SMAD2 overexpression rescues the TGF-β3 null mutant mice cleft palate by increased apoptosis
Introduction
The fetal development of the secondary palate is a tightly regulated process in mammals (Li et al., 2017; Ferguson, 1988). This process requires precise interaction between several cells and tissues (Nakajima, F. Shuler, Gulka & Hanai, 2018; Shuler et al., 1991). Temporal and spatial specialization of extracellular matrix (Nakajima, F. Shuler, Gulka & Hanai, 2018; Brinkley and Morris-Wiman, 1984), mesenchymal cells (Ferguson, 1988), and epithelial cells (Ferguson, 1988) are necessary in order to achieve palatal fusion prior to birth. Failure of this process results in cleft palate.
In a mouse model, palatal development begins with palatal shelves growing from the inner aspect of each maxillary process at embryonic day E11.5. Initially at day E13.5, the shelves protrude vertically along either side of the tongue. As the tongue begins to grow and form, the shelves reorient horizontally above the tongue at day E14.0. The shelves make contact in the midline at E14.5, initiating fusion and forming the temporary midline epithelial seam (MES) (Bush and Jiang, 2012). Histologically, the palatal shelves are composed of mesenchyme which is derived from the neural crest (Li et al., 2017; Bush and Jiang, 2012; Ferguson, 1988; Shuler, 1995). The mesenchyme is surrounded by a layer of oral epithelial cells, derived from the ectoderm. The MES must eventually be eliminated in order to permit mesenchymal confluence of the palatal shelves to complete fusion.
Recent studies have identified possible molecular and cellular mechanisms involved in the critical process of MES disappearance, some aspects remain controversial (Nakajima, F. Shuler, Gulka & Hanai, 2018; Gritli-Linde, 2007). Three different theories of processes regulating the fate of MES have been proposed; 1) apoptosis (DeAngelis and Nalbandian, 1968; Farbman, 1968; Hayward, 1969; Shapiro and Sweney, 1969; Cuervo and Covarrubias, 2004; Vaziri Sani et al., 2005; Dudas et al., 2006; Dudas et al., 2007; Xu et al., 2006); 2) epithelial mesenchymal transdifferentiation (EMT) (Fitchett and Hay, 1989; Griffith and Hay, 1992; Shuler et al., 1991, 1992; Kaartinen et al., 1997; Nawshad and Hay, 2003; Takigawa and Shiota, 2004; Kang and Svoboda, 2005; Jin and Ding, 2006); 3) cell migration (Carette and Ferguson (1992). Recent studies have shown that both apoptosis and EMT may be involved in the disappearance of the MES (Nawshad, 2008; Martinez-Alvarez et al., 2000; Ahmed et al., 2007).
Transforming growth factor beta 3 (TGF-β3) is a cytokine involved in many cellular functions including cell cycle control, cell development, differentiation, hematopoiesis and apoptosis (Zhang et al., 2016; Schuster and Krieglstein, 2002). TGF-β3 is involved in the repression of growth of epithelial cells and is expressed in the MES (Schuster and Krieglstein, 2002; Fitzpatrick et al., 1990). TGF-β3 null mutant mice have a phenotype that includes a cleft secondary palate due to the MES remaining intact during development (Kaartinen et al., 1995; Proetzel et al., 1995). TGF-β3 utilizes SMAD proteins (SMAD2/SMAD3) to transmit regulatory signals inside target cells (Schuster and Krieglstein, 2002). SMAD2 and SMAD3 are extremely homologous sharing nearly 95% of their amino acid sequence but they differ in function (Liu et al., 2016; Shiomi et al., 2006). The concept that SMAD3 null mutant mice develop normally while SMAD2 null mutant mice embryos are non-viable indicate the important role that SMAD2 plays during embryonic development (Brown et al., 2007). SMAD2 and SMAD3 are both expressed in the medial edge epithelium (MEE) but during the process of palatal fusion but only SMAD2 is phosphorylated (Cui et al., 2003). The inhibition and inactivation of SMAD signaling and specifically SMAD2 during palatal fusion by all-trans retinoic acid (atRA) resulted in maintenance of MEE and failure of palatal fusion (Wang et al., 2011). The inhibition of SMAD2 in MEE cells by SMAD2 siRNA maintained the proliferation of MEE and prevented the fusion of the palatal shelves (Shiomi et al., 2006). Furthermore, the addition of exogenous TGF-β3 to the siRNA treated MEE cells failed to rescue the palatal fusion indicating the critical role of SMAD2 signaling pathway during palatal fusion (Shiomi et al., 2006). Interestingly, SMAD2 overexpression in TGF-β3 null mutant mice has been found to rescue palatal fusion (Cui et al., 2005). SMAD2 overexpression has also been found to increase the apoptosis rate in junctional epithelium and downregulates Bcl-2 (Fujita et al., 2012), in prostate epithelial cells (Yang et al., 2009), human gingival epithelial cells (Yoshimoto et al., 2015), and human ocular lens epithelial cells (Lee et al., 2002).
