Transforming growth factor (TGF) -β receptors (TβRs) have been shown to have a critical regulatory role in medial edge epithelium (MEE) during palatal fusion (Sporn and Robert,1990; Gehris et al.,1994; Cui et al.,1998, Taya et al.,1999; Cui and Shuler,2000). The pattern of expression of the TβRs has been characterized in several studies; however, the effects of receptor deficiencies have not been studied in depth. Cleft palate craniofacial defects occur in TGF-β type II receptor (TβR-II) null mutant mice due to a cell proliferation defect within the cranial neural crest-derived palatal mesenchyme (Ito et al.,2003). The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro (Ito et al.,2003). TGF-β type III receptor (TβR-III) has been shown to have a MEE-specific pattern of expression at critical stages of palatal fusion; however, this receptor has been considered nonsignaling (Cui and Shuler,2000). It was hypothesized that TβR-III may modulate TGF-β3 binding to TβR-II in the MEE cells to locally enhance TGF-β3 autocrine signaling through the TβR-I/TβR-II receptor complex. Therefore, the complex of all three TβRs in the MEE may represent an autocrine pathway regulating the fate of the MEE during palatal fusion. Testing these hypotheses regarding the critical role of TβR-III during palatogenesis requires additional investigation of TβR-III gene expression and analysis of the effects on palatogenesis when TβR-III is knocked down.
Previous studies have demonstrated that small interfering RNA (siRNA) could silence gene expression by directing the sequence-specific degradation of mRNA (Elbashir et al.,2001a,b; Caplen et al.,2001). Degradation of the target mRNA reduces the amounts of the protein and leads to a detectable change in phenotype in both cell and organ culture systems (Elbashir et al.,2001a,b; Harborth et al.,2001; Sakai et al.,2003; Davies et al.,2004). There are advantages in the use of siRNA to knock down gene expression that include less toxicity, greater specificity and efficiency, and longer life in in vitro culture systems (Zender and Kubicka,2004). The use of siRNA inhibition in the palatal organ culture system has been reported, and knock down of Smad gene expression alters the process of palatal fusion (Shiomi et al.,2006).
We have hypothesized that TβR-III is required for palatal fusion and that reduction in TβR-III will inhibit the normal program of palatogenesis. The goals of the present study were to precisely characterize the spatial and temporal patterns of TβR-III gene expression in the palatal shelves during palatal fusion. Based on these findings, we examined the effects of knocking down TβR-III gene expression on the progress of palatogenesis in vitro. These studies will provide additional insight into the TGF-β–related mechanisms that are critical during craniofacial development.
TβR-III mRNA Gene Expression In Vivo
At embryonic day (E), 13 TβR-III mRNA could be identified in the palatal shelf epithelium, including the MEE at very low levels (Fig. 1a). The reorientation of the palatal shelves was associated with an increased level of TβR-III, primarily in the midline seam epithelial cells (Fig. 1b). There was a definite change in gene expression and a marked increase in MEE expression coincident with critical events in palatal fusion.
At E14.5, the midline epithelial seam begins to disappear, with some cells migrating to the oral and nasal aspects and some cells going through programmed cell death and some MEE transdifferentiating to mesenchyme (Glucksmann,1951; Saunders,1966; DeAngelis and Nalbandian,1968; Smiley and Dixon,1968; Shapiro and Sweney,1969; Smiley and Koch,1975; Fitchett and Hay,1989; Shuler et al.,1991,1992; Carette and Ferguson,1992; Griffith and Hay,1992; Mori et al.,1994; Taniguchi et al.,1995; Hay,1995,2005; Martinez-Alvarez et al.,2000; Cuervo et al.,2002; Nawshad and Hay,2003; Cuervo and Covarrubias,2004; Nawshad et al.,2004a,b; Vaziri et al.,2005; Jin and Ding,2006). At E14.5, the expression of TβR-III occurred predominantly in the midline epithelium in both the anterior and posterior regions of the palate (Fig. 1c,d).
