Ras signaling and its effector RREB1 are required for the dissociation of MEE cells in palatogenesis

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has largely remained unelucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for the proper MEE removal. Ras Responsive Element Binding Protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental EMT, in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Heme Oxygenase Protects against Placental Vascular Inflammation and Abortion by the Alarmin Heme in Mice

Both infectious as non-infectious inflammation can cause placental dysfunction and pregnancy complications. During the first trimester of human gestation, when palatogenesis takes place, intrauterine hematoma and hemorrhage are common phenomena, causing the release of large amounts of heme, a well-known alarmin. We postulated that exposure of pregnant mice to heme during palatogenesis would initiate oxidative and inflammatory stress, leading to pathological pregnancy, increasing the incidence of palatal clefting and abortion. Both heme oxygenase isoforms (HO-1 and HO-2) break down heme, thereby generating anti-oxidative and -inflammatory products. HO may thus counteract these heme-induced injurious stresses. To test this hypothesis, we administered heme to pregnant CD1 outbred mice at Day E12 by intraperitoneal injection in increasing doses: 30, 75 or 150 μmol/kg body weight (30H, 75H or 150H) in the presence or absence of HO-activity inhibitor SnMP from Day E11. Exposure to heme resulted in a dose-dependent increase in abortion. At 75H half of the fetuses where resorbed, while at 150H all fetuses were aborted. HO-activity protected against heme-induced abortion since inhibition of HO-activity aggravated heme-induced detrimental effects. The fetuses surviving heme administration demonstrated normal palatal fusion. Immunostainings at Day E16 demonstrated higher numbers of ICAM-1 positive blood vessels, macrophages and HO-1 positive cells in placenta after administration of 75H or SnMP + 30H. Summarizing, heme acts as an endogenous “alarmin” during pregnancy in a dose-dependent fashion, while HO-activity protects against heme-induced placental vascular inflammation and abortion.

Anterior Cleft Palate due to Cbfb deficiency and its rescue by folic acid

Core binding factor β (Cbfb) is a cofactor of Runx transcription factors. Among Runx transcription factors, Runx1 is a prerequisite for anterior-specific palatal fusion. However, whether Cbfb serves as a modulator or obligatory factor in Runx signaling that regulates palatogenesis is unclear. We herein report that Cbfb is essential and indispensable in anterior palatogenesis. Palatal fusion in Cbfb mutants is disturbed due to failed disintegration of the fusing epithelium specifically at the anterior portion, as is observed in Runx1 mutants. In this mutants, the Tgfb3 expression is disturbed at the corresponding area of the failed palatal fusion, where phosphorylation of Stat3 is also disturbed. TGFB3 protein rescues the palatal fusion in vitro. Strikingly, the anterior cleft palate in Cbfb mutants is further rescued by pharmaceutical application of folic acid that activates suppressed Stat3 phosphorylation and Tgfb3 expression in vitro. With these findings, we provide the first evidence that Cbfb is a prerequisite for anterior palatogenesis as an obligatory cofactor in the Runx1/Cbfb-Stat3-Tgfb3 signaling axis. Furthermore, the rescue of the mutant cleft palate using folic acid may elucidate potential therapeutic targets by Stat3 modification for the prevention and pharmaceutical intervention of cleft palate.Summary StatementEpithelial deletion of Cbfb results in an anterior cleft palate with impaired fusion of the palatal process and folic acid application rescues the mutant phenotype with Stat3 activation in vitro.

TGF-β Signaling and the Epithelial-Mesenchymal Transition during Palatal Fusion

Signaling by transforming growth factor (TGF)-β plays an important role in development, including in palatogenesis. The dynamic morphological process of palatal fusion occurs to achieve separation of the nasal and oral cavities. Critically and specifically important in palatal fusion are the medial edge epithelial (MEE) cells, which are initially present at the palatal midline seam and over the course of the palate fusion process are lost from the seam, due to cell migration, epithelial-mesenchymal transition (EMT), and/or programed cell death. In order to define the role of TGF-β signaling during this process, several approaches have been utilized, including a small interfering RNA (siRNA) strategy targeting TGF-β receptors in an organ culture context, the use of genetically engineered mice, such as Wnt1-cre/R26R double transgenic mice, and a cell fate tracing through utilization of cell lineage markers. These approaches have permitted investigators to distinguish some specific traits of well-defined cell populations throughout the palatogenic events. In this paper, we summarize the current understanding on the role of TGF-β signaling, and specifically its association with MEE cell fate during palatal fusion. TGF-β is highly regulated both temporally and spatially, with TGF-β3 and Smad2 being the preferentially expressed signaling molecules in the critical cells of the fusion processes. Interestingly, the accessory receptor, TGF-β type 3 receptor, is also critical for palatal fusion, with evidence for its significance provided by Cre-lox systems and siRNA approaches. This suggests the high demand of ligand for this fine-tuned signaling process. We discuss the new insights in the fate of MEE cells in the midline epithelial seam (MES) during the palate fusion process, with a particular focus on the role of TGF-β signaling.