Role of lncRNAs and circRNAs in Orofacial Clefts.

Different modes of gene regulation, such as histone modification, transcription factor binding, DNA methylation, and microRNA (miRNA) expression, are critical for the spatiotemporal expression of genes in developing orofacial tissues. Aberrant regulation in any of these modes may contribute to orofacial defects. Noncoding RNAs (ncRNAs), such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs), have been shown to alter miRNA expression, and are thus emerging as novel contributors to gene regulation.

MicroRNA-Mediated Regulation of BMP Signaling in the Developing Neural Tube.

Background: Neural tube (NT) morphogenesis is reliant on the proper temporospatial expression of numerous genes and synchronized crosstalk between diverse signaling cascades and gene regulatory networks governing key cellular processes. MicroRNAs (miRNAs), a group of small non-coding regulatory RNAs, execute defining roles in directing key canonical pathways during embryogenesis.

Objective: In order to comprehend the mechanistic underpinnings of miRNA regulation of NT morphogenesis, we have identified in the current study various miRNAs and their target mRNAs associated with BMP signaling during critical stages of neurulation.

MicroRNAs as Biomarkers for Birth Defects.

It is estimated that 2-4% of live births will have a birth defect (BD). The availability of biomarkers for the prenatal detection of BDs will facilitate early risk assessment, prompt medical intervention and ameliorating disease severity. miRNA expression levels are often found to be altered in many diseases.

MicroRNAs as epigenetic regulators of orofacial development.

Environmental and genetic factors contribute significantly to the etiology of orofacial clefting, which is one of the most common of human congenital craniofacial malformations. Current biological thought now recognizes that epigenetics represents a fundamental contributing process in embryogenesis. Indeed, many of the mechanisms whereby environmental insults affect key pathways crucial for proper embryonic growth and development are increasingly thought to be mediated via the epigenome.

Spatiotemporal Expression and Functional Analysis of miRNA-22 in the Developing Secondary Palate.

Objective: Normal development of the embryonic orofacial region requires precise spatiotemporal coordination between numerous genes. MicroRNAs represent small, single-stranded, non-coding molecules that regulate gene expression. This study examines the role of microRNA-22 (miR-22) in murine orofacial ontogeny.

MicroRNA targeting of the non-canonical planar cell polarity pathway in the developing neural tube.

MicroRNAs (miRNAs) provide context-dependent transcriptional regulation of genes comprising signalling networks throughout the developing organism including morphogenesis of the embryonic neural tube (NT). Using a high-sensitivity, high-coverage microarray analysis platform, miRNA expression in the murine embryonic NT during the critical stages of its formation was examined. Analysis of a number of differentially expressed (DE) miRNAs enabled identification of several gene targets associated with cellular processes essential for normal NT development.

MicroRNAs as Epigenetic Targets of Cigarette Smoke During Embryonic Development.

The adverse developmental effects of exposure to Cigarette Smoke (CS) during pregnancy are documented in this paper. These include low birth weight, congenital anomalies, preterm birth, fetal mortality and morbidity. The current biological thought now recognizes that epigenetics represents a fundamental contributing process in embryogenesis, and that the environment can have a profound effect on shaping the epigenome.

Developmental toxicity of e-cigarette aerosols.

Maternal smoking during pregnancy represents a major public health concern increasing the risk for low birth weight, congenital anomalies, preterm birth, fetal mortality, and morbidity. In an effort to diminish adverse developmental effects of exposure to cigarette smoking, pregnant women, and women of reproductive age, are increasingly turning to electronic nicotine delivery systems (ENDS), such as e-cigarettes, as an alternative. Given that health risks associated with ENDS use during pregnancy are largely unknown, there is an acute need to determine risks vs.

Nucleic acid methylation and orofacial morphogenesis.

In this review, we highlight the current state of knowledge of the diverse roles nucleic acid methylation plays in the embryonic development of the orofacial region and how aberrant methylation may contribute to orofacial clefts. We also consider the role of methylation in the regulation of neural crest cell function as it pertains to orofacial ontogeny. Changes in DNA methylation, as a consequence of environmental effects, have been observed in the regulatory regions of several genes, potentially identifying new candidate genes for orofacial clefting and opening promising new avenues for further research.

Laser Capture Microdissection of Murine Embryonic Neural Crest Cells.

The purpose of this chapter is to provide a step-by-step protocol to enable performance of laser capture microdissection (LCM) on tissue sections from mammalian embryos or postnatal organism stages in order to collect pure populations of neural crest cells from which sufficient amounts of nucleic acids and/or protein can be obtained for quantitative analysis. The methods (1) define a strategy to genetically and indelibly label mammalian neural crest-derived cells with a fluorescent marker, thus enabling their isolation throughout the pre- and postnatal life span of the organism, and (2) describe subsequent isolation by LCM of the labeled neural crest cells, or their derivatives, from embryonic/postnatal tissue cryosections. Details are provided for using the Arcturus PixCellIIe Laser Capture Microdissection System (Arcturus) and CapSure LCM Caps (Thermo Fisher Scientific), to which the selected cells adhere upon laser-mediated capture.

Impact of prenatal arsenate exposure on gene expression in a pure population of migratory cranial neural crest cells.

Prenatal exposure to arsenic, a naturally occurring toxic element, causes neural tube defects (NTDs) and, in animal models, orofacial anomalies. Since aberrant development or migration of cranial neural crest cells (CNCCs) can also cause similar anomalies within developing embryos, we examined the effects of in utero exposure to sodium arsenate on gene expression patterns in pure populations of CNCCs, isolated by fluorescence activated cell sorting (FACS), from Cre/LoxP reporter mice. Changes in gene expression were analyzed using Affymetrix GeneChip microarrays and expression of selected genes was verified by TaqMan quantitative real-time PCR.

