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. Some of these appear to function as 'miRNA sponges', thereby diminishing the availability of these miRNAs to inhibit the expression of target genes. Such ncRNAs are also termed competitive endogenous RNAs (ceRNAs). Here, we examine emerging data that shed light on how lncRNAs and circRNAs may alter miRNA regulation, thus affecting orofacial development and potentially contributing to orofacial clefting.

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. METHODS: We previously demonstrated the expression of several miRNAs during the critical stages of neurulation (gestational days (GD) 8.5, 9.0, and 9.5) employing high-sensitivity, high-coverage microarrays. In the present study, bioinformatic analyses were used to identify miRNAs differentially expressed (DE) in the embryonic NT that target messenger RNAs (mRNAs) associated with the bone morphogenetic protein (BMP) signaling pathway. RNAs extracted from the developing NT were hybridized to both miRNA and mRNA arrays to evaluate miRNA-mRNA interactions. RESULTS: Bioinformatic analysis identified several DE miRNAs that targeted mRNAs encoding members of (and proteins associated with) the BMP signaling pathway - a signaling cascade central to normal NT development. CONCLUSION: Identification of the miRNAs and their mRNA targets associated with BMP signaling facilitates a better understanding of the crucial epigenetic mechanisms underlying normal NT development as well as the pathogenesis of NT defects. The current study supports the notion that miRNAs function as key regulators of neural tube morphogenesis via modulation of the BMP signaling cascade. Altered expression of these miRNAs during neurulation may therefore result in NT defects.

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. METHODS: Spatiotemporal and differential expression of miR-22 (mmu-miR-22-3p) within the developing secondary palate was determined by in situ hybridization and quantitative real-time PCR, respectively. Bioinformatic approaches were used to predict potential mRNA targets of miR-22 and analyze their association with cellular functions indispensable for normal orofacial ontogeny. An in vitro palate organ culture system was used to assess the role of miR-22 in secondary palate development. RESULTS: There was a progressive increase in miR-22 expression from GD12.5 to GD14.5 in palatal processes. On GD12.5 and GD13.5, miR-22 was expressed in the future oral, nasal, and medial edge epithelia. On GD14.5, miR-22 expression was observed in the residual midline epithelial seam (MES), the nasal epithelium and the mesenchyme, but not in the oral epithelium. Inhibition of miR-22 activity in palate organ cultures resulted in failure of MES removal. Bioinformatic analyses revealed potential mRNA targets of miR-22 that may play significant roles in regulating apoptosis, migration, and/or convergence/extrusion, developmental processes that modulate MES removal during palatogenesis. CONCLUSIONS: Results from the current study suggest a key role for miR-22 in the removal of the MES during palatogenesis and that miR-22 may represent a potential contributor to the etiology of cleft palate.

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. Epigenetic regulators, such as microRNAs (miRNAs), play vital roles in the ontogeny of the orofacial region. Evidence for this comes from conditional knockouts of Dicer or DGCR8, genes encoding key enzymes in the miRNA biosynthetic machinery, in neural crest cells. Such knockouts result in a range of craniofacial/orofacial anomalies, including cleft palate and cleft lip. Epigenetic pathways may thus represent key vehicles in the regulation, and misregulation, of gene expression during normal and abnormal orofacial embryogenesis. Significant strides have been made in the last decade in identifying miRNAs and their target genes involved in lip and palate morphogenesis. Such morphogenetic processes include apoptosis, cell proliferation, cell differentiation, and epithelial-mesenchymal transition (EMT). While some of the miRNA-target gene interactions have been functionally validated, many exhibit causal relationships that await functional confirmation. A plethora of genes associated with cleft palate/cleft lip have now been identified that provides a veritable treasure trove of information that could be harnessed to identify novel miRNA candidates for further analysis. In this review, we summarize studies identifying miRNAs involved in various aspects of lip and palate morphogenesis and whose aberrant expression may result in orofacial clefts.

