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Keywords:

  • cell proliferation;
  • medial edge epithelium;
  • siRNA, palate;
  • SMAD2

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Transforming growth factor (TGF)-beta 3 is known to regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Our previous studies showed that SMAD2, a TGF-beta signaling mediator, was expressed and phosphorylated primarily in the MEE and that SMAD2 phosphorylation in the MEE was temporospatially regulated by TGF-beta 3. The goal of this study was to examine the requirement for SMAD2 to complete the developmental events necessary for palatal fusion. SMAD2 expression was inhibited with Smad2 siRNA transfection into palatal tissues in vitro. The results showed that Smad2 siRNA transfection resulted in the maintenance of MEE cells in the palatal midline. Western blot and immunofluorescence analyses confirmed that the endogenous SMAD2 and phospho-SMAD2 levels were reduced following siRNA transfection. The SMAD3 level was not altered by the Smad2 siRNA transfection. The persistence of the MEE and the decreased SMAD2/phospho-SMAD2 levels were coincident with increased MEE cell proliferation. Addition of exogenous TGF-beta 3 increased p-SMAD2 level but not the total SMAD2 level. Therefore, exogenous TGF-beta 3 was not able to induce p-SMAD2 enough to rescue the palatal phenotype in the Smad2 siRNA group. The results indicated that the endogenous SMAD2 level is crucial in the regulation of disappearance of MEE during palatal fusion. Developmental Dynamics 235:1785–1793, 2006. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

TGF-beta3 plays an important role in the regulation of medial edge epithelium (MEE) disappearance during palatal fusion (Brunet et al., 1995; Kaartinen et al., 1995; Proetzel et al., 1995). Studies on TGF-beta3 null mutant mice revealed that the MEE were not removed from the midline and the mice were born with a cleft secondary palate (Kaartinen et al., 1995; Proetzel et al., 1995). TGF-beta transduces an intracellular signal through TGF-beta receptor regulated SMAD2 and/or SMAD3. SMAD2/SMAD3 are activated through phosphorylation, and then form a complex with the pathway common-mediator SMAD4. The complex translocates into the nucleus where it interacts with DNA in a cell-specific manner to effect transcription of TGF-beta-responsive genes (Heldin et al., 1997; Massague, 1998).

SMAD2 and SMAD3 share more than 95% homology but may function differently during embryonic development (Baker and Harland, 1996; Wu et al., 1997; Nomura and Li, 1998; Weinstein et al., 1998; Zhu et al., 1998; Yang et al., 1999). Smad2 homozygous mutant mice are early embryonic lethal, while Smad3 homozygous null mutants develop normally (Nomura and Li, 1998; Waldrip et al., 1998; Weinstein et al., 1998; Yang et al., 1998; Zhu et al., 1998; Datto et al., 1999). It is believed that SMAD2 plays a more important role in regulating embryonic development and that SMAD3 may be more involved in responses such as wound healing (Ashcroft et al., 1999; Massague et al., 2000).

In our previous study, SMAD2 and SMAD3 were both present in the MEE, whereas only SMAD2 was phosphorylated during palatal fusion. SMAD2 phosphorylation was spatially restricted to the MEE and temporally correlated with the inhibition of MEE cell proliferation and disappearance of the MEE. Phosphorylated SMAD3 was not detectable during normal palatal fusion (Cui et al., 2003). The cleft palate phenotype in TGF-beta3−/− mice could be rescued by overexpression of a Smad2 transgene in the MEE cells (Cui et al., 2005). The results suggested that SMAD2 was crucial for palatal fusion. However, there was no previous direct evidence that inhibition of endogenous Smad2 affected the palatal fusion since Smad2 null mutant mice die at an early embryonic stage before emergence of the palatal shelves (Nomura and Li, 1998; Waldrip et al., 1998; Weinstein et al., 1998).

