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Dev Biol. Author manuscript; available in PMC Oct 15, 2008.
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PMCID: PMC2042108
NIHMSID: NIHMS32395

Deletion of Tgfbr2 in Prx1-cre expressing mesenchyme results in defects in development of the long bones and joints

Abstract

In this study, we address the function of Transforming Growth Factor beta (TGF-β) and its Type II receptor (Tgfbr2) in limb development in vivo. Mouse embryos were generated in which the Tgfbr2 gene was deleted in early limb mesenchyme using Prx1Cre-mediated LoxP recombination. A high level of Tgfbr2 gene deletion was verified in limb mesenchyme by PCR between E9.5 and E10.5 days in Cre expressing mice. RT-PCR assays indicated a significant depletion of Tgfbr2 mRNA by E10.5 days as a result of Cre mediated gene deletion. Furthermore, limb mesenchyme from Cre+;Tgfbr2f/f mice placed in micromass culture did not respond to exogenously added TGF-β1 confirming the functional deletion of the receptor. However, there was an unexpected increase in the number and intensity of Alcian blue stained chondrogenic nodules in micromass cultures derived from Tgfbr2 deleted limbs relative to cultures from control limbs suggesting Tgfbr2 normally limits chondrogenesis in vitro. In vivo, early limb development and chondrocyte differentiation occurred normally in Tgfbr2 depleted mice. Later in development, depletion of Tgfbr2 in limb mesenchyme resulted in short limbs and fusion of the joints in the phalanges. Alteration in the length of the long bones was primarily due to a decrease in chondrocyte proliferation after E13.5 days. In addition, the transition from prehypertrophic to hypertrophic cells was accelerated while there was a delay in late hypertrophic differentiation leading to a reduction in the length of the marrow cavity. In the joint, cartilage cells replaced interzone cells during development. Analysis of markers for joint development indicated that the joint was specified properly and that the interzone cells were initially formed but not maintained. The results suggest that Tgfbr2 is required for normal development of the skeleton and that Tgfbr2 can act to limit chondrogenesis in mesenchymal cells like the interzone.

Keywords: micromass culture, Tgfbr2, chondrocyte, synovial joint, sternum, skull

INTRODUCTION

Transforming Growth Factor β (TGF-β) signaling regulates diverse cellular processes, such as cell growth, differentiation, proliferation, and formation of extracellular matrix during embryonic development (Dunker and Krieglstein, 2000; Mummery, 2001; Serra and Chang, 2003; Verrecchia and Mauviel, 2002). Members of the TGF-β superfamily include TGF-β isoforms (TGF-β1, 2, 3), the Activins and Inhibins, Bone Morphogenetic Protein (BMP), and Growth and Differentiation Factors (GDF). Members of the TGF-β superfamily signal through heteromeric serine threonine kinases composed of Type I and Type II receptors. TGF-β signaling is initiated when ligand binds to the type II receptor (Tgfbr2) on the cell surface (Massague, 1998). Ligand binding recruits the type I receptor (Tgfbr1) to form a heterotetrameric complex of two type I and two type II receptors. Tgfbr2 is a constitutively active serine/threonine kinase. The formation of the complex enables Tgfbr2 to phosphorylate the GS domain of Tgfbr1, activating its serine/threonine kinase. Smad proteins are downstream targets of the type I receptor kinase. Activated Smad proteins translocate into nucleus to induce cellular responses by acting as transcription factors (Feng and Derynck, 2005; Massague, 1998).

Endochondral bone is formed by a series of consecutive and strictly regulated differentiation steps (de Crombrugghe et al., 2000; Goldring et al., 2006; Lefebvre and Smits, 2005). Chondrogenesis is the earliest step for endochondral bone formation. It begins with the condensation of undifferentiated mesenchyme, which is the result of an increase in cell density. During this process, undifferentiated progenitor cells differentiate into chondrocytes expressing Type II collagen as well as cartilage specific proteoglycans. These two cellular processes, in fact, occur almost at the same time and result in the formation of a cartilage template for future bone formation. Differentiated chondrocytes then undergo maturation and hypertrophic differentiation. Eventually, the cartilage template is replaced by bone. One of the most well characterized properties of cartilage is the elaboration of a specific extracellular matrix (ECM) that can stain with Peanut Agglutinin (PNA) and Alcian blue (Bagnall and Sanders, 1989; DeLise and Tuan, 2002; Stringa and Tuan, 1996).

