Runx1-Stat3-Tgfb3 signaling network regulating the anterior palatal development

Abstract

Runx1 deficiency results in an anteriorly specific cleft palate at the boundary between the primary and secondary palates and in the first rugae area of the secondary palate in mice. However, the cellular and molecular pathogenesis underlying such regional specificity remain unknown. In this study, Runx1 epithelial-specific deletion led to the failed disintegration of the contacting palatal epithelium and markedly downregulated Tgfb3 expression in the primary palate and nasal septum. In culture, TGFB3 protein rescued the clefting of the mutant. Furthermore, Stat3 phosphorylation was disturbed in the corresponding cleft regions in Runx1 mutants. The Stat3 function was manifested by palatal fusion defects in culture following Stat3 inhibitor treatment with significant downregulation of Tgfb3. Tgfb3 is therefore a critical target of Runx1 signaling, and this signaling axis could be mediated by Stat3 activation. Interestingly, the expression of Socs3, an inhibitor of Stat3, was specific in the primary palate and upregulated by Runx1 deficiency. Thus, the involvement of Socs3 in Runx1-Tgfb3 signaling might explain, at least in part, the anteriorly specific downregulation of Tgfb3 expression and Stat3 activity in Runx1 mutants. This is the first study to show that the novel Runx1-Stat3-Tgfb3 axis is essential in anterior palatogenesis.

Observation of the Epithelial Cell Behavior in the Nasal Septum During Primary Palate Closure in Mice

“…From our previous results, the epithelial-specific elimination of Runx1 is known to be exhibited at the anterior cleft palate ( Sarper et al, 2018a , b ). It is also known that fewer TUNEL-positive cells were detected on the unfused epithelium in Runx1 mutants ( Sarper et al, 2018b ). We did not detect a significant reduction in the area of protruded cells in the cultured nasal septum of K14-Cre/Runx1 fl/fl mice in comparison to the control group ( Figure 5 ).…”

“…The present and previous findings suggest that the induction of extrusion is critical for the fusion of facial processes and that it can be inhibited by Runx1 depletion ( Martínez-Alvarez et al, 2000 ; Takigawa and Shiota, 2004 , 2007 ; Charoenchaikorn et al, 2009 ; Hu et al, 2015 ). Importantly, our previous report showed that the excision of Runx1 in epithelial tissues takes place at the beginning of explant culture ( Sarper et al, 2018a , b , 2019 ).…”

“…In Runx1 mutant mice, the anterior cleft palate is associated with reduced apoptosis and retained periderm, at the boundary between the primary and secondary palate junction. In these mutants, the expression of Tgfb3, a critical regulator of apoptosis, was significantly disturbed and TGFB3 protein rescued the formation of the anterior cleft ( Sarper et al, 2018a , b , 2019 ). The molecular mechanism through which apoptosis is initiated remains unclear; however, the reduced induction of extrusion is associated with a reduction in the expression of Tgfb3 .…”

“…For generating K14-Cre/Runx1 fl/fl mice, K14-Cre/Runx1 fl/+ male mice were bred with Runx1 fl/fl female mice. Genotyping was performed using the genome DNA extracted from individual tail and conventional polymerase chain reaction (PCR), as previously described ( Sarper et al, 2018b ). The littermates that did not carry the K14-Cre/Runx1 fl/fl genotype were used as control animals.…”

“…Several transcription factors and/or signaling molecules (e.g., Msx1 , Bmp4 , Shh , Fgf10 , Fgf7 , and Shox2 ) are specifically expressed in the anterior regions and some mutant mice also showed anterior cleft palate. Although a different gene mutation caused anterior-specific cleft palate, it was of interest that the posterior border of the anterior cleft is localized at the 1 st rugae ( Dudas et al, 2006 ; Xu et al, 2006 ; Lane et al, 2015 ; Sarper et al, 2018b , 2019 ). However, most of previous investigations have focused on palatal fusion of bilateral secondary palate, and mechanisms of fusion in the anterior region between the primary and secondary palate is still largely elusive.…”