By E15, the MEE had largely disappeared from the midline resulting in mesenchymal confluence. TβR-III mRNA could be identified in some residual MEE islands in the anterior palatal region (Fig. 1e). In the posterior region, TβR-III mRNA was detected in the midline seam area, and the nasal triangle of epithelial cells in the posterior region (Fig. 1f). As development continued, the midline seam was completely disrupted and TβR-III mRNA was only detected in the oral and nasal triangle areas. The expression of TβR-III was characteristic for MEE that retained an epithelial phenotype and were associated with a basement membrane.
To further identify and quantify TβR-III and TGF-β3 gene expression in the MEE during palatal development, we performed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) on MEE cells isolated by laser capture microdissection (LCM) at E13, E14.5, and E15 in vivo palatal tissue, and E13 + 24, 48, and 72 hr palatal organ culture in vitro. The effectiveness of LCM recovery of MEE was shown in Figure 2A-a and A-b. TGF-β3 gene expression occurred from E13 to E15, with negligible changes associated with the critical events in palatal fusion. TβR-III gene expression did demonstrate palatal fusion-related changes in gene expression with low levels at E13, and a peak in expression at E14.5. At E15, TβR-III gene expression decreased and remained in only very defined groups of midline epithelial cells (Fig. 2B). The amount of TβR-III gene expression at E14.5 was significantly higher than other stages of palatal fusion and expressed predominantly in the MEE.
TβR-III Gene Expression in the MEE In Vitro
To determine whether the palatal organ culture model replicated the TβR-III spatial, temporal, and quantitative patterns of gene expression observed in vivo, we performed real-time RT-PCR and Western blot analysis on samples isolated from palatal shelves maintained in vitro. At the initiation of organ culture, the expression of TβR-III was low; however, by E13 + 24 hr, the TβR-III expression had markedly increased. The change in TβR-III expression was coincident with the initial stages of midline seam disintegration. The TβR-III mRNA and protein peaked at E13 + 24 hr. After 48 hr of organ culture, the level of TβR-III gene expression began to decrease from the levels observed at E13 + 24 hr; however, there was no significant difference between the quantities at these two culture points. The palatal shelves were completely fused following 72 hr of organ culture, and cells with an epithelial morphology could not be identified in the midline. TβR-III expression was significantly decreased compared with E13 + 24 hr (Figs. 2C, 3). The observed TβR-III gene expression and protein quantity were correlated in vitro with the sample mRNA and protein profiles observed in vivo at the same developmental stages. These findings provided a foundation for intervention studies to knock down TβR-III expression because the in vitro and in vivo findings were similar.
Effects of Knocking Down TβR-III in MEE Cells Using siRNA
The highly defined pattern of TβR-III gene expression during palatal fusion leads to the hypothesis that elimination of TβR-III would alter patterns of palatal fusion. We examined the effects of knocking down TβR-III using siRNA. Western blot analysis of palatal organ culture tissue at the E13 + 24 hr point was completed to determine whether the TβR-III siRNA had been effective. There was a dose-dependent effect of the siRNA with the greatest reduction in TβR-III occurring with 500 nM (Fig. 4A,B). The 500 nM dose of siRNA resulted in a 75% decrease in TβR-III when compared with control siRNA. There was no significant difference in TβR-III between nontreatment control and control siRNA groups. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not affected in either the experimental or control siRNA treatment groups (Fig. 4A,B).
Immunohistochemistry provided additional evidence that treatment with 500 nM TβR-III siRNA reduced the amounts of the protein. TβR-III was strongly expressed in the MEE of the control palatal organ cultures at E13 + 24 hr in comparison to the very low levels detected in the siRNA-treated palatal shelf organ cultures (Fig. 4C-a,C-b). TGF-β3 and TβR-II were detected in the siRNA-treated organ cultures and were not effected by either the siRNA or by the reduction in TβR-III (Fig. 4C-c,C-d). The anterior and posterior regions of control siRNA-treated palatal organ cultures were completely fused, without any residual MEE in the midline seam at E13 + 72 hr in vitro (Fig. 5a,c), whereas the 500 nM siRNA-treated palatal organ cultures retained either one or two cell layers of MEE in the anterior region (Fig. 5b) and in the posterior regions MEE remained in the midline position (Fig. 5d).