Previously it was shown that BMPs are critical for early stages of endochondral bone formation. The majority of skeletal elements were absent in mice deficient in both BMP type I receptors, Bmpr1a and Bmpr1b (Yoon et al., 2005). It was determined that BMP is required for chondrocyte proliferation, survival, and differentiation. Based on gain-of-function studies in cell culture models, it has been suggested that TGF-β triggers chondrogenesis and that BMP maintains and promotes the differentiated phenotype (Carrington and Reddi, 1990; Chimal-Monroy and Diaz de Leon, 1997; Kulyk et al., 1989; Leonard et al., 1991; Macias et al., 1999; Roark and Greer, 1994; Verrecchia and Mauviel, 2002). The conclusions were based on the observation that treatment with TGF-β resulted in increased Alcian blue staining and expression of Type II collagen even though the cells did not obtain chondrocyte morphology, meaning treatment with TGF-β resulted in a continuous sheet of Alcian blue staining and the cells remained spindle shaped (Chimal-Monroy and Diaz de Leon, 1997). In contrast, treatment with BMP or GDF5 resulted in the formation of discreet Alcian blue stained nodules containing cells with typical round chondrocyte morphology (Denker et al., 1999; Hatakeyama et al., 2004; Kulyk et al., 1989). These results led us to investigate the function of TGF-β in limb development using a loss of function model. For these experiments, the Tgfbr2 gene was deleted in mouse limb mesenchyme using Prx-1-cre mediated recombination. The expression pattern of Cre recombinase in Prx1-cre mice was previously shown using the Z/AP reporter mouse line in which the histochemical marker human placental alkaline phosphatase is transcriptionally activated following Cre-mediated recombination (Logan et al., 2002). At 9.5 days of gestation, the first expression of alkaline phosphatase reporter was shown in the forelimb mesenchyme. By E10.5 days, transgenic embryos showed complete recombination of the reporter in both forelimb and hind-limb mesenchyme. Cre recombinase activity was not detected in the apical ectodermal ridge (AER) or any of the limb ectoderm. Activity was also detected in cranial mesoderm as well as ventral mesoderm in the trunk. Here, we show maximal deletion of Tgfbr2 exon 2 DNA in limb mesenchyme from Prx1Cre+;Tgfbr2f/f mice between E9.5 and E10.5 days. Furthermore, cells from Tgfbr2-deleted limbs did not respond to TGF-β1 indicating functional deletion of the receptor. Nevertheless, early limb development occurred normally. That is, the mesenchyme condensed in the correct pattern and Sox9 protein was expressed normally. However, by E15.5 days, defects in the development of the limbs were apparent. The limbs were shorter than controls, the deltoid tuberosity was missing, and the joints of the phalanges were fused. Reduced limb length was shown to be primarily the result of reduced chondrocyte proliferation after E13.5 days. In the developing joint, cartilage replaced the interzone cells leading to joint fusion. Marker analysis indicated that the interzone was initially formed but was not maintained. Since interzone cells were replaced with cartilage and mesenchyme from Tgfbr2-depleted limbs grown in micromass culture formed more discreet Acian blue stained chondrogenic nodules than Tgfbr2-containing controls, we propose that Tgfbr2 normally limits chondrogenic differentiation in mesenchymal cells including the interzone. Together the results suggest that Tgfb2 is required for normal skeletal development.

MATERIALS AND METHODS

Mouse crosses

Mice in which exon2 of Tgfbr2 was flanked with loxP sites (Tgfbr2f/f) were obtained from Dr. H.L Moses, Vanderbilt University, Nasshville, TN (Chytil et al., 2002). Tgfbr2f/f mice were mated to transgenic mice that express Cre under the control of the Prx1 promoter (obtained from Dr. Clifford J. Tabin, Harvard Medical School, Boston, Massachusetts; (Logan et al., 2002) to create a mouse in which Tgfbr2 is deleted in early limb mesenchyme. The genotype of adult transgenic mice was determined by PCR analysis of genomic DNA isolated from tail biopsies. Embryos were genotyped using DNA isolated from one of the limb buds by proteinase K digestion. PCR for the Cre transgene was performed using two different primer sets: Cre5V(TGC TCT GTC CGT TTG CCG), Cre3V(ACT GTG TCC AGA CCA GGC), Prx1CreF (AGG AGG TAG GAG ATT GTG ATG GAG), and Prx1 CreR (ACC GGC AAA CGG ACA GAA GCA TTT). Genomic DNA was amplified for 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 60 s, and elongation for 90 s at 72 °C in reaction buffer containing 2.5 mM MgCl2, 1X PCR buffer (HotMaster Taq buffer, Eppendorf), 0.2 mM dNTPs (Pharmacia, Uppsala, Sweden), 0.2 μM each primer. The loxP allele and deletion of the target gene were identified using the following primers (Figure 5A): 8w-a (TAA ACA AGG TCC GGA GCC CA), and LA-LoxP (ACT TCT GCA AGA GGT CCC CT). Two bands can be detected with these primers. One band represents the wild-type allele (420 bp); another band represents the loxP allele (540 bp). The presence of both bands indicates a mouse that is heterozygous for the loxP allele. To detect recombination and the subsequent loss of the floxed allele (Tgfbr2), CldelR (AGA GTG AAG CCG TGG TAG GTG AGC TTG) was added to reactions as a third primer. Successful recombination was confirmed by the presence of a 610-bp band (Baffi et al., 2004).

Figure 5
Characterization of the growth plate phenotype in Prx1Cre+/Tgfbr2f/f mice

RT PCR

mRNA was extracted from limbs of E9.5, 10.5, 11.5 12.5 days mice using the standard Trizol method. Cells were lysed with Trizol and RNA was precipitated in ethanol. RNA concentration was determined by UV spectrophotometry. For RT-PCR analysis, cDNA was synthesized from 1μg of total mRNA using random primers. PCR for tgfb1, 2, 3, Tgfbr2 and 18S was performed using the following primers:

tgfb1 primers (forward 5′-GCT AAT GGT GGA CCG CAA CAA C - 3′ and reverse 5′-CAC TGC TTC CCG AAT GTC TGA C - 3′), Tgfb2 primers (forward 5′-CTT CAC CAC AAA GAC AGG AAC CTG G - 3′ and reverse 5′-CCT GCT AAT GTT GTT GCC CTC CTA C -3′), tgfbr2 exon2 primers (forward 5′-TTA ACA GTG ATG TCA TGG CCA GCG - 3′ and reverse 5′-AGA CTT CAT GCG GCT TCT CAC AGA - 3′), tgfbr2 exon2-3 primers (5′-CCA CTT GCG ACA CCG AGA AGT - 3′ and reverse 5′-GCA CAC ATG AAG AAA GTC TCG C - 3′), Sox9 primers (forward 5′-GAG AAA AGC TAT GGT GAC AGA GC - 3′ and reverse 5′-GTC CTC CAT GTT AAC TCT GAA GG - 3′), 18S primers (forward 5′-ACG GAA GGG CAC CAC CAG G - 3′ and reverse 5′ – CAC CAA CTA AGA ACG GCC ATG C -3′). 10–200 ng of cDNA was amplified for 20–35 cycles in reaction buffer containing 2.5 mM MgCl2, 1 X PCR buffer (HotMaster Taq buffer, Eppendorf), 0.2 mM dNTPs (Pharmacia, Uppsala, Sweden), 0.2 μM each primer (Pharmacia, Uppsala, Sweden). The linear range of product formation was determined by varying the number of cycles for each primer pair. Unless indicated, product formation within the linear range is shown.

Limb micromass culture

Limb micromass cultures were set up as previously described (Kulyk et al., 2000). Briefly, limb buds were separated from E11.5 day mouse embryos. Embryos from crosses of Prx1cre;Tgfbr2f/wt X Tgfbr2 f/f were used to generate cultures deleted for Tgfbr2 and controls. Mesenchymal cells were dissociated into a single cell suspension with incubation in 1mg/ml collagenase D at 37 °C for 1–2 h and reconstituted at a density of 1 × 107 cells/ml. Twenty microliters of cell suspension was dropped into each well of a 24 well plate. After a pre-incubation time of 1 h at 37 °C to allow cells to attach, the cultures were then flooded with F-12:DMEM (3:2) containing 10 % FBS, 50 μg/ml ascorbic acid, 10 mM β-glycerolphosphate, 2 mM glutamine, antibiotics with or without 5ng/ml of TGFβ1 (R&D Systems). Cultures were incubated at 37 °C in CO2 incubator up to 3 days without further medium change. Following incubation, cultures were processed for Alcian blue staining, immunofluorescence staining, western blotting or RT-PCR.

Alcian blue staining

Micromass cultures were rinsed with PBS and fixed with 4 % paraformaldehyde for 15 minutes at room temperature at which time cells were incubated in Alcian blue staining solution (75 %ethanol Alcian blue solution: 0.1 M HCl = 4:1) at 37 °C overnight. Finally, cells were washed with 70 % ethanol.

Whole limb buds from embryos at E12.5, E13.5 days of gestation were also stained with Alcian blue to detect the cartilaginous skeleton (Kimmel and Trammell, 1981). Limbs were fixed in 95 % ethanol overnight and then, placed in 95 % ethanol-Alcian blue solution over night to stain chondrocytes, followed by a 95 % ethanol washes for 6 h, and maceration in 1 % KOH for 1–2h. Samples were cleared in 20, 50 and 80 % glycerol in 1 % KOH for 1h for each step. Samples were stored in 100 % glycerol.

Whole mount Alizarin red - Alcian blue stains were used to determine the skeletal structure in older embryos. Skeletons from 15.5, 18.5 days in gestation were double-stained for cartilage and bone with Alcian blue and Alizarin red (Kimmel and Trammell, 1981; McLeod, 1980). All carcasses were skinned and fixed in 95% ethanol for 24 h. Samples were then placed in 95% ethanol-Alcian blue and Alizarin red solution (24, 48 h respectively) for cartilage and bone staining, followed by a 95% ethanol wash (8 h), and maceration in 1% KOH overnight at 4C. Samples were cleared in 20, 50 and 80% glycerol in 1% KOH for 12h for each step. Samples were stored in 100% glycerol..

Histology and Immunofluorescence

Limb buds from E12.5, 13.5, 14.5 15.5 16.5 day-embryos were fixed in 4% paraformaldehyde overnight. Specimen were dehydrated in ethanol solution and embedded in paraffin. Sections were cut at a thickness of 5μm and mounted on Superfrost Plus slides (Fisher). Sections were stored at 4°C. For histological analysis, sections were stained with hematoxylin and eosin. For immunofluorescence, sections were dewaxed and hydrated and then incubated with blocking buffer (PBS with 10 % goat serum) for 1 h. To examine chondrogenic differentiation, SOX9 rabbit antibody (Santa Cruz Biotechnology) (diluted 1:1000 with blocking buffer) was added, and incubated for 1 h at room temperature. A secondary antibody consisting of goat biotinylated anti-rabbit IgG (Vector) (diluted 1:2000 in blocking buffer) was added, and the mixture was incubated for another 1 h. For fluorescence detection, Cy3-conjugated streptavidin (diluted 1:2000 in block solution) was added and incubated for 1 h at room temperature. Cells were washed three times with PBS with 0.1% Tween 20 after each step. In last step, cells were supplemented with 300nM of the DNA dye, YoPro, for counterstaining. For PNA staining, sections were dewaxed and hydrated and then incubated with PNA according to the protocol of Baffi et al. 2006. Slides were washed in PBS then incubated with 10 μg/ml rhodamine-conjugated PNA (Vector Laboratories) for 2 hours at room temperature. The slides were washed and counterstained with YoPro (Molecular Probes). Slides were mounted with Aqua poly/Mount (Polyscience Inc.). Images were captured with an Olympus BX-51 fluorescent microscope and macrofire digital camera.