We hypothesized that: SMAD2 overexpression rescues the TGF-β3 null mutant mice by increased apoptosis of MEE. The aim of this study was to investigate the TGF-β3 and SMAD2 regulated mechanism of MES disappearance in a rescue mouse model [K14-SMAD2/TGF-β3(−/−)] compared to wild type mice, null mutant [TGF-β3(−/−)] and SMAD2 overexpression [K14-SMAD2/TGF-β3(+/-)].
Section snippets
Increased MEE cell apoptotic rate
Cleaved caspase3 (Ccaspase3), an apoptosis marker, was used to detect apoptotic cells associated with SMAD2 overexpression in the palatal MEE cells. Ccaspase-3 protein was localized in the palatal MEE cells, in the anterior, middle and posterior regions of the secondary palate at day E14.5 (Fig. 1). Our results showed that Ccaspase-3 positive cells were detected in the nasal and oral MES triangles. The K14-SMAD2/TGF-β3(+/-) mice also had a much higher ratio of Ccaspase3 positive MEE (16.14%
Discussion
TGF-β3 has been known to play a role in the fate of MEE cells during palatal fusion (Kaartinen et al., 1995). TGF-β3 null mutant mice are born with a clefting of the secondary palate due to failure in MEE adhesion (Taya, O'Kane & Ferguson, 1999; Kaartinen et al., 1995). SMAD2 over expression in TGF-β3 null mutant mice rescued the cleft palatal fusion and mesenchymal confluence was established (Cui et al., 2005). Little is known about the precise MEE fate induced by SMAD2 overexpression in this
Conclusion
The present study has focused on the effect of SMAD2 over expression on medial edge epithelial cells during palatal fusion. The mechanism of MEE disappearance during palatal fusion was examined. This study is the first to have studied specifically the fate of MEE during palatal fusion in the K14-SMAD2/TGF-β3(−/−) mouse model. The available data indicate that the SMAD2 over expression rescues the palatal fusion in the TGF-β3(−/−) mice by increased MEE apoptosis. The same effect was seen in the
Animal breeding and genotyping
Methods within this study followed the guidelines of the Animal Care Committee of the University of British Columbia. To identify the K14-SMAD2 transgene a polymerase chain reaction (PCR) primer set was used to detect the cytokeratin 14 promoter region. To detect the TGF-β3 knockout gene another PCR primer set was used to detect the mutated allele (discussed in detail in section 3.2). Mice heterozygous with respect to the TGF-β3(+/−) gene were mated to produce TGF-β3(−/−) null embryos, as well
Acknowledgements
The research was supported by funding from the UBC Faculty of Dentistry research support mechanism. The authors wish to declare no potential conflicts of interests with respect to the authorship and publication of this article.
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