Matings of TβR-III+/− mice resulted in offspring with the following genotypes: 33.1% wild-type mice, 66.6% heterozygous mice, and 0.3% homozygous null mice, indicating that the null mutants do not survive to term (Stenvers et al.,2003). The homozygous null mice of TβR-III developed lethal proliferative defects in heart muscle (50%) and apoptosis in the liver (75%). We treated 34 palatal organ cultures with TβR-III siRNA and examined all of these histologically for evidence of palatal fusion. Inhibition of palatal fusion occurred in 23 palatal organ cultures coincident with a reduction in TβR-III. Therefore, the overall outcome of siRNA treatment was that two thirds of the exposed palatal organ cultures had an alteration in the program of fusion when the level of TβR-III function was reduced by 75%. The present experimental approach precluded an assessment of the roles of other ligands that bind to TβR-III such as TGF-β1, 2, and Inhibin.
Effects on Smad2 Phosphorylation Following TβR-III Knockdown in MEE Cells
After TGF-β binding to TβRs, activated phospho-Smad2/3 forms a complex with Smad4 and translocates to the nucleus to propagate TGF-β signaling (Massague,1998). In a previous study, Smad2 and Smad3 were both present in the MEE, whereas only Smad2 was phosphorylated during palatal fusion (Cui et al.,2003). To identify TGF-β downstream signaling following TβR-III knockdown during palatal fusion, we observed total Smad2 and phospho-Smad2 protein expression using Western blot analysis at E13 + 24 hr organ culture. Although total Smad2 protein expression was unchanged, the phospho-Smad2 was decreased when compared with control and the changes in phospho-Smad2 had a dose-dependent relationship with the concentration of the siRNA (Fig. 6A). GAPDH was unchanged all in all experimental groups (Fig. 6A). Immunohistochemistry examination of phospho-Smad2 in the palatal tissues demonstrated that treatment with TβR-III siRNA resulted in a lower level at E13 + 24 hr than control (Fig. 6B-a,B-b). Thus, the reductions in TβR-III following siRNA treatment altered the generation of phospho-Smad2 and, consequently, the downstream signaling events in the TGF-β pathway.
Cell Proliferation and Cell Death in MEE Cells Following TβR-III Knockdown
To determine whether TβR-III inhibition by siRNA altered the normal pattern of MEE cell proliferation, bromodeoxyuridine (BrdU) incorporation was used to assess active DNA replication. MEE cell proliferation at E13 + 24 hr was assessed following TβR-III siRNA treatment. BrdU-positive cells were infrequently detected in control MEE cell; however, the TβR-III siRNA-treated palates had increased numbers of BrdU-positive cells in the MEE (Fig. 7A-a,A-b). The ratio of BrdU-positive cells in MEE in E13 + 24 hr palatal organ cultures was quantified and compared between the TβR-III siRNA-treated and control siRNA groups. The ratio of BrdU-positive to total MEE cells was significantly greater in TβR-III siRNA-treated groups (P* < 0.05; n = 10; Fig. 7B). These results provided evidence that TβR-III siRNA treatment resulted in persistent MEE cell proliferation, which has been shown to be linked to a failure to complete palatal fusion events.
To identify the fate of the remaining MEE, we also performed BrdU for cell proliferation and in situ terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) to assess apoptosis at E13 + 72 hr palatal organ culture. Both BrdU-positive and TUNEL-positive cells were identified in the remaining MEE cells (n = 10, respectively; Fig. 7A-c,A-d). This result provided evidence that, even in the late stages of palatal fusion in vitro, the MEE cells had multiple phenotypic fates, either persisting as proliferating cells or undergoing programmed cell death.