RNA interference (RNAi) is a recently developed technique that can elicit down-regulation of gene expression (Fountaine et al., 2005). RNAi is a conserved system through which double-stranded RNA (dsRNA) guides sequence-specific mRNA degradation (Fire et al., 1998; Montgomery et al., 1998). The RNAi apparatus may be artificially triggered by delivery of naked small interfering RNA (siRNA) molecules or by plasmid-based expression of dsRNA (Fountaine et al., 2005). Previous studies have demonstrated that siRNA could be transfected into mammalian cells, silencing gene expression by directing the sequence-specific degradation of mRNA containing the siRNA sequence, and bypassing the non-specific interferon response (Elbashir et al., 2001a, b; Caplen et al., 2001). Degradation of the target mRNA led to reduction in the cognate protein, and ultimately led to a detectable change in phenotype in both cell and organ culture system (Elbashir et al., 2001a, b; Harborth et al., 2001; Sakai et al., 2003; Davies et al., 2004). There are many advantages in the use of siRNA compared with antisense oligonucleotides. The siRNA advantages include much less toxicity, greater specificity and efficiency, and longer life in in vitro culture systems (Zender and Kubicka, 2004). The use of siRNA inhibition in palatal organ culture system has not been reported.

In the present study, we utilized the siRNA technique to specifically knock down the Smad2 gene in the palate in vitro during a critical palatal fusion time window that has SMAD2 expression. We hypothesized that the inhibition of SMAD2 expression by Smad2 siRNA would inhibit palatal fusion by specifically decreasing the endogenous SMAD2 level and consequently result in a failure to inhibit MEE cell proliferation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Smad2 siRNA Inhibited Palatal Fusion

To determine whether Smad2 siRNA inhibited palatal fusion, E13 palatal shelves were transfected with 100 and 500 nM Smad2 siRNA or pooled control siRNA, respectively. The organ cultures were maintained for 48 h after siRNA transfection. Palatal fusion is normally complete after 72 hr of organ culture with only limited numbers of MEE remaining in the midline position. Therefore, organ cultured palatal tissues were harvested following 72 hr of organ culture and processed for histological examination. Palatal fusion was characterized by quantifying the MEE remaining in the midline position in semi-serial sections from defined region of the palate. The patterns of palatal fusion were compared for the different treatment groups: 100 nM siRNA Smad2 group (n = 6), 500 nM siRNA Smad2 group (n = 19), and pooled control siRNA group (n = 30). Smad2 siRNA at 100 and 500 nM inhibited palatal fusion resulting in MEE remaining in the palatal midline seam at E13+72h. Treatment with Smad2 siRNA at concentrations of either 100 or 500 nM resulted in greater numbers of MEE persisting in the midline than observed in the pooled control siRNA groups (Fig. 1A–C). Statistical analysis indicated that Smad2 siRNA treatment groups had significantly more persistent MEE (Scheffe's F, P < 0.01), and that the number of MEE remaining had a dose-dependent relationship (Scheffe's F, P < 0.05) (Fig. 1D). Based on the results of these studies, a Smad2 siRNA concentration of 500 nM was used for all subsequent experiments examining changes in cell differentiation and alterations of the intracellular signaling pathway.

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Figure 1. Comparison of MEE remaining between pooled control siRNA (A), 100 nM of Smad2 siRNA (B) and 500 nM of Smad2 siRNA (C) groups at E13+72 hr of palate culture. A: Palate MEE transfected with control siRNA fused completely with no MEE remaining in this section. B,C: Palates transfected with Smad2 siRNA had persistent MEE in the midline. D: Quantitative comparison of the MEE remaining between pooled control siRNA and each Smad2 siRNA group (100 and 500 nM). The results demonstrated that Smad2 siRNA caused MEE to remain in a dose-dependent manner. Control: 5.38 ± 2.96, n = 30. 100 nM: 10.68 ± 3.99, n = 6. 500 nM: 15.68 ± 6.50, n = 19. Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

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Smad2 siRNA Reduced SMAD2 and Phospho-SMAD2 Level But Did Not Result in an Alteration of the SMAD3 Level