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. There is, thus, a growing interest in determining whether miRNAs, particularly extracellular miRNAs, can predict, diagnose, or monitor BDs. These miRNAs, typically encapsulated in exosomes, are released by cells (including those of the fetus and placenta) into the extracellular milieu, such as blood, urine, saliva and cerebrospinal fluid, thereby enabling interaction with target cells. Exosomal miRNAs are stable, protected from degradation, and retain functionality. The observation that placental and fetal miRNAs can be detected in maternal serum, provides a strong rationale for adopting miRNAs as noninvasive prenatal biomarkers for BDs. In this mini-review, we examine the current state of research involving the use of miRNAs as prognostic and diagnostic biomarkers for BD.

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. Using computational pathway analysis, interactive biologic networks and functional relationships connecting DE miRNAs with their targeted messenger RNAs (mRNAs) were identified. Potential mRNA targets and a key signal transduction pathway governing critical cellular processes indispensable for normal mammalian neurulation were also identified. RNA preparations were also used to hybridize both miRNA arrays and mRNA arrays allowing miRNA-mRNA target analysis using data of DE miRNAs and DE mRNAs - co-expressed in the same developing NT tissue samples. Identification of these miRNA targets provides key insight into the epigenetic regulation of NT development as well as into potential mechanistic underpinning of NT defects. SIGNIFICANCE OF THE STUDY: This study underscores the premise that microRNAs are potential coordinators of normal neural tube (NT) formation, via regulation of the crucial, planar cell polarity pathway. Any alteration in their expression during neurulation would result in abnormal NT development.

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. benefits of e-cigarette use by pregnant women. While the most recent Surgeon General's Report on the "Health Consequences of Smoking" states that "the evidence is sufficient to infer that nicotine adversely affects maternal and fetal health during pregnancy, contributing to multiple adverse outcomes," it remains unclear whether use of ENDS represents a "safer alternative" to tobacco smoking during pregnancy. This is due, in part, to the lack of sufficient and conclusive evidence concerning whether or not maternal e-cigarette use adversely affects embryonic/fetal development. While several recent developmental studies have challenged the safety of nicotine inhalation via ENDS, the true risks of smoking e-cigarettes during the first trimester of pregnancy-the period of organogenesis-are largely unknown. Moreover, evidence is emerging that even nicotine-free e-cigarette aerosols may harm the developing conceptus, suggesting that components of e-cigarette liquid, including flavorings, may be developmentally toxicity. Focused human epidemiological analyses, and carefully designed animal studies are critically needed to address the question of the safety of ENDS use during pregnancy.

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. We report, for the first time, arsenate-induced alterations in the expression of a number of novel candidate genes and canonical cascades that may contribute to the pathogenesis of orofacial defects. Ingenuity Pathway and NIH-DAVID analyses revealed cellular response pathways, biological themes, and potential upstream regulators, that may underlie altered fetal programming of arsenate exposed CNCCs.

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. Results from several studies indicate that the effect of AzaD on gene expression is highly context-dependent and can differ for the same gene under different environmental settings. This may, in part, be due to the nature of the silencing mechanism(s) involved - DNA methylation, repressive histone modifications, or a combination of both. The varied effects of AzaD on such context-dependent regulation of gene expression may underlie some of the diverse responses exhibited by patients undergoing AzaD therapy. In this review, we describe the salient properties of AzaD with particular emphasis on its diverse effects on gene expression, aspects that have barely been discussed in most reviews of this interesting drug.

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. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect.

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. Several genes critical for normal palatogenesis were found to be upregulated in 1-BA, 12h after AzaD exposure. MethylCap-Seq (MCS) analysis identified several differentially methylated regions (DMRs) in DNA extracted from AzaD-exposed 1-BAs. Hypomethylated DMRs did not correlate with the upregulation of genes in AzaD-exposed 1-BAs. However, most DMRs were associated with endogenous retroviral elements. Expression analyses suggested that interferon signaling was activated in AzaD-exposed 1-BAs. Our data, thus, suggest that a 12-h in utero AzaD exposure demethylates and activates endogenous retroviral elements in the 1-BA, thereby triggering an interferon-mediated response. This may result in the dysregulation of key signaling pathways during palatogenesis, causing CP.