Effects of Locally Enhanced TGF-β3 Autocrine Signaling on Palatal Organ Cultures Exposed to TβR-III siRNA
Previously, it has been reported that TGF-β3 accelerated palatal fusion in vitro (Brunet et al.,1993). TβR-III binds TGF-β with high affinity and modulates the association of these ligands with their signaling receptors. We examined the effects of exogenous TGF-β3 on the process of palatal fusion in organ culture following exposure to TβR-III siRNA. Recombinant human (rh) TGF-β3 was added to E13 palatal organ cultures 6 hr after the transfection of the TβR-III siRNA. Palatal shelves were harvested at E13 + 72 hr. The palatal fusion was examined by quantifying the MEE remaining in the midline and was compared with the results from the different examination groups at E13 + 72 hr: TβR-III siRNA (500 nM) + rhTGF-β3 treatment (10 ng/ml and 50 ng/ml), TβR-III siRNA only, and control siRNA only (500 nM; n = 10, respectively).
The treatment of palatal organ cultures with TβR-III siRNA resulted in a reduction of TβR-III at E13 + 24 hr in all experimental groups as assessed with Western blot approaches. The addition of exogenous rhTGF-β3 (10 ng/ml and 50 ng/ml) did not rescue the levels of TβR-III such that all groups had significantly lower levels than the palatal organ cultures treated with control siRNA (Fig. 8A). The palatal organ cultures treated with TβR-III siRNA + 10 ng/ml TGF-β3 treatment palates were analyzed for palatal fusion and MEE cells remained in the midline position at E13 + 72 hr (n = 8; Fig. 8B-a). In palatal organ cultures treated with TβR-III siRNA + 50 ng/ml TGF-β3 MEE cells could not be detected in the midline position (Fig. 8B-b). These results provide evidence that endogenous TβR-III is necessary for the normal TGF-β3 autocrine signaling pathway but that this requirement can be overcome with increased levels of TGF-β3 that interact directly through the TβR-I/TβR-II receptor complex and complete the normal process of palatal development.
The role of the TGF-β signaling pathway has been shown to be important during palatogenesis, and continued investigation into this pathway as a potential target for the molecular etiology of cleft palate is warranted. The TGF-β family members initiate their cellular action by binding to specific cell surface receptors. There are three major types of receptors (TβRs), which have been identified by receptor affinity labeling assays (Massague,1998). Both type I (TβR-I) and type II receptors (TβR-II) are transmembrane serine/threonine kinases indispensable for TGF-β signaling (Brown et al.,1996,1999). TβR-III lacks a cytoplasmic protein kinase domain and, thus, had been considered to be a nonsignaling receptor (Brown et al.,1999). TβR-III expression has a characteristic pattern that is specifically localized to the MEE at very precise stages of palatal development (Cui and Shuler,2000). A TβR-III null mutant model has been developed (Stenvers et al.,2003). The mice with null mutations in TβR-III developed lethal proliferative defects in the heart and excess apoptosis in the liver, indicating that, although TβR-III is considered nonsignaling, there is a requirement for TβR-III during murine development.
TβR-III in the MEE has not yet been linked to a precise mechanism for TGF-β3 regulation of palatal fusion. In the present study, we completed a thorough evaluation of the spatial, temporal, and quantitative expression of TβR-III during palatogenesis both in vivo and in vitro. Based on the similarity between the TβR-III patterns of expression in vivo and in vitro, we evaluated the effect of knocking down TβR-III using a siRNA-based approach. The reduction in TβR-III resulted in a delay in the process of palatal fusion in vitro and supports the hypothesis that TβR-III has an important role in TGF-β signaling during the process of palatal fusion.