SMAD2 and phospho-SMAD2 (p-SMAD2) level were examined by Western blot analysis in the palatal midline following transfection with Smad2 siRNA (500 nM). The palatal tissues were collected at E13+24h, and the MEE midline region was dissected to extract the proteins (Saito et al., 2005; Yamamoto et al., 2003). To demonstrate the consistent reduction of SMAD2/p-SMAD2 level, three independent specimens from each group were processed together and run in the same gel. The results showed that the both SMAD2 and p-SMAD2 bands were greatly reduced in the Smad2 siRNA treated group (500 nM) compared to the control siRNA group (Fig. 2A). The signal intensities of the bands were normalized to GAPDH and analyzed statistically. Smad2 siRNA-transfected palatal tissue had significantly lower levels of SMAD2 (P < 0.01, n = 7) (Fig. 2B) and p-SMAD2 (P < 0.01, n = 7) (Fig. 2C) than control siRNA-transfected palatal tissue. The GAPDH level was unchanged in both Smad2 siRNA and control siRNA-transfected groups (Fig. 2A). To demonstrate the specificity of the siRNA for SMAD2 in this study, SMAD3 was also characterized. The result showed that there was no significant difference in SMAD3 levels between the Smad2 siRNA-transfected and control siRNA-transfected groups (500 nM) (n = 8) (Fig. 2D,E).

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Figure 2. Comparison of SMAD2, p-SMAD2 (A–C) and SMAD3 (D, E) levels between the control siRNA-transfected (500 nM) groups and Smad2 siRNA-transfected (500 nM) groups. A: Western blotting of SMAD2, p-SMAD2, and GAPDH isolated from MEE at E13+24 hr of organ culture. B,C: Quantitative comparison for SMAD2 (B) and p-SMAD2 (C) level between control siRNA-transfected groups and Smad2 siRNA-transfected groups. D: Western blotting of SMAD3 and GAPDH isolated from MEE at E13+24 hr of organ culture. E: Quantitative comparison for SMAD3 level between the control siRNA-transfected groups and Smad2 siRNA-transfected groups. The results demonstrated that Smad2 siRNA transfection reduced the SMAD2 and p-SMAD2 levels but did not result in an alteration of the SMAD3 level. The vertical axis is expressed as the ratio of each protein normalized with GAPDH. Data are presented as mean ± SD (SMAD2: n = 7, p-SMAD2: n = 7, SMAD3: n = 8). **P < 0.01.

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Evidence that Smad2 siRNA treatment had effectively targeted Smad2 and p-SMAD2 was further provided by immunofluorescence. The Smad2 siRNA treatment substantially decreased the localization of SMAD2 and p-SMAD2 in the MEE. The intensity of fluorescence representing either SMAD2 or p-SMAD2 localization was weaker in the Smad2 siRNA group (Fig. 3B,D) than in the control siRNA group (Fig. 3A,C).

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Figure 3. Effect of control siRNA (500 nM) (A, C, E, G) and Smad2 siRNA (500 nM) (B, D, F, H) on cultured palates was demonstrated by immunofluorescence: SMAD2 (green, A, B), p-SMAD2 (red, C, D), and nuclear DAPI (blue, E, F). A–D: The intensity of SMAD2 and p-SMAD2 fluorescence in palate was weaker in the Smad2 siRNA group (B, D) than in the control siRNA group (A, C), respectively. E,F: Nuclear DAPI staining showed the equal number of cells in both groups. G,H: Three fluorescence images of the same section were merged for each individual group. White color represented the outcome of merged green, red, and blue florescence. There were less white color in H than in G as the result of reduction of SMAD2 and p-SMAD2 in the Smad2 siRNA group.

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Thus, inhibition of palatal fusion following Smad2 siRNA transfection could be attributed to the reduction of both total endogenous SMAD2 and p-SMAD2. The change in SMAD2 and inhibition of palatal fusion was not associated with any compensatory change in either total SMAD3 or phospho-SMAD3.