In situ hybridization

In situ hybridization was performed as described (Pelton et al., 1990). Briefly, sections were hybridized to 35S-labeled anti-sense probes of Gdf5 and Wnt9a. Gdf5 and Wnt9a probes were generated by RT-PCR and subcloned into pGEM-T easy (Promega) cloning vector. The primers and resulting size of each fragment are as follows: Gdf5, 248 bp, forward 5′-TGA ATA TTT GTT CAG CCA GCG GCG - 3′, reverse: 5′-TTA GGG TCT GAA TGA CTG CGT GGT - 3′, Wnt9a, 237 bp, forward 5′-TTG CAA ATG CCA TGG TGT GTC TGG – 3′, reverse 5′-TCC AGG TGT ACA AGC TCT GGT GTT - 3′. All inserts were sequence verified.

Slides were exposed to KODAK Autoradiography Emulsion Type NTB (Cat. No. 889 5666, Kodak) at 4 °C for 2 weeks and then developed by KODAK Developer D-19 (Cat. No. 146 4593, Kodak). Sections were counterstained with 0.2% Toluidine blue. Images were obtained using an Olympus BX51 microscope and Magnafire digital camera. Images were taken under dark field illumination (35S signal).

Cell Proliferation and TUNEL Labeling

To measure proliferation, BrdU staining was carried out. 60μg/g (BrdU/body weight) of BrdU was injected intraperitoneally into the mouse in 13.5 days gestation, 3 hours before sacrifice and removal of embryos. Rat anti-BrdU antibody (Abcam) was used as described above for immunostaining. TdT-Frag EL Fragmentation Detection Kit (Calbiochem) was used according to the manufacturer’s instructions.

RESULTS

Deletion of Tgfbr2 by Prx1Cre

To investigate the requirement of Tgfbr2 for limb development in vivo, Tgfbr2 was deleted in mouse limb buds using Cre-mediated recombination (Sauer, 1998). Mice homozygous for the floxed Tgfbr2 allele, Tgfbr2f/f, (Chytil et al., 2002) were crossed with mice carrying the Cre recombinase gene under control of the Prx1 promoter (Logan et al., 2002) to generate Prx1Cre;Tgfbr2f/wt mice. Prx1Cre;Tgfbr2f/wt mice were then crossed to Tgfbr2f/f mice to generate Prx1Cre;Tgfbr2f/f mice. Cre-negative mice from this cross were used as controls. To confirm activity of Cre recombinase and estimate the level Cre-mediated recombination and deletion of the Tgfbr2 gene, genomic DNA isolated from the limbs of E9.5, 10.5, 11.5, and 12.5 day Prx1Cre;Tgfbr2f/f and control embryos was used as a template in a PCR assay that allowed for the simultaneous detection of the wild-type (420 bp), loxP (540 bp), and deleted (610 bp) Tgfbr2 alleles (Figure 1A) (Baffi et al., 2004). The presence of the Cre transgene was also determined by PCR analysis. In the presence of Cre, significant gene deletion was detected by E9.5 days and was maximal by E10.5 days as indicated by the ratio of the bands for the deleted and the floxed alleles (Figure 1B). Only a very small amount of intact (floxed) receptor DNA was detected after E10.5 days, likely from the limb ectoderm, which does not express Cre. To address this issue, condensed mesenchyme and interdigit mesenchyme were removed from sections of limbs from E12.5 day embryos by laser capture microdissection. The DNA was used in the PCR assay described above. Essentially all of Tgfbr2 exon 2 was deleted in the both condensed and non-condensed mesenchyme (Figure 1C). Finally, we determined the relative level of Tgfbr2 mRNA in total limb buds from control and Prx1Cre;Tgfbr2f/f embryos at E10.5 days using semi-quantitative RT-PCR (Figure 1D). Primer pairs within exon 2 (e2) or spanning exon 2 and exon 3 (e2–e3) were used. The level of 18S was used as an internal control. Tgfbr2 mRNA was significantly reduced in Prx1Cre;Tgfbr2f/f limbs relative to the controls.

Figure 1
Tissue specific deletion of Tgfbr2 by Prx1-Cre

To confirm that the receptor was functionally deleted we used a biological assay. Cells from E11.5 day Prx1Cre+;Tgfbr2f/f and control limbs were placed in micromass culture and treated with 5ng TGF-β1/ml for three days then stained with Alcian blue (Figure 2). Under our culture conditions, nodules of Alcian blue stained chondrocytes can be seen within 3 days. In control cultures grown without TGF-β1, Alcian blue staining was observed only in discrete nodules (Figure 2A). As described previously, treatment of control cultures with TGF-β1 resulted in diffuse staining with Alcian blue so that discrete stained nodules were not easily detected (Figure 2C). In contrast, the cells from Prx1Cre+;Tgfbr2f/f limbs did not respond to exogenously added TGF-β1 and discrete Alcian blue stained nodules similar to that seen in the untreated samples were observed (Figure 2B, D) suggesting functional deletion of the receptor by E11.5 days. Furthermore, the nodules that formed in the Prx1Cre+;Tgfbr2f/f cultures were consistently larger and stained more intensely than in the control cultures suggesting that Tgfbr2 is not required for induction of chondrocyte differentiation and that Tgfbr2 normally acts to limit chondrogenesis in limb mesenchyme (Figure 2A, B).