Smad2 is a major TGF-β signalling mediator (Derynck and Zhan,2003). Previous studies have shown that Smad2 phosphorylation was regulated by TGF-β3 and directly linked to the disappearance of the MEE during palatal fusion (Cui et al.,2003). Additionally, overexpression of Smad2 in the MEE was capable of rescuing the cleft palate phenotype in TGF-β3 null mutant mice (Cui et al.,2005). The use of a K14 promoter to drive Smad2 expression in the MEE resulted in greatly increased level of the protein reaction, which led to increases in phospho-Smad2 sufficient to complete the process of palatal fusion. Smad2 inhibition with siRNA resulted in persistence of the MEE in the midline seam with continued MEE cell proliferation, and exogenous TGF-β3 failed to rescue those phenotypic effects (Shiomi et al.,2006). Additionally, it has been shown that a requirement exists for a critical amount of endogenous Smad2 in the intracellular TGF-β3–associated signaling pathway regulating palatal fusion (Shiomi et al.,2006). In the TβR-III null mutant, it was demonstrated that an alteration in phospho-Smad2 quantities occurred that altered the signaling pathway and the mechanisms of heart and liver development (Stenvers et al.,2003). Knocking down TβR-III using siRNA also reduced phospho-Smad2 levels during palatal fusion in organ culture without any change in the total amounts of Smad2. The TβR-III null mutant model was also examined for changes in other potential TGF-β signal transduction molecules. No significant differences in the phosphorylation levels of several molecules (Erk1/2, p38, and SAPK/JNK) were detected when TβR-III +/+, +/−, and −/− genotypes were compared (Stenvers et al.,2003). Based on a reduction of TβR-III, our siRNA treatment results suggested that TβR-III could be an important modulator of TGF-β function and that this signaling pathway was strongly associated with the palatal fusion phenotype.
TGF-β3 has a critical role in palatogenesis (Fitzpatrick et al.,1990; Pelton et al.,1990; Shuler et al.,1991,1992; Kaartinen et al.,1995,1997; Shuler,1995; Sun et al.,1998; Cui et al.,1998,2003,2005; Taya et al.,1999; Cui and Shuler,2000; Martinez-Alvarez et al.,2000; Gato et al.,2002; Nawshad and Hay,2003; Nawshad et al.,2004a,b). The timing of TGF-β3 expression is temporally correlated with the critical events surrounding palatal shelf adhesion. In the TGF-β3 null mutant mice the palatal shelves fail to adhere properly, the basement membrane is not degraded and the MEE do not disappear from the midline seam (Kaartinen et al.,1995,1997). TGF-β3 belongs to a family of growth factors that have a broad range of regulatory activities, including control of cell proliferation, regulation of extracellular matrix deposition, matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases, cell migration, differentiation, and epithelial mesenchymal transformation (Miettinen et al.,1994; Morris-Wilman et al.,2000; Blavier et al.,2001; Brown et al.,2002; Kang and Svoboda,2002,2005; Nawshad and Hay,2003; Nawshad et al.,2004a,b). TGF-β3 thus has a critical role during palatal fusion, and the localized expression of TGF-β3 to the MEE suggests either a paracrine or autocrine signaling mechanism. It has been shown that TβR-I and TβR-II are located in most regions of the palatal epithelium and mesenchyme; however, TβR-III is specifically localized to the MEE in a distribution similar to TGF-β3 and at the critical stages of palatal fusion. The high correlation between patterns of TGF-β3 and TβR-III expression in the MEE during palatogenesis presents a situation where TβR-III may play an essential role in the mechanisms leading to palatal fusion. Our results demonstrated that knocking down TβR-III could affect the downstream signaling pathway of TGF-β such as phosphorylation of Smad2 during palatogenesis. Additionally, the effects of exogenous rhTGF-β3 demonstrated that the palatal fusion phenotype could be rescued in organ cultures treated with TβR-III siRNA and that the effect on MEE was dependent on the rhTGF-β3 dose. Thus, TβR-III plays a role in normal palatal fusion that can be circumvented in conditions with high levels of TGF-β3, which supports a role of TβR-III in increasing the local concentrations of TGF-β3 in the region of the MEE. The ability to identify and recover MEE cells during palatal fusion will provide the opportunity to more closely evaluate the different mechanistic events regulated by TβR-III during palatogenesis.