The Inhibition of SMAD2 Maintained MEE Cell Proliferation

To determine whether SMAD2 inhibition by siRNAs altered the normal pattern of MEE cell proliferation, BrdU incorporation was analyzed to assess active DNA replication. BrdU-labeled cells were present in the MEE in both the experimental (Fig. 4B,D) and control (Fig. 4A,C) groups at E13+24 hr. We quantified the BrdU-labeled cells in the MEE in semi-serial sections from defined regions of the organ cultured palate and compared the numbers of BrdU-positive cells between the Smad2 siRNA (500 nM) and control siRNA (500 nM) groups. The Smad2 siRNA treated group (n = 11) had significantly more BrdU-positive cells than the control siRNA group (n = 10) (P < 0.01) (Fig. 4E). This result provides evidence that siRNA inhibition of SMAD2 resulted in the maintenance of MEE cell proliferation. Persistent MEE cell proliferation has been linked to mechanisms that result in a failure of the normal program for palatal fusion (Cui et al., 2003; Yamamoto et al., 2003; Saito et al., 2005).

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Figure 4. Comparison of MEE proliferation between control siRNA-transfected (500 nM) (A, C) groups and Smad2 siRNA-transfected (500 nM) (B, D) groups at E13+24 hr of palate culture. A,B: When bilateral palatal shelves contacted, the BrdU positive cells were increased in the Smad2 siRNA group (B) compared to the control group (A). C,D: When palatal shelves remained apart, the BrdU positive cells were also increased more in the Smad2 siRNA group (D) than in the control group (C). E: Comparison of BrdU-positive rate in the MEE between control siRNA transfection group (n = 10) and Smad2 siRNA transfection group (n = 11). Quantitative analysis confirmed that there were more BrdU-positive MEE cells in the Smad2 siRNA treated group than in the control siRNA group (E) (P < 0.01). The vertical axis is expressed as BrdU-positive cells as a percentage of the total MEE cells. Data are presented as mean ± SD. *P < 0.01.

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Exogenous TGF-beta3 Failed to Rescue Smad2 siRNA-Induced MEE Remaining

Previously, it has been reported that TGF-beta3 accelerated palatal fusion in vitro (Brunet et al., 1993). TGF-beta3-regulated palatal fusion in vivo has been correlated with SMAD2 phosphorylation in the MEE (Cui et al., 2003). To examine whether TGF-beta3 could rescue the failure of palatal fusion resulting from Smad2 siRNA treatment, we added recombinant human (rh) TGF-beta3 into E13 palatal culture 6 hr after the transfection. Palatal tissues were harvested at E13+72 hr. The palatal fusion was examined by quantifying the MEE remaining in the midline and compared the results from the different examination groups: Smad2 siRNA (500 nM) + rhTGF-beta3 treatment (50 ng/ml) group, Smad2 siRNA only (500 nM) group, and control siRNA only (500 nM) group. Both the Smad2 siRNA (n = 10) group and the Smad2 siRNA + TGF-beta3 treatment groups (n = 11) had significantly (P < 0.05) more MEE remaining in the midline than the control groups (n = 9) at E13 + 72h (Fig. 5). The palatal tissue treated with Smad2 siRNA + rhTGF-beta3 was not statistically different from the SMAD2 siRNA group (Fig. 5). Exogenous TGF-beta3 failed to improve palatal fusion following Smad2 siRNA inhibition in palatal organ cultures.

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Figure 5. Comparison of the MEE that remain between the control siRNA (500 nM), Smad2 siRNA (500 nM), and Smad2 siRNA (500 nM) + rTGF-beta3 (50 ng/ml) groups at E13+72 hr of culture. Both Smad2 siRNA and Smad2 siRNA + rTGF-beta3 groups had more MEE remaining than the control siRNA group (P < 0.01). However, there was no difference between the Smad2 siRNA and Smad2 siRNA + rTGF-beta3 groups. The results indicated that exogenous TGF-beta3 could not rescue Smad2 siRNA-induced persistence of the MEE. Control: 3.68 ± 1.80, n = 9. Smad2 siRNA: 14.58 ± 7.14, n = 10. Smad2 siRNA + rTGF-beta3: 13.58 ± 7.05, n = 11. Data are presented as mean ± SD. **P < 0.01.