Figure 2
Functional assay for the deletion of Tgfbr2

Deletion of Tgfbr2 results in alterations in the development of the long bones after E13.5 days

Next, limb buds from embryos of each genotype at E12.5 days were stained with Alcian blue to examine the developing skeletal elements (Figure 3A–C). Interestingly, limbs in Prx1Cre+;Tgfbr2f/f embryos looked similar to those from Prx1Cre+;Tgfbr2f/w and control (Prx1Cre;Tgfbr2f/w) embryos (Figure 3). The size, shape, and pattern of the limb, the thickness, and Alcian blue staining in Prx1Cre+;Tgfbr2f/f limbs was very similar to the Prx1Cre-negative control limbs at this stage (Figure 3A–C). Alterations were not detected in the organization of the developing cartilage as determined by H&E staining (Figure 3D–F). Furthermore, alterations in the localization or expression of Sox9 were not detected by immunofluorescent staining in sections from control and Prx1Cre+/Tgfbr2f/f limbs (Figure 3G–I). The data suggest Tgfbr2 is not uniquely required in the limb mesenchyme after E10.5 days for the formation of cartilage or the patterning of the limb.

Figure 3
Early skeletal development in Prx1Cre+/Tgfbr2f/f mice

Next, we examined Prx1Cre+;Tgfbr2f/f mice at later stages of development to see if Tgfbr2 was required for any of the subsequent steps of skeletal development (Figure 4, ,5).5). Prx1Cre+;Tgfbr2f/f mice were dead at birth with short limbs, open sternum, and open skull (Figure 4A). Alcian blue and Alzarin red stained skeletons from E18.5 day embryos showed the details of these defects in skeletal development (Figure 4B). In the skull, the parietal bones and frontal bones were either missing or reduced in size, suggesting a defect in intramembraneous bone formation (Figure 4E, F). Since Prx1-Cre is not expressed in the base of the skull, or the axial skeleton, these bones developed normally. Prx-1-Cre is expressed in the ventral mesoderm where the sternum forms and thus Prx1Cre+;Tgfbr2f/f embryos had a split sternum, a phenotype similar but more severe than mice with a null mutation in the Tgfb2 ligand (Figure 4C, Sanford et al., 1997). At E18.5 days, limbs of the mutant embryos were obviously shorter than controls (Figure 4B and Figure 5A). Shortening of the limbs was variable at E15.5 days (Figure 4D) but was consistently observed by E16.5 days (data not shown). Furthermore, the deltoid tuberosity did not develop on the humerus of mutant mice (Figure 4D and Figure 5A).

Figure 4
Analysis of the skeletal phenotype of in Prx1Cre+/Tgfbr2f/f embryos

Since an overall reduction in the length of the mutant long bones was observed starting at E15.5 days and obvious by E16.5 days (Figure 5), we tested the hypothesis that deletion of Tgfbr2 results in alterations in proliferation and/or hypertrophic differentiation. The level of proliferation from control and mutant limbs was measured by BrdU incorporation. At E13.5 days, the number of proliferating chondrocytes was comparable in control and mutant mice (Figure 5B). However, by E15.5 days, proliferation was significantly reduced in mutant cartilage (Figure 5C) relative to controls. These data suggest that reduced proliferation in chondrocytes contributes to the decreased length of the limbs in Prx1Cre+; Tgfbr2f/f mice.

Next, we looked at hypertrophic differentiation. Within the growth plate, prehypertrophic chondrocytes stop proliferating, and enlarge their size generating the hypertrophic zone of the growth plate. By E16.5 to 18.5 days, alterations were readily detected in the growth plates of mutant embryos (Figure 5A, D, E). Mutants demonstrated an expanded zone of hypertrophy relative to controls. That is, the hypertrophic zone represented a greater portion of the total growth plate in mutants (Figure 5D arrows, 5E solid lines; (c/b+c) ×100 = percentage of the growth plate occupied by the hypertrophic zone). At E16.5 days, the hypertrophic zone represented an average of 29% of the total growth plate while 43% of the total growth plate was occupied by the hypertrophic zone in mutant mice (T-test p=1.2 × 10−4). At E 18.5 days, the hypertrophic zone represented 19% of the total growth plate in controls and 41% in mutants (T-test p= 2.9 × 10−5). The expanded hypertrophic zone appeared to be the result of a combination of accelerated conversion of chondrocytes from the prehypertrophic to the hypertrophic zone and a failure of late hypertrophic differentiation. Accelerated early hypertrophic differentiation is suggested by the decrease in the distance from the top of the bone to the beginning of the hypertrophic zone (see line b in Figure 5e). A delay in the formation of the marrow cavity and trabecular bone is suggested by a decrease in the length of the alizarin red stained portion of the bones starting at E15.5 days (Figure 4D, Figure 5A, E). Percent mineralization was measured as the length of the alizarin red stained portion over the total length of the bone. At E16.5 days, 31% of the bone was mineralized in controls while only 14% was mineralized in mutants (T-test p= 7.6 × 10−4; this can also be seen in Figure 5E line d/a). At E18.5 days, an average of 51% of the control and 35% of the mutant long bones were mineralized (T-test p= 1.2 × 10−7). This result suggests that TGFβ signaling is required for proper hypertrophic differentiation.