In Situ Hybridization
Tissue from mouse embryos at defined developmental stages (E13, E14.5, and E15) was frozen sectioned at 15 μm thickness and then fixed using 4% paraformaldehyde. Murine-specific DIG-oxigenin–labeled cRNA probes for TβR-III were used. For each antisense probe tested, a sense probe was also generated for use as a negative control. These probes were hybridized with the tissue sections at 65°C, and then the sections were washed extensively, dehydrated, and air-dried. Thereafter, the sections were incubated with blocking solution (Roche) for 1–1.5 hr at 4°C, washed and incubated with anti DIG-oxigenin overnight at 4°C. The distribution of the probe was determined with a secondary antibody and appropriate staining mixture NTMT (1 M Tris, 5 M NaCl, 1 M MgCl2) in 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT; Roche) signal development. These sections were covered with aquamount and cover slips and examined by light microscopy (Fig. 1).
Sampling and Quantitative Evaluation of TβR-III Receptor and TGF-β3 Expression in MEE Cells Isolated by LCM
We isolated MEE cells from the palatal shelf tissue using LCM (Arcturus, Mountain View, CA; Fig. 2). Palatal tissue section thickness was 10 μm. After microdissection, the collecting tube containing the cells in RLT buffer (Qiagen, CA) with 10% β-metocaptanol was capped and homogenized. RNeasy mini kit (Qiagen, CA) was used for total RNA extraction and purification. Extracted RNA was purified and quantified by spectrophotometry.
Total RNA samples from MEE cells at different stages of palatogenesis were prepared and reverse transcribed into cDNA, and real-time RT-PCR was performed as described by Scanlan et al. (2002). Aliquots containing equal amounts of mRNA were subjected to RT-PCR. First-strand cDNA synthesis was carried out using 1 μg of DNase-treated total RNA in 20 μl of a solution containing first-strand buffer, 50 ng of random primers, 10 mM dNTP mixture, 1 mM dithiothreitol, and 0.5 U of reverse transcriptase at 42°C for 60 min. The cDNA mixtures were diluted fivefold in sterile distilled water, and 2-μl aliquots were subjected to real-time RT-PCR using SYBR Green I dye. The real-time RT-PCR was performed in 25 μl of a solution containing 1× real-time PCR buffer, 1.5 mM dNTP mixture, 1× SYBR Green I, 15 mM MgCl2, 0.25 U of ExTaq polymerase RT-PCR version (TaKaRa, Tokyo Japan), and 20 μM specific primers. The primer pairs were as follows: the primers for TβR-III were 5′-TCG CCT AGC TGA ACC AAG AT-3′ and 5′-CAA GCT ACA CAG GGG GAC AT-3′ with a product size of 105 bp; TGF-β3 were 5′-TTG CAA GGG CTC TGG TAGT-3′ and 5′-ATG GCT TCC ACC TCT TCTT -3′ with a product size of 103 bp. GAPDH was used as a control with primer pairs 5′-GAC AAG CTT CCC GTT CTC AG-3′ and 5′-GAC TCA ACG GAT TTG GTC CT-3′ with a product size of 106 bp.
The primers were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). PCR was carried out in a thermal cycler (Smart Cycler, Cepheid, Sunnyvale, CA), and the data were analyzed using Smart Cycler software (version 2.0d). The PCR conditions were 95°C for 3 sec and 60°C for 20 sec for 35 cycles, and measurements were taken at the end of the annealing step at 60°C in each cycle. All real-time RT-PCR reactions were performed in triplicate, and the levels of mRNA expression were calculated and normalized to the level of GAPDH mRNA at each time point (E13, E14.5, and E15). An analysis of variance (ANOVA), followed by Tukey's honest significant difference (HSD) was completed for the data using SPSS software.
