distalized expression of <i>Ctgf</i> was detected in the middle part of the developing limbs.</p

Analysis of postnatal bone parameters of <i>Col2a1-SHOX</i>-transgenic mice.

<p>(A): Alcian Blue/Alizarin Red S staining at different developmental (E14.5, E18.5) and postnatal (P28) stages does not reveal apparent differences between transgenic and wildtype skeletal elements. (B): Postnatal <i>in vivo</i> time-course analysis of bone growth in 65 animals of two transgenic lines by μ-CT analysis. Tibiae and femora of wildtype and <i>Tg(Col2a1-SHOX)</i> littermates at the age of 4, 12 and 24 weeks were scanned, female and male individuals were evaluated separately. Total bone length, cortical bone thickness and bone volume do not show significant differences between wildtype and transgenic females or males. Some transgenic animals presented longer bones and weaker structures of the cortical bone in the subcartilaginous region (indicated in the μ-CT images). Other micromorphological parameters (bone mineral density (BMD), trabecular volume and thickness) showed no significant differences. Statistical analyses were performed using student's t-test. (C): hematoxilin and eosin (H&E) stainings of the growth plate in wildtype and transgenic tibiae. Consistent differences between wildtype and <i>Tg(Col2a1-SHOX)</i> adult growth plates (24 weeks of age) did not exist (N = 8), but some transgenic tibiae showed a buckling, and the columns of chondrocytes became shorter and were not strictly oriented in a parallel assembly compared to the wildtype (right image).</p

Analysis of <i>CTGF</i> as a direct transcriptional target of SHOX.

<p>(A): Genomic structure of the human <i>CTGF</i> region. ChIP-Seq analysis in ChMM cultures revealed an accumulation of Shox binding in the <i>Ctgf</i> promoter region (grey peaks), especially in a region 3–4 kb from the transcriptional start site (TSS) where an evolutionary conserved sequence (ECR) of 597 bp (human chr6:132317086-132318077) was identified (green bar). (B): Location of the pGL3 ECR and pGL3 ECR+ reporter constructs (grey bars) within the <i>CTGF</i> upstream region. The ECR+ construct encompasses the ECR and an upstream region including ATTA/TAAT motifs and palindromes. SHOX binding motifs (ATTA/TAAT sites and palindromes) in the <i>CTGF</i> 5′ region around the ECR are indicated by asterisks. Red bars represent the location of the generated oligonucleotides for EMSA. (C): Luciferase reporter gene assays in NHDF and U2OS cells. pcDNA4/TO <i>SHOX</i> was cotransfected with a luciferase reporter vector harbouring either the ECR or the ECR+ sequence. Transfections and measurements were carried out in triplicates. A significant activation in the luciferase activity was observed 24 h after <i>SHOX</i> transfection in NHDF cells using both reporter constructs (1.7-fold/2.5-fold with <i>p</i> = 0.02/0.007 for ECR/ECR+). In U2OS cells, an alteration was not observed for the ECR reporter, but a significant reduction was demonstrated for the ECR+ reporter construct (1.0-fold/2.8-fold with <i>p</i> = 0.1/0.003 for ECR/ECR+). (D): EMSA. The SHOX wildtype (Wt) and the mutant p.R153L proteins bind to oligonucleotides 1 and 2, whereas the defective proteins p.Y141D and p.A170P cannot. All fragments of oligonucleotides 1 and 2 containing an ATTA/TAAT site are sensitive to SHOX binding (1a–c, 2a–b). The fragment lacking this motif does not bind (oligonucleotide 2c). Using the SHOX-3 antibody (Ab), we demonstrate that the binding is SHOX-specific. (E): Immunohistochemistry performed on pubertal tibial growth plates. Staining was performed using preimmune serum as a negative control, SHOX antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098543#pone.0098543-Marchini1" target="_blank">[19]</a> and a CTGF-specific antibody. Both the SHOX and CTGF proteins were detected in growth plate chondrocytes.</p

Generation and expression analysis of <i>SHOX</i>-transgenic mice.

<p>(A): The <i>SHOXa</i> cDNA was tagged with a Lumio and SV40 Poly(A) sequence and cloned under the control of a murine <i>Col2a1</i> promotor/enhancer expression cassette. (B): Genotyping was performed using specific primers spanning the first 409 nucleotides of the <i>SHOXa</i> cDNA. No PCR product was detected in wildtype animals. (C):-Southern Blot analysis of the two transgenic lines (1 and 2) used for our investigations. Genomic DNA was digested with <i>BamHI</i>, <i>EcoRV</i> and <i>Hind III</i>. <i>BamHI</i> digestion results in a 1.3 kb fragment that corresponds to the Lumio/SV40-tagged <i>SHOX</i> cDNA, which was flanked by <i>BamHI</i> sites. The presence of only one signal per lane indicates a single integration site of the transgene. (D): Relative quantitative expression of <i>Col2a1</i> and <i>SHOXa</i> transcripts in limbs of wildtype and transgenic littermates (N = 5–8 per litter) at E12.5, E13.5 and E14.5. The expression of the transgene corresponds to the expression dynamics of <i>Col2a1</i>. <i>SHOX</i> levels are generally low with highest expression at E12.5. Values are variable among individual animals as indicated by the standard deviation (SD). (E): WISH of wildtype (Wt) and transgenic (Tg) embryonic limbs from E11.5-E14.5 (N = 20 for each stage). The transgene is weakly expressed in the developing limb at E11.5 and becomes defined around the cartilaginous anlagen at E12.5. From E13.5 onwards, the expression is mainly seen in the mesenchyme around the developing cartilage and in the perichondrium and decreases during later stages.</p