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Exogenous TGF-Beta3 Induced Phospho-SMAD2 But Not SMAD2

The palatal shelves treated with rhTGF-beta3 were examined to determine whether the growth factor had an effect. The Western blot analysis of the midline region indicated that treatment with exogenous rhTGF-beta3 did induce phosphorylation of SMAD2 (Fig. 6A,C), but not the total SMAD2 level (Fig. 6A,B), in normal palatal culture. The same result was seen in the palates that were treated with Smad2 siRNA+TGF-beta3 (Fig. 6D–F). TGF-beta3 had no effect on the total amount of SMAD2 when Smad2 siRNA presented (Fig. 6D,E). With the decline of endogenous SMAD2 knocked-down by the Smad2 siRNA, the TGF-beta3 induced p-SMAD2 level (Fig. 6D,F) was not sufficient to induce the completion of palatal fusion (Fig. 5). The effectiveness of signaling by the exogenous rhTGF-beta3 was assessed by measuring the changes in MMP-13, which has been shown to be induced by the growth factor (Fig. 6G,H) (Kaartinen et al., 1995). These results provide evidence that the amount of p-SMAD2 induced by TGF-beta3 can still result in molecular regulation associated with the growth factor and also demonstrate that the absolute magnitude of p-SMAD2 is critical for different events. The completion of palatal fusion requires a significant increase in p-SMAD2 and when the endogenous levels are reduced the cells are incapable of generating the response necessary for the removal of the MEE from the midline region. This information indicates that reduction in endogenous SMAD2 could be associated with a mechanism for cleft palate when other aspects of the TGF-beta3 signaling mechanism are at the normal levels.

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Figure 6. SMAD2, p-SMAD2 levels between control and TGF-beta3 (50 ng/ml) supplemented groups (A–C), and between control siRNA, Smad2 siRNA and Smad2 siRNA + TGF-beta3 (50 ng/ml) supplemented groups (D–F). MMP-13 levels between control and TGF-beta3 (50 ng/ml) supplemented groups (G, H). A: Western blotting of SMAD2, p-SMAD2, and GAPDH isolated from MEE at E13+24 hr of organ culture. B,C: Quantitative comparison for SMAD2 (B) and p-SMAD2 (C) levels. The results showed that although rhTGF-beta3 induced SMAD2 phosphorylation (P < 0.05), it did not alter the total SMAD2 level. D: Western blotting of SMAD2, p-SMAD2, and GAPDH. E,F: Quantitative comparison for SMAD2 (E) and p-SMAD2 (F) levels. The results showed that rhTGF-beta3 induced a limited small amount of SMAD2 phosphorylation under the Smad2 siRNA transfection because it did not induce the total SMAD2 level. G: Western blotting of MMP-13 and GAPDH. H: Quantitative comparison for MMP-13 level between control and TGF-beta3 supplemented groups. The result showed an increase in the MMP-13 level (P < 0.01), which confirmed the effectiveness of rhTGF-beta3 in the culture system. The vertical axis is expressed as the ratio of each protein normalized with GAPDH. Data are presented as mean ± SD (each graph, n = 8). *P < 0.05, **P < 0.01.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

SMAD2 is a major TGF-beta signaling mediator (Derynck and Zhang, 2003). It has been known that SMAD2 inhibits epithelial cell proliferation and SMAD2 expression was shown to be reduced in some carcinoma cells (Uchida et al., 1996; Yakicier et al., 1999; Sjoblom et al., 2004). Our previous studies have suggested that SMAD2 was spatially phosphorylated in the MEE at critical stages of palatal development. The SMAD2 phosphorylation was regulated by TGF-beta3 and correlated with 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-beta3-null mutant mice (Cui et al., 2005). The use of a K14 promoter to drive Smad2 expression in the MEE resulted in greatly increased levels of the protein, which lead to increases in p-SMAD2 sufficient to complete the process of palatal fusion. These previous studies did not investigate the relationship of endogenous levels of SMAD2 with the TGF-beta3-associated mechanism for palatogenesis. The goal of the present study was to investigate the relationship of endogenous levels of SMAD2 in the MEE and the effect of reducing those levels on the process of palatogenesis. These studies have shown that reduction in the amount of SMAD2 in the MEE will alter the TGF-beta3 signaling pathway sufficiently to inhibit palatal fusion.