Palatal Shelf Organ Culture
Palatal shelves were cultured according to the methods previously described (Shuler et al.,1991; Cui and Shuler,2000, Yamamoto et al.,2003; Saito et al.,2005; Shiomi et al.,2006). Timed-pregnant Swiss-Webster mice were used for these studies. The females were mated for 2 hr, and the presence of a vaginal plug was used to determine E0. The palatal shelves were dissected from the fetal murine heads under sterile conditions and placed in pairs on Millipore filters with correct anterior–posterior orientation and with the medial edges in contact. The palatal shelves were cultured at the air–fluid interface in BGjb medium using Grobstein organ culture dishes (GIBCO, Grand Island, NY) at 37°C in 8% CO2 for up to 72 hr. Cultures were analyzed at the developmental stages determined by previous studies (Shuler et al.,1991; Cui and Shuler,2000; Yamamoto et al.,2003; Saito et al.,2005; Shiomi et al.,2006). Analysis of the organ cultures was performed at three key time points that are equivalent to critical stages in palatogenesis in vivo: (1) 24 hr of organ culture (equivalent to E14), when palatal shelves become adherent and an epithelial seam forms in the midline; (2) 48 hr of organ culture (equivalent to E15), when palatal fusion begins and the midline epithelial seam begins to breakdown; and (3) 72 hr of organ culture (equivalent to E16), when palatal fusion is completed with disappearance of the MEE and continuity of the mesenchyme. All experiments used this time schedule and were completed in triplicate.
Experimental groups were treated with siRNA (Invitrogen: Stealth siRNA) at final concentrations of 300, 400, and 500 nM, respectively. The sequences of siRNA pairs were 5′-UUA CCA AUU UGG UCA CUG UCA UGGA-3′ and 5′-UCC AUG ACA GUG ACC AAA UUG GUAA-3′ (Invitrogen Japan, Stealth). The culture medium was changed at the 48-hr culture point (Shiomi et al.,2006), and the process of palatal fusion was examined by characterizing the midline region at E13 + 72 hr (Shiomi et al.,2006). Based on the results of these studies a concentration of 500 nM siRNA was used for subsequent experiments examining changes in cell differentiation and alteration of the intracellular signaling pathway.
For the exogenous TGF-β3 experiment, 10 and 50 ng/ml of recombinant human (rh) TGF-β3 (R&D Systems, Inc., Minneapolis, MN) dissolved in sterile 4 mM HCl containing 1 mg/ml bovine serum albumin (BSA). The rhTGF-β3 was added to the organ cultures 6 hr after the siRNA transfection. Control groups were treated only with the sterile 4 mM HCl containing 1 mg/ml BSA. Cultures were maintained for up to 72 hr in medium containing rhTGF-β3.
Frozen sections, 10 μm thick, were prepared from the cultured palatal shelves at E13 + 72 hr. After sectioning, these slides were processed for hematoxylin–eosin staining following the standard procedures previously described (Saito et al., 2004; Shiomi et al.,2006). The region of the palate examined was restricted between the third ruga and the posterior boundary of maxillary first molar tooth organ. The fifth ruga was identified as the point separating anterior and posterior regions of the palate. Every fifth section from each cultured palate was examined by microscopy.
Immunofluorescent Analysis of TβR-III and TGF-β3 in Palatal Organ Culture
Frozen sections, 10 μm thick, were prepared from the cultured palatal shelves at E13 + 24 hr. We performed immunohistochemistry using anti-TβR-III (Santa Cruz Biotechnology, Santa Cruz, CA), TGF-β3 (Santa Cruz), TβR-II (Santa Cruz), p-Smad2 (Cell Signaling Technology, Inc., Beverly, MA) primary antibodies following procedures previously described. Secondary antibodies labeled with fluorescein isothiocyanate (FITC) were used to localize primary antibody binding. FITC fluorescence was identified with a Zeiss fluorescent microscope at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.
MEE Cell Proliferation Assay
The organ-cultured palates were analyzed for incorporation of 5-bromo-2′-deoxyuridine (BrdU, 100 μM, SIGMA, St. Louis, MO) to examine the patterns of MEE proliferation reflected by the numbers of cells in S-phase of the cell cycle. Briefly, the organ-cultured palates were grown in medium containing BrdU for 2 hr before they were harvested at E13 + 24 hr and 72 hr. The incorporation of BrdU was examined in both siRNA-treated and control palatal culture groups.
