| Background:Craniofaical malformation, is the most common congenital birth defects in humans, including cleft lip with or without cleft palate and cleft palate only, with a rate of approximately 1 in 700 births. These defects cause significant dysfunction and require medical intervention. Current research to uncover the etiology of CL/P or CPO has largely focused on possible genetic causes. Wihle more and more convincing evidence from animal models and humans studies indicates genetic and environmental factors are part of the equation.Gain-of- and loss-of-function studies have demonstrated that changes in BMP signaling during embryo development causes craniofacial malformations, including cleft palate. It remains uncertain whether BMP signaling could be targeted pharmacologically to affect craniofacial morphogenesis.Objective:The purpose of this research is to determine whether BMP signaling can be targeted pharmacologically by LDN-193189 to affect craniofacial morphogenesis and mimic the effects caused by genetic mutations: 1. We analyzed the phenotypic spectrum resulting from in utero BMP signaling inhibition by LDN-193189 in mice. 2.In the current study, we focused on the etiology of the cleft palate resulting from pharmacological inhibition of BMP signaling.Material and Methods: 1.Pregnant C57Bl/6J mice were treated with the BMP type I receptor inhibitor LDN-193189 at doses of 3,6 and 9mg/kg twice a day by intraperitoneal injection from embryonic day 10.5(E10.5) to E 15.5. E16.5. Embryos were investigated for malformations by facial measurement analysis and histology to determine the optimal concentration. Subsequent embryonic phenotype was analyzed in details using histology, masson staining, whole mount skeletal staining, Micro-CT. 2. Another 24 pregnant females at E10.5 were randomly divided into eight groups. Four groups were treated with LDN-193189 at the optimal concentration and the other four groups were treated with sterile water as a control.Embryos of treated and control groups were collected at E12.5, E13.5, E14, and E14.5 for histology,in vitro culture, and immunohistochemistry studies.We further analyzed protein expression of the BMP-mediated canonical(p-Smad1/5/8),non-canonical signaling components(p-p38,p-Erk1/2),and cell proliferation(p-H3) using immunohistochemistry.Results: 1. Considering that embryos exposed to the dose of 6mg/kg had a highest rate of visible facial defects, tolerable rate of early embryonic lethality with no obvious embryo toxicity, we chose this dose for the optimal concentration. LDN-193189 caused multiple craniofacial defects in the developing embryos, including cleft palate, micrognathia, and cleft of the lower lip. 2. No apparent difference in the shape and size of palatal shelves at E12.5 and E13.5 between vehicle- and LDN-exposed embryos was observed. However, by E14 and E14.5 palatal shelf elevation appeared delayed which was caused by reduced cell proliferation in the secondary palate and delayed palatal elevation caused by micrognathia. Analysis of signal transduction in palatal shelves at E12.5 and E13.5 identified a significant reduction of BMP/Smad signaling(p-Smad1/5/8),unchanged BMP non-canonical signaling(p-p38, p-Erk1/2) and a significant reduction of p-H3 after treatment with LDN-193189.Conclusions: 1. In the present study,we attempted for the first time to demonstrate that LDN-193189, a small molecular inhibitor of BMP type I receptor, caused multiple craniofacial defects in the developing embryos,recapitulating the phenotype of loss of BMP type I receptor(ALK2, ALK3). 2.Thus a reduced cell proliferation rate in the palate shelves and delay palate shelves elevation is a major mechanism defect that may cause cleft palate in LDN-exposed embryos. LDN-193189 can be used to manipulate BMP signaling by selectively targeting BMP/Smad signaling pathway to affect palatal morphogenesis. This work established a novel mouse model for teratogen-induced cleft palate. |
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