Deletion of Tgfbr2 results in defects in the maintenance of the interzone

Another defect observed in Prx1Cre+/Tgfbr2f/f limbs was that the joints in the phalanges of the forelimb and some in the hindlimb were fused while other more proximal joints in the limb were normal (Figure 6A–C). The difference in the fore and hind limb joints is likely a result of delayed and reduced Prx1-Cre expression in the hindlimb as previously described (Logan et al., 2002). At E13.5 days a clear line within the alcian blue stained phalange representing the interzone was apparent in both control and mutant mice (Figure 6A, arrows); however, by E15.5 days the interzone was not detected and the joints were fused (Figure 6B arrows). In the autopod, the joint develops within a continous sheet of chondrocytes. The joint site is specified and the cells within this area form the interzone. The interzone consists of condensed flat cells that can be easily distinguished from the round cells of the cartilage (Figure 6D arrow). Specification of the joint accurs between E12.5 and E13.5 days and is thought to involve the activity of Wnt9a (reviewed in Pacifici et al. 2005). The interzone is characterized by distinct gene expression patterns. One of the markers of interzone is GDF5, which is also required to maintain the developing joint. The next step, cavitation, occurs between E14.5 and E15.5 (Figure 6E). Some of the cells in the interzone undergo apoptosis to form the joint space. Next, morphogenesis of the joint to the proper shape occurs (Figure 6F). Cells that resembled flat cells of the interzone were visible in Prx1Cre+; Tgfbr2f/f limbs at E13.5 days (Figure 6D arrow); however, by E14.5 days the presence of the cells was variable and they were difficult to find (Figure 6E). In most cases the darkly staining cells were small and round like immature cartilage. In some cases the cells were large and appeared hypertrophic (Figure 6E and Figure 8B, C). By E16.5 days, the joints were completely fused.

Figure 6
Characterization of the joint phenotype in Prx1Cre+/Tgfbr2f/f mice
Figure 8
Cartilage replaces interzone in E15.5 day Prx1Cre+/Tgfbr2f/f embryos

Next, to determine at which step development of the joint was disrupted we looked at markers of early joint formation (Figure 7). To examine whether Tgfbr2 is required for the specification, initiation, or maintenance of the interzone, we analyzed the expression of Wnt9a and GDF5 in the digits of control and Prx1Cre+; Tgfbr2f/f embryos at E12.5, E13.5, E.14.5, and E15.5 day limbs (Figure 7). Sections from control and Prx1Cre+; Tgfbr2f/f embryos were hybridized to 35S-labeled probes for Gdf5 and Wnt9a. In control joints, Gdf5 mRNA was detected as early as E12.5 days (not shown) and expression persisted until at least E15.5 days (Figure 7A–E). Expression was limited to a very discreet line of cells representing the interzone. Although Gdf5 expression was comparible in control and mutant limbs up to E13.5 days (Figure 7A,B) by E14.5 days, Gdf5 expression was reduced in the interzone of mutant limbs and ectopic expression was found in the perichondrium along the area where the joint had been specified (Figure 7C,D). This pattern was even more pronounced by E15.5 days when GDF5 expression in the presumptive joint area was barely detectable (Figure 7E, F). Similar results were found for the expression of Wnt9a mRNA (Figure 7G, H). These results suggest that Tgfbr2 is not required for specification or initiation of the joint but may be required to maintain the interzone.

Figure 7
GDF5 and Wnt9a expression in Prx1Cre+/Tgfbr2f/f mice

Previously, we showed that deletion of Tgfbr2 in the mesenchyme of the axial skeleton results in defects in the formation of the intervertebral disc (IVD) (Baffi et al., 2004). Specifically, markers of the annulus fibrosus phenotype were lost and peanut agglutinin (PNA) stained cartilage was found in the area of the developing IVD (Baffi et al., 2004). PNA is a lectin that recognizes β-galactose (1–3) N-acetyl galactosamine residues. It is commonly used to detect chondrogenic tissues and can detect chondrogenic cells before the overt deposition of cartilage specific matrix and staining with Alcian blue (Bagnall and Sanders, 1989; DeLise and Tuan, 2002; Stringa and Tuan, 1996). To determine if the defects in development of the joint were due to cartilage differentiation in the interzone, which normally contains non-cartilagenous cells, PNA staining was used (Figure 8). As described above, in control cells, the interzone was clearly seen as a strip of lightly stained cells in the region where the new joint will form (Figure 8A–C). Cavitation was evident at this stage (inset Figure 8A). In contrast, the lightly stained cells of the interzone were not detected in the autopods of Prx1Cre+/Tgfbr2f/f mice (Figure 8B, C). When control cells were stained with PNA, a clear, unstained section of cells corresponding to the interzone was observed inside a rectangle of PNA stained cells representing the cartilage on either side of the joint (Figure 8D). A clear boundary between the stained cartilage and unstained interzone was seen. In contrast, PNA staining was observed throughout this area in the Prx1Cre+/Tgfbr2f/f limbs. Clear boundaries were not detected (Figure 8E, F). The results suggest that Tgfbr2 is required to maintain the interzone by preventing the mesenchymal cells of the interzone from differentiating into cartilage and/or by forming a boundary between cartilage and interzone.

Cells in the interzone undergo apoptosis as part of cavitation to form the joint space. Apoptosis was not detected in this area in control or mutant limbs at E12.5 or E13.5 days (data not shown). Apoptosis was detected by TUNEL staining in the interzone of control limbs at E14.5 and E15.5 days (Figure 9A, C). TUNEL positive cells were not detected in this area within mutant limbs (Figure 9B, D) although normal staining was observed in the interdigit mesenchyme. Since non-cartilagenous interzone cells normally undergo apoptosis, the lack of apoptosis in this region is likely a consequence of the fact that the interzone is not maintained and that this zone has been replaced with cartilage. Together the results suggest that TGF-β regulates joint formation at a point after the specification and initiation of the interzone that blocks the subsequent cavitation of the joint.

