Functional role of TGF-β receptors during palatal fusion in vitro

Abstract

Objective Reported expression patterns for TGF-β receptors (TβR-I, -II, and -III) during palatogenesis suggest that they play essential roles in the mechanisms leading to palatal fusion. The purpose of this study was to compare the functions of the three TβRs during palatal fusion. Methods Using organ culture of mouse palatal shelves, expression levels of TβR-I, -II, and -III were suppressed by transfecting the siRNAs siTβR-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signaling via each TβR. Linkage between TGF-β signaling and critical events in palatal fusion led to the use of, MMP-13 expression as an outcome measure for the function of the TGF-β receptors. Results The siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siTβR-I or -II, palatal shelves at E13 + 72 h were not fused, with complete clefting in the anterior and posterior regions. The middle palatal region following treatment with either siTβR-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siTβR-III resulted in a persistent midline seam of medial edge epithelium (MEE) in the anterior region with islands of persistent MEE in the middle and posterior regions of the midline. Treatment with all three siTβRs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siTβR-I or -II, but not -III, showed altered MMP-13 expression levels. Conclusion The ability to identify and recover MEE and palatal mesenchymal cells during palatal fusion will aid in the evaluation of the different mechanistic events regulated by each TβR during palatogenesis.

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

“…In contrast with TβR1 and TβR2, TβR3 is strongly expressed in the MEE only during the palatal fusion stage [ 32 , 33 , 35 ] ( Figure 5 A(c)). TβR3 mutations in mice manifest in lethal proliferative defects in heart tissue and apoptosis in liver tissue at E13.5, indicating that TβR3 is required for somatic development in mice [ 86 ].…”

“…Among these, TGF-β3 is strongly expressed in the medial edge epithelium (MEE) ( Figure 1 a,b) [ 21 ]. All three TGF-β receptor isoforms are expressed in the MEE; two are serine/threonine receptor kinases, type I receptor (TβR1) and type II receptor (TβR2), and the third is an accessory receptor TGF-β type III receptor (TβR3, βglycan) [ 20 , 21 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. Recently, TGF-β signaling has been shown to be regulated by both Smad-dependent and non-Smad dependent pathways ( Figure 2 ).…”

Nicotine Exposure During Pregnancy Results in Persistent Midline Epithelial Seam With Improper Palatal Fusion

“…37 The height and width of the palates in offspring of nicotine-treated dams were found to be significantly decreased ( Figure 1C). The observed smaller palate size of nicotine-treated fetuses might simply be a reflection of the smaller size of the pups.…”

“…Newborn pups were also examined visually under the dissecting microscope for existence of any OFC and palatal fusion phenotypes were classified from type A to F as described in Supplementary Data (S2). 37 Following the sacrifice of all newborn pups, palatal shelves were collected using a dissecting microscope, and analyzed in three sections. The middle one-third of the palate underwent fixation in 10% formalin and was prepared for hematoxyline and eosin staining using a Tissue Tek VIP processor (GMI Inc, Ramsey, MN).…”

Activation of Notch1 inhibits medial edge epithelium apoptosis in all-trans retinoic acid-induced cleft palate in mice

“…H&E staining was used to detect the mesenchymal confluence rate of cultured palatal shelves as previously described [13,14]. Briefly, serial sections (more than 15 sections) from the middle region of the cultured palates were randomly selected and processed for H&E staining.…”