There are multiple approaches to inhibit endogenous Smad2 expression. The technique of RNAi mediated by siRNA has the potential to inhibit gene expression at a time of the experimenter's choosing. siRNA has been used for both cell culture (Elbashir et al., 2001a, b; Harborth et al., 2001) and organ culture model systems (Sakai et al., 2003; Davies et al., 2004). In this study, we used concentrations of either 100 or 500 nM Smad2 siRNA and found that 500 nM Smad2 siRNA caused the greatest alteration of MEE cell fate without affecting tissue viability. The efficiency and specificity of Smad2 siRNA inhibition was confirmed by Western blot and immunofluorescence analyses on the transfected palates. The results showed that both SMAD2 and p-SMAD2 level were reduced, but the SMAD3 level remained unchanged after Smad2 siRNA transfection. The decreased SMAD2 and phospho-SMAD2 level coincided with persistent MEE cell proliferation. The results supported our hypothesis that inhibition of SMAD2 expression by siRNA transfection prevented palatal fusion by maintaining MEE cells in a proliferation state.

A previous study emphasized the critical role of SMAD2 phosphorylation in the MEE during palatal fusion (Cui et al., 2003). The importance of the endogenous SMAD2 level, however, has not been evaluated. In this study, we showed that exogenous recombinant TGF-beta3 in culture was not able to rescue palatal fusion if the levels of endogenous SMAD2 were reduced by Smad2 siRNA transfection. However, exogenous TGF-beta3 was able to induce MMP-13 expression demonstrating that the signaling pathway was active. In TGF-beta3 homozygous null mutant mice, the endogenous SMAD2 level was unchanged but SMAD2 phosphorylation was lost in the MEE cells (Cui et al., 2003). Overexpression of a Keratin-14 promoter-directed Smad2 in the MEE rescued the cleft palate phenotype in the TGF-beta3-null mutant mice. The success of rescue could be attributed to an elevation of both SMAD2 and phosphorylated SMAD2 in the MEE (Cui et al., 2005). Exogenous rhTGF-beta3 rescued palatal fusion in TGF-beta3−/− palatal organ cultures in vitro (Kaartinen et al., 1997; Taya et al., 1999), as there was sufficient endogenous SMAD2 available to be converted to p-SMAD2.

Although exogenous TGF-beta3 increased the amount of p-SMAD2, the overall decrease in total SMAD2 resulting from the siRNA treatment established a condition in which the TGF-beta3 binding was not capable of rescuing palatal fusion. Thus, the endogenous SMAD2 level is critical to the TGF-beta signaling pathway and the program for the palatal fusion. It is possible that reduction in endogenous SMAD2 could represent a mechanism for cleft palate induction when the TGF-beta growth factors and receptors are unchanged.

It is evident that the developmental events are affected by the amount of endogenous SMAD2 (Nomura and Li, 1998; Ito et al., 2001). In Smad2 heterozygous mice with half of the normal amount of Smad2, 20% of embryos and newborn pups showed various abnormalities including craniofacial defects (Nomura and Li, 1998). In K14-driven Smad2 transgenic mice, lower levels of Smad2 transgene expression were associated with a less severe phenotype, and a higher level of Smad2 transgene expression correlated with severe changes in skin and ear (Ito et al., 2001). The extent of the improvement of palatal fusion in the TGF-beta3−/− genetic background with a K14-Smad2 transgene was also associated with the level of SMAD2 and p-SMAD2. The p-SMAD2 level was statistically significantly correlated with the palatal fusion outcomes (Cui et al., 2005). In this study, we showed that about 60% reduction of SMAD2 protein by 500 nM Smad2 siRNA (Fig. 2B) caused the failure of palatal fusion in organ culture. It is likely that there is a threshold with regard to the level of SMAD2 necessary to transduce TGF-beta signals in the cell to accomplish different developmental events (Ito et al., 2001). In palatal tissues, the amount of SMAD2 required to complete the disappearance of the MEE from the midline is greater than needed to alter the expression of gene associated with basement membrane degradation such as MMP-13.