Spatio-Temporal Expression and Functional Analysis of miR-206 in Developing Orofacial Tissue.

Background: Development of the mammalian palate is dependent on precise, spatiotemporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs, function as crucial modulators of cell and tissue differentiation, regulating expression of key downstream genes.

Observations: Our laboratory has previously identified several developmentally regulated miRNAs, including miR-206, during critical stages of palatal morphogenesis.

Effects of 5-Aza-2'-deoxycytidine (decitabine) on gene expression.

5-Aza-2'-deoxycytidine (AzaD), also known as Decitabine, is a deoxycytidine analog that is typically used to activate methylated and silenced genes by promoter demethylation. However, a survey of the scientific literature indicates that promoter demethylation may not be the only (or, indeed, the major) mechanism by which AzaD affects gene expression. Regulation of gene expression by AzaD can occur in several ways, including some that are independent of DNA demethylation.

Determinants of orofacial clefting II: Effects of 5-Aza-2'-deoxycytidine on gene methylation during development of the first branchial arch.

Defects in development of the secondary palate, which arise from the embryonic first branchial arch (1-BA), can cause cleft palate (CP). Administration of 5-Aza-2'-deoxycytidine (AzaD), a demethylating agent, to pregnant mice on gestational day 9.5 resulted in complete penetrance of CP in fetuses.

Determinants of orofacial clefting I: Effects of 5-Aza-2'-deoxycytidine on cellular processes and gene expression during development of the first branchial arch.

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses.

Developmental cigarette smoke exposure II: Hepatic proteome profiles in 6 month old adult offspring.

Utilizing a mouse model of 'active' developmental cigarette smoke exposure (CSE) [gestational day (GD) 1 through postnatal day (PD) 21] characterized by offspring low birth weight, the impact of developmental CSE on liver proteome profiles of adult offspring at 6 months of age was determined. Liver tissue was collected from Sham- and CSE-offspring for 2D-SDS-PAGE based proteome analysis with Partial Least Squares-Discriminant Analysis (PLS-DA). A similar study conducted at the cessation of exposure to cigarette smoke documented decreased gluconeogenesis coupled to oxidative stress in weanling offspring.

Developmental cigarette smoke exposure II: Hippocampus proteome and metabolome profiles in adult offspring.

Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. Utilizing a murine experimental model of "active" inhalation exposure to cigarette smoke spanning the entirety of gestation and through human third trimester equivalent hippocampal development [gestation day 1 (GD1) through postnatal day 21 (PD21)], we examined hippocampus proteome and metabolome alterations present at a time during which developmental cigarette smoke exposure (CSE)-induced behavioral and cognitive impairments are evident in adult animals from this model system. At six month of age, carbohydrate metabolism and lipid content in the hippocampus of adult offspring remained impacted by prior exposure to cigarette smoke during the critical period of hippocampal ontogenesis indicating limited glycolysis.

Developmental cigarette smoke exposure II: Kidney proteome profile alterations in 6 month old adult offspring.

Cigarette smoke exposure (CSE) during gestation and early development suppresses the growth trajectory in offspring. In prior studies utilizing a mouse model of 'active' developmental CSE (GD1-PD21), low birth weight induced by CSE persisted throughout the neonatal period and was present at the cessation of exposure at weaning with proportionally smaller kidney mass that was accompanied by impairment of carbohydrate metabolism. In the present study, littermates of those characterized in the prior study were maintained until 6 months of age at which time the impact of developmental CSE on the abundance of proteins associated with cellular metabolism in the kidney was examined.

Inhibition of p300 histone acetyltransferase activity in palate mesenchyme cells attenuates Wnt signaling via aberrant E-cadherin expression.

p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate.

Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl CpG-/CpG domain-binding proteins in embryonic orofacial cells.

Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins.

Temporal Expression of miRNAs in Laser Capture Microdissected Palate Medial Edge Epithelium from Tgfβ3(-/-) Mouse Fetuses.

Clefting of the secondary palate is the most common birth defect in humans. Midline fusion of the bilateral palatal processes is thought to involve apoptosis, epithelial to mesenchymal transition, and cell migration of the medial edge epithelium (MEE), the specialized cells of the palate that mediate fusion of the palatal processes during fetal development. Data presented in this manuscript are the result of analyses designed to identify microRNAs that are expressed and regulated by TGFβ3 in developing palatal MEE.

Methylated microRNA genes of the developing murine palate.

Environmental factors contribute to the etiology of cleft palate (CP). Environmental factors can also affect gene expression via alterations in DNA methylation suggesting a possible mechanism for the induction of CP. Identification of genes methylated during development of the secondary palate provides the basis for examination of the means by which environmental factors may adversely influence palatal ontogeny.

Deciphering TGF-β3 function in medial edge epithelium specification and fusion during mouse secondary palate development.

Background: Transforming growth factor-β3 (TGF-β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-β3 functions during secondary palate fusion are still poorly understood.

Results: We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.

MicroRNA expression profiling of the developing murine upper lip.

Clefts of the lip and palate are thought to be caused by genetic and environmental insults but the role of epigenetic mechanisms underlying this common birth defect are unknown. We analyzed the expression of over 600 microRNAs in the murine medial nasal and maxillary processes isolated on GD10.0-GD11.

Developmental cigarette smoke exposure: hippocampus proteome and metabolome profiles in low birth weight pups.

Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. However, brain region specific biomolecular alterations induced by developmental cigarette smoke exposure (CSE) remain largely unexplored. In the current molecular phenotyping study, a mouse model of 'active' developmental CSE (serum cotinine > 50 ng/mL) spanning pre-implantation through third trimester-equivalent brain development (gestational day (GD) 1 through postnatal day (PD) 21) was utilized.