Robert M. Greene

An animal model of cigarette smoke-induced in utero growth retardation

Maternal/fetal genetic constitution and environmental factors are vital to delivery of a healthy baby. In the United States (US), a low birth weight (LBW) baby is born every minute and a half. LBW, defined as weighing less than 5.5 lbs at birth, affects nearly 1 in 12 infants born in the US with resultant costs for the nation of more than 15 billion dollars annually. Infant birth weight is the single most important factor affecting neonatal mortality. Various environmental and genetic risk factors for LBW have been identified. Several risks are preventable, such as cigarette smoking during pregnancy. Over one million babies are exposed prenatally to cigarette smoke accounting for over 20% of the LBW incidence in the US. Cigarette smoke exposure in utero results in a variety of adverse developmental outcomes with intrauterine growth restriction and infant LBW being the most well documented. However, the mechanisms underlying the causes of LBW remain poorly understood. The purpose of this study was: (1) to establish an animal model of cigarette smoke-induced in utero growth retardation and LBW using physiologically relevant inhalation exposure conditions which simulate active and passive tobacco smoke exposures, and (2) to determine whether particular stages of development are more susceptible than others to the adverse effects of in utero smoke exposure on embryo/fetal growth. Pregnant C57BL/6J mice were exposed to cigarette smoke during three periods of gestation: pre-/peri-implantation (gestational days [gds] 1-5), post-implantation (gds 6-18), and throughout gestation (gds 1-17). Reproductive and fetal outcomes were assessed on gd 18.5. Exposure of dams to mainstream/sidestream cigarette smoke, simulating active maternal smoking, resulted in decreases in fetal weight and crown-rump length when exposed throughout gestation (gds 1-17). Similar results were seen when dams were exposed only during the first 5 days of gestation (pre-/peri-implantation period gds 1-5). Exposure of dams from the post-implantation period through gestation (gds 6-18) did not result in reduced fetal weight, although a significant reduction in crown-rump length remained evident. Interestingly, maternal sidestream smoke exposure, simulating exposure to environmental tobacco smoke (ETS), during the pre-/peri-implantation period of development also produced significant decreases in fetal weight and crown-rump length. Collectively, results from the present study confirm an association between prenatal exposure to either active or passive cigarette smoke and in utero growth retardation. The data also identify a period of susceptibility to in utero cigarette smoke exposure-induced growth retardation and LBW during pre-/peri-implantation embryonic development.

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two‐hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par‐3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S‐transferase) pull‐down assays, Erbin, Par‐3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full‐length Smad 3 protein. Failure of Erbin, Par‐3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par‐3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor β (TGFβ) has been suggested to alter cell polarity through a Smad‐independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFβ could alter cell polarity via a Smad‐dependent signaling mechanism.

Palate morphogenesis: current understanding and future directions.

In the past, most scientists conducted their inquiries of nature via inductivism, the patient accumulation of pieces of information in the pious hope that the sum of the parts would clarify the whole. Increasingly, modern biology employs the tools of bioinformatics and systems biology in attempts to reveal the big picture. Most successful laboratories engaged in the pursuit of the secrets of embryonic development, particularly those whose research focus is craniofacial development, pursue a middle road where research efforts embrace, rather than abandon, what some have called the pedestrian qualities of inductivism, while increasingly employing modern data mining technologies. The secondary palate has provided an excellent paradigm that has enabled examination of a wide variety of developmental processes. Examination of cellular signal transduction, as it directs embryogenesis, has proven exceptionally revealing with regard to clarification of the facts of palatal ontogeny-at least the facts as we currently understand them. Herein, we review the most basic fundamentals of orofacial embryology and discuss how functioning of TGFbeta, BMP, Shh, and Wnt signal transduction pathways contributes to palatal morphogenesis. Our current understanding of palate medial edge epithelial differentiation is also examined. We conclude with a discussion of how the rapidly expanding field of epigenetics, particularly regulation of gene expression by miRNAs and DNA methylation, is critical to control of cell and tissue differentiation, and how examination of these epigenetic processes has already begun to provide a better understanding of, and greater appreciation for, the complexities of palatal morphogenesis.