The palatal samples were fixed in 4% paraformaldehyde–phosphate buffered saline (PBS) at 4°C for 15 min followed by routine procedures to prepare frozen sections (10 μm). Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide in methanol. The sections were heated in citrate buffer for epitope retrieval. Nonspecific backgrounds were eliminated by incubating with nonimmune goat serum. The sections were then incubated with biotinylated mouse anti-BrdU monoclonal antibody. Streptavidin–peroxidase was used as a signal generator, the procedure was completed by using diaminobenzidine/H2O2 as a chromagen (BrdU staining kit, Zymed). Counterstaining was performed with hematoxylin (Yamamoto et al.,2003; Saito et al.,2005; Shiomi et al.,2006).
The number of BrdU-positive cells in the MEE and the total number of MEE cells were both counted in the midline seam region in every fifth section. The number of labeled cells was calculated and compared between the siRNA-treated group and the control group at E13 + 24 hr. An ANOVA, followed by Tukey's HSD was completed for the data using SPSS software. The acceptable level of significance was set at P* < 0.05.
Cell Death Detection
The organ-cultured palates were harvested at E13 + 72 hr to identify the fate of the remaining MEE cells. TUNEL staining was completed to detect cell death in MEE using the in situ Cell Death Detection kit, Fluorescein (Roche, Indianapolis, IN). The palatal samples were fixed in 4% paraformaldehyde–PBS at 4°C for 14 min, followed by routine procedures to prepare frozen serial sections (10 μm). Sections were treated with 20 μg/ml proteinase K (Roche, Indianapolis, IN) at room temperature for 15 min and then washed in PBS, and fixed in 4% paraformaldehyde–PBS for 10 min. After washing, the sections were incubated with the TUNEL reaction mixture at 37°C for 2 hr under a coverslip. After the TUNEL reaction, sections were washed in PBS and covered with antifade (Molecular Probes) for observation under a fluorescence microscope with filters.
Quantitative protein analysis was performed for TGF-β type III receptor using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting following the standard methods for the Boehringer Mannheim Chemiluminescence Blotting Kit (Roche, Indianapolis, IN). To identify the time course dependency of TβR-III expression during palatal development in vitro, we performed palatal organ culture and harvested the tissue for analysis at E13 + 24, 48, and 72 hr. The midline region of each pair of palatal shelves was dissected to study the changes.
The midline region from a single pair of palatal shelves was homogenized at 85°C in lysis buffer (50 mM Tris-HCl, 2% SDS, 10 glycerol). The homogenates were then boiled for 10 min and centrifuged, and the supernatants were recovered for determination of protein content by using the DC protein Assay Kit (Bio-Rad Labs, Hercules, CA). For each lane, 25 μg of protein was resolved by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membrane (Roche). The blots were blocked for 1 hr at room temperature with 1% blocking solution (Roche) in Tris-buffered saline and incubated overnight at 4°C with the specific polyclonal antibody (anti–TβR-III [Santa Cruz]; 1: 250, anti–TGF-β3 [Santa Cruz]; 1:250, anti-Smad2 [Cell Signaling Technology, Inc.]; 1: 1,000, anti-Smad2 [Transduction Laboratories, Lexington, KY]; 1:1,000, anti-GAPDH [Chemicon International, Temecula, CA]; 1:1,000). The blots were exposed to horseradish peroxidase–conjugated goat anti-rabbit IgG for 30 min at room temperature and reacted with substrate (Luminol/H2O2) according to the manufacturer's protocol, and thereafter exposed to X-ray films. The signal intensities were quantified by densitometric analysis using Scion Image Release Beta 184.108.40.206 (Scion Corporation, Frederick, MD). The ratio of GAPDH intensity was calculated and compared between siRNA TβR-III–treated (500 nM) and control groups.
The results from multiple groups were compared with ANOVA and Tukey's HSD multiple comparison tests. The acceptable level of significance was set at P* < 0.05. Data were analyzed with the SPSS software.
The authors thank our colleagues at the Center for Craniofacial Molecular Biology, University of Southern California, and at Department of Orthodontics and Pathology, Nihon University School of Dentistry, for their continuous strong support. This work was supported by MEXT Grant for multidisciplinary research projects, Nihon University Research Individual Grant for 2005, and Sato Funds from Nihon University School of Dentistry.