Figure 9
Reduced apoptosis in Prx1Cre+/Tgfbr2f/f joints

Discussion

Chondrogenesis is a series of consecutive and strictly regulated steps that start with condensation of mesenchyme followed by differentiation of cells into chondrocytes. The factors that regulate the early stages of chondrogenesis are just beginning to be elucidated. Several previous reports suggested that TGF-β plays a critical role in early chondrogenesis including mesenchymal condensation (Carrington and Reddi, 1990; Chimal-Monroy and Diaz de Leon, 1997; Kulyk et al., 1989; Leonard et al., 1991; Macias et al., 1999; Roark and Greer, 1994; Verrecchia and Mauviel, 2002). It was shown that TGF-β is sufficient to induce the accumulation of Alcian blue stained matrix in micromass cultures derived from chick or mouse limbs. Even though the cells did not obtain a typical chondrocyte morphology like that observed in BMP treated cultures (Hatakeyama et al., 2004) it was concluded that TGF-β acts as a pro-chondrogenic factor. Furthermore, the effects of TGF-β were initiated in a specific window of time during culture suggesting that TGF-β affected the earliest stages of chondrogenesis including mesenchymal condensation (Carrington and Reddi, 1990; Chimal-Monroy and Diaz de Leon, 1997; Kulyk et al., 1989; Leonard et al., 1991; Macias et al., 1999; Roark and Greer, 1994; Verrecchia and Mauviel, 2002). Since the previous conclusions were based on gain-of-function experiments using in vitro systems, we wanted to determine the normal role TGF-β in limb development using loss-of function experiments. To this end we used conditional deletion of Tgfbr2 in limb mesenchyme. Mice that express the Cre recombinase under control of Prx1 promoter were crossed to mice in which exon 2 of Tgfbr2 was flanked with loxP sites, resulting in conditional deletion of Tgfbr2 primarily in limb mesenchyme (Chytil et al., 2002; Logan et al., 2002). Previous studies have shown that this Cre strain results in complete deletion of a reporter gene in the forelimb mesenchyme by E9.5 days and the hind-limb mesenchyme by E10.5 days (Logan et al., 2002). We showed significant deletion of Tgfbr2 exon 2 DNA by E9.5 days, and maximal deletion of receptor DNA by E10.5 days. The half-life of the TGF-β type II receptor protein has been estimated to be between 1 and 3 hours (Koli and Arteaga, 1997; Wells et al., 1997) while the half-life of the mRNA is estimated between 1 and 5 hours (Jiang et al., 1997). RT-PCR studies also indicated a significant reduction in the level of Tgfbr2 mRNA in the limb of Prx1Cre+/Tgfbr2f/f mice relative to controls at E10.5 days. Deletion of the receptor protein was confirmed using a functional assay. Limb mesenchyme from E11.5 day mice did not respond to exogenously added TGF-β1 confirming the functional deletion of the receptor by E11.5 days. Type II collagen and limb cartilage are first detected in the limb between E11.5 and E12.5 days (Ovchinnikov et al., 2000). Since the receptor protein is expressed at very low levels in vivo and difficult to detect even in wild type mice, the functional assay is more sensitive than looking at protein levels alone. Furthermore, Activin and Activin receptors are present in limb mesenchyme (Nohno et al., 1993) and are known to activate Smad2 and Smad3, therefore, the phosphorylation level of Smad2/3 cannot be used as a specific assay for TGF-β receptor function in the limb (Hoodless et al., 1996; Kretzschmar et al., 1997; Macias-Silva et al., 1996; Moustakas et al., 2001; Nakao et al., 1997).

Mice in which Tgfbr2 was deleted in Prx1-Cre expressing cells demonstrated defects in the development of the long bones of the limb as well as defects in the development of the sternum and parietal bones of the skull. In addition to expression in the limb, Prx-1 Cre is expressed in the anterior cranial mesoderm as well as the ventral mesoderm in the trunk explaining defects in the skull and sternum (Logan et al., 2002). Defects in the parietal bones in the skull suggest a defect in intramembranous bone formation. It has long been suggested that TGF-β plays and important role in osteoblast differentiation. It was previously shown that mice with germ-line deletion of Tgfb2-ligand demonstrate defects in the development of the sternum that are similar to but less severe than the defect described here (Sanford et al., 1997. Similar mild defects were seen in mice expressing a dominant-negative form of Tgfbr2 (Serra et al., 1997). It is not clear how TGF-β regulates the development of the sternum. This study focused on defects in the development of the long bones, which include shortened bones, absence of the deltoid tuberosity, and fusion of the joints in the autopod.