Cross‐talk between the TGFβ and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells

The transforming growth factor beta (TGFβ) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFβ mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of β‐catenin. Previous studies have demonstrated several points of cross‐talk between the TGFβ and Wnt signaling pathways. In yeast two‐hybrid and GST‐pull down assays, Dishevelled‐1 and Smad 3 have been shown to physically interact through the C‐terminal one‐half of Dishevelled‐1 and the MH2 domain of Smad 3. The current study demonstrates that co‐treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt‐3a and TGFβ leads to enhanced reporter activity from TOPflash, a Wnt‐responsive reporter plasmid. Transcriptional cooperation between TGFβ and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFβ‐responsive reporter plasmid (p3TP‐lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFβ while overexpression of dominant‐negative Smads 2 and 3 inhibited this effect. Co‐stimulation with TGFβ led to greater nuclear translocation of β‐catenin, providing explanation for the effect of TGFβ on Wnt‐3a reporter activity. Wnt‐3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFβ. In addition, Wnt‐3a and TGFβ in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFβ and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Stimulation of palatal glycosaminoglycan synthesis by cyclic AMP

Abstract Intracellular levels of cyclic AMP in primary cultures of mouse embryo palate mesenchyme cells were elevated by exogenous administration of dibutyryl cAMP, 8Br-cAMP, prostaglandin E2 or prostacyclin. Glycosaminoglycan synthesis was stimulated in a dose-dependent manner. Qualitative analysis by DEAE anion exchange chromatography and sensitivity to hyaluronidase digestion indicated preferential stimulation of hyaluronic acid synthesis. Cyclic AMP may thus play a role in regulating the synthesis of palatal glycosaminoglycans known to be requisite for normal development of the palate.

Intracellular dynamics of Smad-mediated TGFβ signaling

The transforming growth factor‐β (TGFβ) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFβ on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFβ action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFβ. In the current study, elements of the Smad component of the TGFβ intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFβ1, TGFβ2, and TGFβ3 was demonstrated. TGFβ‐initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFβ‐inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad‐mediated TGFβ signaling system in cells of the developing murine palate. J. Cell. Physiol. 197: 261–271, 2003.

Alcohol modulates expression of DNA methyltranferases and methyl CpG-/CpG domain-binding proteins in murine embryonic fibroblasts.

Fetal alcohol syndrome (FAS), presenting with a constellation of neuro-/psychological, craniofacial and cardiac abnormalities, occurs frequently in offspring of women who consume alcohol during pregnancy, with a prevalence of 1-3 per 1000 livebirths. The present study was designed to test the hypothesis that alcohol alters global DNA methylation, and modulates expression of the DNA methyltransferases (DNMTs) and various methyl CpG-binding proteins. Murine embryonic fibroblasts (MEFs), utilized as an in vitro embryonic model system, demonstrated ∼5% reduction in global DNA methylation following exposure to 200mM ethanol. In addition, ethanol induced degradation of DNA methyltransferases (DNMT-1, DNMT-3a, and DNMT-3b), as well as the methyl CpG-binding proteins (MeCP-2, MBD-2 and MBD-3), in MEF cells by the proteasomal pathway. Such degradation could be completely rescued by pretreatment of MEF cells with the proteasomal inhibitor, MG-132. These data support a potential epigenetic molecular mechanism underlying the pathogenesis of FAS during mammalian development.

Developmental MicroRNA Expression Profiling of Murine Embryonic Orofacial Tissue

BACKGROUNDnOrofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs involved in gene silencing, represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development.nnnMETHODSnTo identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore microarrays from Miltenyi Biotech. Quantitative real-time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis.nnnRESULTSnExpression of over 26% of the 588 murine miRNA genes examined was detected in murine orofacial tissues from GD-12-GD-14. Among these expressed genes, several clusters were seen to be developmentally regulated. Differential expression of miRNAs within such clusters wereshown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development.nnnCONCLUSIONSnUsing miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs.

Prenatal exposure to environmental tobacco smoke alters gene expression in the developing murine hippocampus

BACKGROUNDnLittle is known about the effects of passive smoke exposures on the developing brain.nnnOBJECTIVEnThe purpose of the current study was to identify changes in gene expression in the murine hippocampus as a consequence of in utero exposure to sidestream cigarette smoke (an experimental equivalent of environmental tobacco smoke (ETS)) at exposure levels that do not result in fetal growth inhibition.nnnMETHODSnA whole body smoke inhalation exposure system was utilized to deliver ETS to pregnant C57BL/6J mice for 6 h/day from gestational days 6-17 (gd 6-17) [for microarray] or gd 6-18.5 [for fetal phenotyping].nnnRESULTSnThere were no significant effects of ETS exposure on fetal phenotype. However, 61 expressed genes in the gd 18.5 fetal hippocampus were differentially regulated (up- or down-regulated by 1.5-fold or greater) by maternal exposure to ETS. Of these 61 genes, 25 genes were upregulated while 36 genes were down-regulated. A systems biology approach, including computational methodologies, identified cellular response pathways, and biological themes, underlying altered fetal programming of the embryonic hippocampus by in utero cigarette smoke exposure.nnnCONCLUSIONSnResults from the present study suggest that even in the absence of effects on fetal growth, prenatal smoke exposure can alter gene expression during the early period of hippocampal growth and may result in abnormal hippocampal morphology, connectivity, and function.

MSX-1 GENE EXPRESSION AND REGULATION IN EMBRYONIC PALATAL TISSUE

SummaryThe palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.