In summary, SMAD2 inhibition affects the program for palatal fusion in organ culture. SMAD2 inhibition results in persistence of MEE in the midline seam and continued MEE cell proliferation. The presence of SMAD3 in the tissues fails to prevent the effects caused by siRNA Smad2 inhibition. Exogenous TGF-beta3 failed to rescue the effects resulting from Smad2 siRNA treatment due to a decrease in the amount of endogenous SMAD2. These results demonstrate the critical role of the amount of endogenous SMAD2 in the intracellular TGF-beta3-associated signaling pathway regulating palatal fusion.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Animal and Palatal Shelf Organ Culture

Palatal shelves were cultured according to the methods previously described (Cui et al., 1998; Cui and Shuler, 2000; Shuler et al., 1991). Briefly, timed-pregnant Swiss-Webster mice were sacrificed on embryonic day 13 (E13). 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 their medial edges in contact. The palatal shelves were cultured using Grobstein organ culture dishes in BGJb medium (GIBCO, Grand Island, NY) at 37°C in 5% CO2. The palatal tissues were harvested at two key time points that are equivalent to critical stages in palatogenesis in vivo: (1) 24 hr of organ culture, when palatal shelves become adherent and an epithelial seam forms in the midline (equivalent to the initial contact stage in vivo); (2) 72 hr of organ culture, when the mesenchyme becomes confluent and there is a disappearance of the MEE (equivalent to complete palatal fusion in vivo). All experiments were completed in triplicate.

For the exogenous TGF-beta3 study, 50 ng/ml of recombinant human (rh) TGF-beta3 (R & D systems Inc., Minneapolis, MN) dissolved in sterile 4 mM HCl containing 1 mg/ml BSA was added 6 hr after the transfection. Control groups were treated only with the sterile 4 mM HCl containing 1 mg/ml BSA. Cultures were maintained for up to 48 hr using the same medium with the addition of rhTGF-beta3.

Transfection of siRNA

RNA interference using siRNA was performed as previously described (Davies et al., 2004; Sakai et al., 2003). The 100- and 500-nM siRNA solutions were prepared by diluting a siRNA stock (50 μM) in BGJb medium containing Oligofectamine (0.2%) (Invitrogen, Carlsbad, CA). The siRNA media solutions were prepared in 1-ml aliquots and 1 ml was used in the Grobstein organ culture plates. Four sets of paired palatal shelves were cultured together in each organ culture plate with the BGJb medium. The Smad2 siRNA oligonucleotides are produced by and purchased from Dharmacon Research, which also provided scrambled duplex control siRNA. After 48 hr, the medium of the cultures was changed.

Assessment of Palatal Fusion

Assessment of palatal fusion was performed as previously described (Saito et al., 2005). Briefly, cultured palates were harvested at E13+72 hr and fixed in 4% paraformaldehyde-PBS, followed by routine procedures for embedding in paraffin. Coronal serial sections (6 μm) were mounted on albumin-coated slides and stained with hematoxylin and eosin (Lerner Laboratories, Pittsburgh, PA). The region of the palate examined was restricted between the 3rd ruga and the posterior boundary of 1st molar tooth organ. Every fifth section from each cultured palate was examined microscopically. The MEE remaining in the midline epithelial seams were traced by camera lucida (Olympus, Tokyo, Japan), and included the MEE covering opposing palatal shelves, 1- and 2-cell layer MEE seam, MEE islands, MEE in the oral and nasal triangle areas. The traced image was scanned and measured by computer software (Image-Pro Plus 4.0, Media Cybernetics, Inc, Silver Spring, MD) to generate an area value (μm2). Total values were divided by the total number of sections that were examined. The mean of the MEE remaining area was calculated for each sample.