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. In the current study, exposure throughout development to cigarette smoke resulted in impaired hepatic carbohydrate metabolism, decreased serum glucose levels, and increased gluconeogenic regulatory enzyme abundances during the fed-state coupled to decreased expression of SIRT1 as well as increased PEPCK and PGC1α expression. Together these findings indicate inappropriately timed gluconeogenesis that may reflect impaired insulin signaling in mature offspring exposed to 'active' developmental CSE.

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. Kidney protein abundances were examined by 2D-SDS-PAGE based proteome profiling with statistical analysis by Partial Least Squares-Discriminant Analysis. Key findings of this study include a persistence of impact of developmental CSE past the original exposure period on the nucleic acid and carbohydrate metabolism networks and oxidant scavenging pathways.

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. These findings indicate developmental CSE-induced systemic glucose availability may limit both organism growth and developmental trajectory, including the capacity for learning and memory.

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. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic ß-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the 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. Primary cultures of 1-BA cells, exposed to 80µg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting.

Temporal Expression of miRNAs in Laser Capture Microdissected Palate Medial Edge Epithelium from Tgfß3(-/-) 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 TGFß3 in developing palatal MEE. The expression of 7 microRNAs was downregulated and 1 upregulated in isolated MEE from wildtype murine fetuses on gestational day (GD) 13.5 to GD14.5 (prior to and during epithelial fusion of the palatal processes, respectively). Among this group were miRNAs linked to apoptosis (miR-378) and epithelial to mesenchymal transformation (miR-200b, miR-205, and miR-93). Tgfß3(-/-) fetuses, which present with a complete and isolated cleft of the secondary palate, exhibited marked dysregulation of distinct miRNAs both in the palatal MEE and mesenchyme when compared to comparable wild-type tissue. These included, among others, miRNAs known to affect apoptosis (miR-206 and miR-186). Dysregulation of miRNAs in the mesenchyme underlying the palatal MEE of Tgfß3(-/-) fetuses is also discussed in relation to epithelial-mesenchymal transformation of the MEE. These results are the first systematic analysis of the expression of microRNAs in isolated fetal palatal epithelium and mesenchyme. Moreover, analysis of the Tgfß3 knockout mouse model has enabled identification of miRNAs with altered expression that may contribute to the cleft palate phenotype.

Deciphering TGF-ß3 function in medial edge epithelium specification and fusion during mouse secondary palate development.

BACKGROUND: Transforming growth factor-ß3 (TGF-ß3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-ß3 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 14.5, and this expression was completely abolished in Tgf-ß3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down-regulated in the medial edge epithelium during palatal fusion. Jagged2 down-regulation was regulated by TGF-ß3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf-ß3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf-ß3 null mutant palatal shelves. CONCLUSIONS: Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF-ß3 mediates palate fusion in part by down-regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF-ß3 downstream target genes in palate medial edge epithelium differentiation.

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. Hippocampus tissue collected at the time of cessation of exposure was processed for gel-based proteomic and non-targeted metabolomic profiling with partial least squares-discriminant analysis (PLS-DA) for selection of features of interest. Ingenuity pathway analysis was utilized to identify candidate molecular and metabolic pathways impacted within the hippocampus. CSE impacted glycolysis, oxidative phosphorylation, fatty acid metabolism, and neurodevelopment pathways within the developing hippocampus.

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. We previously characterized the methylome of the developing murine secondary palate focusing primarily on protein- encoding genes. We now extend this study to include methylated microRNA (miRNA) genes. A total of 42 miRNA genes were found to be stably methylated in developing murine palatal tissue. Twenty eight of these were localized within host genes. Gene methylation was confirmed by pyrosequencing of selected miRNA genes. Integration of methylated miRNA gene and expression datasets identified 62 miRNAs, 69% of which were non-expressed. For a majority of genes (83%), upstream CpG islands (CGIs) were highly methylated suggesting down-regulation of CGI-associated promoters. DAVID and IPA analyses indicated that both expressed and non-expressed miRNAs target identical signaling pathways and biological processes associated with palatogenesis. Furthermore, these analyses also identified novel signaling pathways whose roles in palatogenesis remain to be elucidated. In summary, we identify methylated miRNA genes in the developing murine secondary palate, correlate miRNA gene methylation with expression of their cognate miRNA transcripts, and identify pathways and biological processes potentially mediated by these miRNAs.