It was previously shown that muscle activity is required for the formation of the deltoid tuberosity (Reviewed in Herring 1994). Denervation and subsequent paralysis of the forelimb in rats resulted in a smaller deltoid tuberosity compared to the contra-lateral mobile side (Dysart et al. 1989). The results suggested that mechanical tension induced by the muscle is required to maintain the deltoid tuberosity. Furthermore, mouse embryos lacking striated muscle as a result of disruption of Myf5 and MyoD, master regulators of muscle differentiation, demonstrate defects in several skeletal structures including the deltoid tuberosity suggesting an important role for mechanical force in the development of the deltoid tuberosity (Rot-Nikcevic et al., 2006). Although the muscle in the limb does not express Prx-1 Cre ((Logan et al., 2002) and our unpublished observations), it is expressed in the connective tissue cells that pattern the muscle leaving alterations in muscle activity a possible cause for the absence of the deltoid tuberosity. In addition to the expression described in the skeletal elements, Prx1-Cre is also expressed in the tendon that attaches the bone to the muscle so that the defect in the deltoid tuberosity could also be due to alterations in the attachment of the muscle to the bone as well as in the morphogenesis of the skeleton itself. Additional experiments looking at markers of tendon and muscle differentiation will address this issue.

Although Tgfbr2 was significantly depleted by E10.5 days in Cre expressing mice, the size and shape of the early skeletal elements were similar to controls. The organization of the cartilage and expression of Sox9 were also comparable. The results suggest that Tgfbr2 is not uniquely required for the initiation of limb chondrogenesis or patterning of the limb at least after E10.5 days when we detect very high levels of Tgfbr2 deletion. Previously, we showed that deletion of Tgfbr2 in the sclerotome of the axial skeleton also does not affect the initiation of chondrogenesis (Baffi et al., 2006).

Loss of Tgfbr2 in the limb mesenchyme resulted in distinctly shorter limbs. This is in contrast to previous studies in which Tgfbr2 was deleted using Col2a-Cre (Baffi et al. 2004). Alterations in bone length, area of mineralization, hypertrophic differentiation, or chondrocyte proliferation were not detected in Col2acre:Tgfbr2lox/lox newborn mice. The differences in phenotypes observed in the two mouse models are likely due to the localization and timing of Prx1- and Col2a-Cre-mediated recombination. Prx1- Cre is expressed in early limb mesenchyme starting at E9.5 days of gestation and thus recombination is targeted to both chondrocytes and perichondium (Logan et al 2002). Col2a-Cre is expressed later in the limbs, after cells have committed to the chondrocyte lineage, and the perichondrium is not efficiently targeted (Ovchinnikov et al., 2000; Baffi et al 2004). There is considerable evidence indicating the importance of the perichondrium in coordinating development of the long bone and specifically implicating a role for TGF-β in the perichondrium (Alvarez et al., 2001; Alvarez et al., 2002; Di Nino et al., 2001; Long and Linsenmayer, 1998; Serra et al., 1997). Comparisons of the phenotype in these two mouse models also supports a role for the perichondrium in mediating the effects of TGF-β on embryonic endochondral bone formation.

We also show here that loss of Tgfbr2 results in a failure of the joints to form in the autopod. Marker analysis suggests that the joint is specified and that the interzone initially forms but is not maintained since GDF5 expression is reduced and PNA stained cells are present in the area of the interzone. Furthermore, apoptosis is not seen in the interzone at the initiation of cavitation. Likewise, we previously showed that deletion of Tgfbr2 in the axial skeleton results in a defect in the formation and maintenance of the IVD (Baffi et al., 2006; Baffi et al., 2004). Specifically, loss of Tgfbr2 in the axial skeleton resulted in the loss of the boundary between the vertebrae and IVD so that the IVD space contained cartilage. The previous results suggested that Tgfbr2 has two potential roles in the axial skeleton 1) to promote the differentiation of cells in the IVD and 2) to limit cartilage differentiation in the IVD space (Baffi et al., 2006). Similarly, we propose that TGF-β normally acts in development of the autopod joint by limiting the formation of cartilage in the interzone space and/or by maintaining the boundary between the cartilage and the interzone so that the joint can ultimately form (Archer et al., 2003; Lamb et al., 2003; Pacifici et al., 2005).

Along these lines, micromass cultures of limb mesenchyme from Prx1Cre+/Tgfbr2f/f mice consistently demonstrated an increase in the number and intensity of Alcian blue stained nodules when compared to cultures from Cre-negative mice. The results were at first surprising and suggest that Tgfbr2 normally acts to limit chondrogenesis in limb mesenchyme. This result does not contradict the previous findings from gain-of-function experiments but offer a new interpretation of the data based on loss-of function experiments using both in vitro and in vivo models. We propose that TGF-β is not pro-chondrogenic at the stages of development examined here, due to the timing of Prx-Cre expression our results do not exclude a role for Tgfbr2 in initiating chondrogenesis before E10.5 days. But instead, we propose that TGF-β normally acts to limit chondrogenesis and promote maintenance of connective tissues that are similar but distinct from cartilage, for example, annulus fibrosus of the intervertebral disc and interzone of the synovial joint (Lamb et al., 2003). The model in which TGF-β limits chondrogenesis and promotes other connective tissue phenotypes would also help to explain why TGF-β1 treated limb mesenchyme does not obtain chondrocyte morphology or discreet Alcian blue stained nodules like those observed in BMP treated cultures (Chimal-Monroy and Diaz de Leon, 1997). In summary, the results presented here provide information regarding signaling through Tgfbr2 and mechanisms of development in mouse limb development.

Acknowledgments

We would like to thank Ms. Anna Chytil and Dr. H. Moses for the kind gift of the Tgfbr2LoxP/LoxP mice and Dr. Cliff Tabin for the use of the Prx-1 cre strain. Dr. Serra is supported by NIH R01 AR053860 and R01 AR045605.

Footnotes

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