Western Blot Analysis

Quantitative protein analysis was performed as described (Yamamoto et al., 2003; Saito et al., 2005) using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting following the standard method for the BM Chemiluminescence Blotting Kit (Roche, Indianapolis, IN). The primary antibody against SMAD2 (mouse monoclonal IgG, 1:1,000) was purchased from Transduction Laboratories™ (San Jose, CA). The primary antibody against p-SMAD2 (rabbit polyclonal IgG, 1:1,000) was purchased from Cell Signaling Technology, Inc. (Beverly, MA). The primary antibody against SMAD3 (rabbit polyclonal IgG, 1:1000) was purchased from Upstate Biotechnology (Lake Placid, NY). The primary antibody against MMP-13 (mouse monoclonal IgG, 1:100) was purchased from Lab Vision Corp. (Fremont, CA). The primary antibody against GAPDH (rabbit polyclonal IgG, 1:1,000) was purchased from Chemicon International Inc. (Temecula, CA). The signal intensities were quantitated by densitometric analysis using Image Quant (Molecular Dynamics, Sunnyvale, CA).

Immunohistochemistry

Immunostaining was completed by following standard procedures (Chai et al., 1999; Hosokawa et al., 2005). Briefly, serial sections (6 μm) of cultured palates transfected with control or Smad2 siRNA were cut and mounted onto the same slide to ensure exposure to the same antibody concentration. The source of primary antibody against SMAD2 (1:10) or p-SMAD2 (1:10) used here was the same as used for Western blot analysis. Fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG (1:100) (Invitrogen) was used as the secondary antibody for SMAD2, and Alexa Fluor® 568 labeled goat anti-rabbit IgG (1:100) (Invitrogen) was used for p-SMAD2. Vectashield® Mounting Medium with DAPI (Vector Laboratories. Burlingame, CA) was used to prevent rapid loss of fluorescence during microscopic examination and for nuclear staining.

MEE Cell Proliferation Assay

This assay was performed as previously described (Yamamoto et al., 2003; Saito et al., 2005). Briefly, the organ-cultured palates were grown in media containing 5-bromo-2-deoxyuridine (BrdU, 100 μM, Sigma, St. Louis, MO) for 2 hr before they were harvested at E13+24 hr day. Incorporation of BrdU was analyzed to examine the patterns of MEE cell proliferation (cells in S-phase of the cell cycle).

The palate tissues were fixed in 4% Paraformaldehyde-PBS, followed by routine procedures for embedding in paraffin. Coronal serial sections (6 μm) were mounted on poly-L-lysine-coated slides. The region of the palate examined was restricted between the 3rd ruga and the posterior boundary of 1st molar tooth organ (Saito et al., 2005). The number of BrdU-positive cells in the MEE and the total number of MEE cells were counted in every fifth section. The criteria used to identify MEE were based on the morphology of the cultured palatal shelves. If a normal process of fusion was occurring after 24 hr of culture, the MEE were easily defined as the cells in the midline and either oral or nasal triangles (Fig. 7A, page 4). If the palatal shelves were not undergoing fusion, two different medial edge margins were observed (Fig. 7B,C). The MEE counted in those palatal shelves with the Figure 7B morphology were identified as the epithelial cells in the straight-line region and these cells were counted. In cases where there was a large curvature of the medial edge, a mid point was identified and 10 cells on the either side of the midpoint counted (Fig. 7C).

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Figure 7. Schematic of MEE cell counting method to analyze BrdU incorporation. Palatal shelves had 3 configurations at E13+24 hr in organ culture. A: Contacted palatal shelf. B: Straight-shaped palatal shelf non-adherent. C: Curvature-shaped palatal shelf non-adherent. Red lines show border of MEE. Red dots show mid point.

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Statistical Analysis

The results from multiple groups were compared with ANOVA and Scheffe's multiple comparison tests. The results from two groups were compared with the Student's t-test. Values of P < 0.05 were considered significant. The data are presented as mean ± standard deviation (SD).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Dr. Akira Yamane, Turumi University, for statistical analysis. We thank Dr. Ryoichi Hosokawa for discussions. We also acknowledge Drs. Yoji Murayama, Shogo Takashiba, and Fumio Myokai, Okayama University, for their advice and kindness.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES