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The role that nutritional factors play in mammalian development has received renewed attention over the past two decades as the scientific literature has exploded with reports that folic acid supplementation in the periconceptional period can protect embryos from a number of highly significant malformations. As is often the case, the relationship between B vitamin supplementation and improved pregnancy outcomes is more complicated than initially perceived, as the interaction between nutritional factors and selected genes must be considered. In this review, we attempt to summarize the complex clinical and experimental literature on nutritional factors, their biological transport mechanisms, and interactions with genetic polymorphisms that impact early embryogenesis. While not exhaustive, our goal was to provide an overview of important gene-nutrient interactions, focusing on folic acid and vitamin B12, to serve as a framework for understanding the multiple roles they play in early embryogenesis. [ABSTRACT FROM AUTHOR]

Congenital heart defects (CHD) are the most common congenital defects worldwide, and perigestational folate supplementation (PFS) is the most effective large-scale intervention to date for reducing CHD. This review is based upon the following premises: that the majority of CHD result from disruption of development of the cardiac neural crest (CNC); and that the CNC is highly responsive to folate and homocysteine. The following roles of folate are discussed in relation to CNC development: one-carbon metabolism in support of mitosis and gene methylation; and gene regulation via direct activity of the folate receptor. The following roles of hyperhomocysteinemia are discussed in the same context: increased oxidative stress; disruption of gene methylation; homocysteinylation of key proteins; and NMDA receptor binding. It is proposed that well-focused advances in folate-CNC research could lead to development of strategies, in addition to PFS, to facilitate normal CNC and heart development, and thereby further reduce CHD., (Copyright © 2013 Wiley Periodicals, Inc.)

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high-affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site-directed short-interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long-term consequences for the development of the PAA and outflow tract.

Antagonists of the N-methyl-d-aspartate receptor (NMDAR) may disrupt the development of the cardiac neural crest (CNC) and contribute to conotruncal heart defects. To test this interaction, a loss-of-function model was generated using small interfering RNAs (siRNA) directed against the critical NR1-subunit of this receptor in avian embryos. The coding sequence of the chicken NR1 gene and predicted protein sequences were characterized and found to be homologous with other vertebrate species. Analysis of its spatiotemporal expression demonstrated its expression within the neural tube at pre-migratory CNC sites. siRNA targeted to the NR1-mRNA in pre-migratory CNC lead to a significant decrease in NR1 protein expression. However, embryo survival and heart development were not adversely affected. These results indicate that the CNC may function normally in the absence of functional NMDAR, and that NMDAR antagonists may have a complex impact upon the CNC that transcends impairment of a single receptor type., (Copyright 2009 Elsevier Inc. All rights reserved.)

Background: The reduced folate carrier (RFC1) is a ubiquitously expressed integral membrane protein that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. In this study, embryonic/fetal development is characterized in an RFC1 knockout mouse model in which pregnant dams receive different levels of folate supplementation., Methods: RFC1(+/-) males were mated to RFC1(+/-) females, and pregnant dams were treated with vehicle (control) or folic acid (25 or 50 mg/kg) by daily subcutaneous injection (0.1 mL/10 g bwt), beginning on E0.5 and continuing throughout gestation until the time of sacrifice., Results: Without maternal folate supplementation, RFC1 nullizygous embryos die shortly postimplantation. Supplementation of pregnant dams with 25 mg/kg/day folic acid prolongs survival of mutant embryos until E9.5-E10.5, but they are developmentally delayed relative to wild-type littermates, display a marked absence of erythropoiesis, severe neural tube and limb bud defects, and failure of chorioallantoic fusion. Fgfr2 protein levels are significantly reduced or absent in the extraembryonic membranes of RFC1 nullizygous embryos. Maternal folate supplementation with 50 mg/kg/day results in survival of 22% of RFC1 mutants to E18.5, but they develop with multiple malformations of the eyelids, lungs, heart, and skin., Conclusions: High doses of daily maternal folate supplementation during embryonic/fetal development are necessary for early postimplantation embryonic viability of RFC1 nullizygous embryos, and play a critical role in chorioallantoic fusion, erythropoiesis, and proper development of the neural tube, limbs, lungs, heart, and skin., ((c) 2008 Wiley-Liss, Inc.)

The amino acid homocysteine increases in the serum when there is insufficient folic acid or vitamin B(12), or with certain mutations in enzymes important in methionine metabolism. Elevated homocysteine is related to increased risk for cardiovascular and other diseases in adults and elevated maternal homocysteine increases the risk for certain congenital defects, especially those that result from abnormal development of the neural crest and neural tube. Experiments with the avian embryo model have shown that elevated homocysteine perturbs neural crest/neural tube migration in vitro and in vivo. Whereas there have been numerous studies of homocysteine-induced changes in gene expression in adult cells, there is no previous report of a homocysteine-responsive transcriptome in the embryonic neural crest. We treated neural crest cells in vitro with exogenous homocysteine in a protocol that induces significant changes in neural crest cell migration. We used microarray analysis and expression profiling to identify 65 transcripts of genes of known function that were altered by homocysteine. The largest set of effected genes (19) included those with a role in cell migration and adhesion. Other major groups were genes involved in metabolism (13); DNA/RNA interaction (11); cell proliferation/apoptosis (10); and transporter/receptor (6). Although the genes identified in this experiment were consistent with prior observations of the effect of homocysteine upon neural crest cell function, none had been identified previously as response to homocysteine in adult cells.

Although the beneficial effects of maternal folate supplementation in the periconceptional period have been shown to prevent neural tube defects, congenital heart defects and orofacial clefts, the exact protective mechanism of folates remains unknown. Folates affect DNA synthesis, amino acid metabolism and methylation of genes, proteins and lipids via S-adenosylmethionine-mediated one-carbon transfer reactions. Our laboratory has created several mouse knock out models of folate transport using gene targeting to inactivate folate receptor 1 (Folr1), folate receptor 2 (Folr2) and reduced folate carrier 1 (Slc19a1) genes. Gene ablation of both Folr1 and Slc19a1 leads to lethality, but with maternal folate supplementation, nullizygous embryos for both genes present with neural tube defects (NTDs) and congenital heart defects (CHDs). Folr1 nullizygous mice also exhibit orofacial clefts when the dams are provided with low folate supplementation during pregnancy. Finally, women with NTD-affected pregnancies have been reported to have high autoantibody titers against the folate receptor, potentially inhibiting the transport of folate to the developing embryo. This may be an explanation for some of the folate-responsive NTDs and perhaps other congenital malformations. Herein, we propose how homocysteinylation of the folate receptor may contribute to generation of these autoantibodies against the folate receptor.

It has been suggested that homocysteine-induced defects are mediated by the inhibition of the N-methyl-d-aspartate (NMDA) receptor on neural crest cells. However, the majority of this work has been performed using the chicken embryo model. In an effort to better understand the molecular events involved a murine model of homocysteine-induced defects was sought. However, it has been previously shown that homocysteine failed to induce congenital defects in several strains of mouse. Therefore, in an effort to better understand the difference in the susceptibility between these two species we investigated the ontogeny of the NMDA receptor in the mouse and chicken. To determine the expression of the NMDA receptor we performed Western blot analysis using an antibody to the NR1-subunit of the NMDA receptor in both the chicken and mouse embryos. Further, we used RT-PCR to determine the temporal expression of this subunit in the murine embryos from gestational day 8.5 to 18.5 to confirm our Western blot analysis. Results from these studies demonstrated that the expression of the NMDA receptor was expressed during the early stages of development in the chick embryo but neither the transcript nor the protein was detected in mouse embryos until later in development. These results demonstrate that during the stages of neurulation and/or early heart development the expression of the NR1-subunit of the NMDA receptor was not detected. The expression of this gene increased and was detectable by gestational days 14.5-15.5 and continued to increase in its expression until term. Therefore, these experiments suggest that homocysteine-induced defects may be mediated via the NMDA receptor.

Background: Folate deficiencies have been associated with many adverse congenital abnormalities. It is not clear, however, whether these defects are due to a folate deficiency or to an increase in homocysteine. Homocysteine has been shown to be teratogenic in the chicken-embryo model and it has been suggested that homocysteine-induced defects are mediated by inhibiting the N-methyl-D-aspartate (NMDA) receptor on neural crest cells. The majority of the teratology studies have been carried out using the chicken embryo model. In an effort to develop a murine model of homocysteine-induced neural tube defects, several inbred mouse strains were treated with homocysteine or the NMDA inhibitor MK801 and the fetuses examined for any induced-NTD., Methods: Several in-bred mouse strains were administered homocysteine once on gestational day (GD) E8.5 or once daily on GD 6.5-10.5. Additionally, because homocysteine was been reported to mediate its effects through the NMDA receptor, the effect of MK801, an antagonist of this receptor, was also investigated., Results: Regardless of the mouse treatment time, homocysteine failed to induce neural tube defects in our in-bred mouse strains. Homocysteine also failed to increase the number of neural tube defects in the splotch strain, regardless of the genotype., Conclusions: Irrespective of the mouse strain or treatment, homocysteine failed to induce neural tube defects in our mouse models, which is in contrast to what has been reported in the chicken embryo models., (Birth Defects Res (Part B) 77:89-94, 2006. (c) 2006 Wiley-Liss, Inc.)

A strong association exists between pregnancy loss and maternal elevations of the sulfur-containing amino acid, homocysteine. Because extra-embryonic vascular growth is critical to maintaining a normal pregnancy, we examined the effects of homocysteine on vessel development by exposing avian embryos to exogenous homocysteine during critical periods of vascular growth. These experiments demonstrated that homocysteine significantly reduced survival and decreased angiogenesis in the extra-embryonic vasculature. Homocysteine was also found to reduce mRNA and protein expression of vascular endothelial growth factor (VEGF), a key molecule for vascular development. Moreover, in cultured human umbilical vein endothelial cells, homocysteine increased the synthesis of nitric oxide, an important regulatory molecule for VEGF. Inhibiting the homocysteine-induced up-regulation of nitric oxide restored normal VEGF expression and vascular development. These results suggest that homocysteine may impair the development of the extra-embryonic vasculature by reducing the expression of VEGF., ((c) 2005 Wiley-Liss, Inc.)

The role that nutritional factors play in mammalian development has received renewed attention over the past two decades, as the scientific literature exploded with reports of retinoid compounds disrupting craniofacial development, and with other reports that folic acid supplementation in the periconceptional period can protect embryos from highly significant malformations. As was often the case, the situation became far more complicated, as the interaction between nutritional factors with selected genes was recognized. In this review, we attempt to summarize a complex clinical and experimental literature of nutritional factors, their biological transport mechanisms, and the impact that they have during early embryogenesis. Although not exhaustive, our goal was to provide an overview of important gene-nutrient interactions and a framework for their investigation.

Background: The mechanisms by which folic acid may contribute to reductions in risk of several congenital anomalies are unknown. The data gap includes a lack of information on possible effect modification between maternal folic acid use and other maternal exposures. We hypothesized that effects of congenital anomalies associated with maternal fever, cigarette smoking or alcohol use would be modified by intake of vitamins., Methods: We explored case-control data that showed risk reductions among infants and fetuses whose mothers consumed vitamins. Data were from California deliveries of infants and fetuses in the period 1987-1989. Maternal telephone interviews were completed for 207 (87%) conotruncal cases, 489 (85%) orofacial cleft cases, 265 (84%) neural tube defect cases, 165 (82%) limb anomaly cases, and 734 controls (nonmalformed infants)., Results: Considering women who reported vitamin use and no periconceptional fever as referents, for each anomaly group we observed elevated effects for the combinations of maternal vitamin use/fever, no use/no fever and no use/fever. Effects were most elevated for the combination of no vitamin use and fever. Adjusted for maternal body mass index, education and race/ethnicity, odds ratios were 2.4 (95% confidence inter-val = 1.0-5.9) for conotruncal defects, 2.9 (1.4-5.8) for cleft lip with or without cleft palate, 1.3 (0.4-3.9) for cleft palate, 3.1 (1.4-6.8) for neural tube defects, and 2.6 (1.0-6.4) for limb-deficiency defects. These interactions were further investigated relative to maternal use of fever-reducing medications. Effects tended to be highest among those women who did not use vitamins, had fevers, and did not use fever-reducing medications. Compared with women who used vitamins and did not smoke periconceptionally, anomaly risks tended to be highest among women who did not use vitamins and smoked. No specific pattern emerged involving alcohol intake., Conclusions: These data further suggest that the underlying mechanisms of folic acid associated with congenital anomalies may be complex.

A positive correlation between elevated maternal homocysteine (Hcys) and an increased risk of neural tube, craniofacial, and cardiac defects is well known. Studies suggest Hcys perturbs neural crest (NC) development and may involve N-methyl-D-aspartate (NMDA) receptors (Rosenquist et al., 1999). However, there is no direct evidence that Hcys alters NC cell behavior. Here, we evaluated the effect of Hcys on cardiac NC cell migratory behavior in vitro. Neural tube segments from chick embryos treated in ovo with or without Hcys were placed in culture and the migratory behavior of emigrating NC cells was monitored. Hcys significantly increased in vitro NC cell motility at all embryonic stages examined. NC cell surface area and perimeter were also increased. However, the relative distance NC cells migrated from their original starting point only increased in NC cells treated in ovo at stage 6 or at the time neural tube segments were cultured. Cysteine had no effect. NMDA mimicked Hcys' effect on NC motility and migration distance but had no effect on cell area or perimeter. The noncompetitive inhibitor of NMDA receptors, MK801+, significantly inhibited NC cell motility, reduced migration distance, and also blocked the effects of NMDA and Hcys on NC motility and migratory distance in vitro. A monoclonal antibody directed against the NMDA receptor immunostained NC cells in vitro and, in western blots, bound a single protein with the appropriate molecular weight for the NMDA receptor in NC cell lysates. These data are consistent with the hypothesis that a Hcys-sensitive NMDA-like receptor is expressed by early emigrating NC cells or their precursors, which is important in mediating their migratory behavior. Perturbation of this receptor may be related to some of the teratogenic effects observed with elevated Hcys., (Copyright 2002 Wiley-Liss, Inc.)

Exposure of the developing conceptus to selected environmental agents can lead to deleterious and often times lethal birth defects. These malformations result in serious emotional and financial consequences to families and societies worldwide. As we continue to progress technologically, we face challenges from the introduction of new pharmacological agents and chemical compounds into the environment. This results in a concomitant need to more fully understand the relationship between in utero exposure to environmental teratogens and the risk of congenital malformations. The goal of this review is to provide a current perspective of the major concepts related to the molecular basis of environmentally induced birth defects. Starting with a discussion of commonly occurring birth defects, we consider important fundamental facets of embryonic development, teratology, and gene-environment interactions. The review then summarizes our current understanding of the molecular mechanisms involved in selected birth defects following exposure to pharmacological compounds, including thalidomide, retinoids, and valproic acid. Understanding these signaling pathways may lead to the development of safer pharmaceutical compounds and a reduction in the number of infants born with preventable birth defects.

The recent identification of numerous matrix genes and gene products has allowed a detailed examination of their roles in development. Two of these extracellular matrix proteins, fibrillin-1 and fibrillin-2, are components of the elastin-associated microfibrils. Given what is known about the distribution of the fibrillins in normal tissues and the abnormalities that result when mutations occur, a basic hypothesis has emerged: fibrillin-1 is primarily responsible for load bearing and providing structural integrity, whereas fibrillin-2 may be a director of elastogenesis. Nevertheless, examination of phenotypes in disorders caused by mutations in fibrillin-1 or fibrillin-2 suggests some common functions. To better understand these similar and diverse roles, it would be helpful to examine these proteins during chick development. To accomplish this goal, it is first necessary to characterize the chick homologs of the known fibrillins. In this study, the partial chick FBN1 cDNA was identified by polymerase chain reaction-aided cloning as a first step toward elucidating these goals. Sequence analysis indicated that there is striking conservation between chick and mammalian fibrillin-1 at the DNA and protein levels. Antisense and sense riboprobes were synthesized and used in in situ hybridization in stage 14 chick embryos and high levels of FBN1 transcripts were observed in the heart.

We showed previously that the induction of neural crest (NC) and neural tube (NT) defects is a general property of N-methyl-D-aspartate receptor (NMDAR) antagonists. Since homocysteine induces NC and NT defects and can also act as an NMDAR antagonist, we hypothesized that the mechanism of homocysteine-induced developmental defects is mediated by competitive inhibition of the NMDAR by homocysteine. If this hypothesis is correct, homocysteine-induced defects will be reduced by NMDAR agonists. To test the hypothesis, we treated chicken embryos during the process of neural tube closure with sufficient homocysteine thiolactone to induce NC and NT defects in approximately 40% of survivors or with homocysteine thiolactone in combination with each of a selected set of NMDAR agonists in 0. 05-5000 nmol doses. Glutamate site agonists selected were L-glutamate and N-methyl-D-aspartate. Glycine site agonists were glycine, D-cycloserine, and aminocyclopropane-carboxylic acid. Glycine was the most effective overall, reducing defects significantly at two different doses (each P>0.001). These results support the hypothesis that homocysteine may affect NC and NT development by its ability to inhibit the NMDAR. One potentially important consequence of this putative mechanism is that homocysteine may interact synergistically with other NMDAR antagonists to enhance its effect on development.

Homocysteine at abnormally high levels is an independent risk factor for atherosclerosis and may be a key factor in atherogenesis. Since homocysteine (Hcys) has been shown to promote cell proliferation and induction of the gene transcription factor c-fos in vascular smooth muscle cells (VSMCs), effects which can be mediated by MAP kinase, we hypothesized that homocysteine activates a MAP kinase-dependent signal transduction pathway. In this study, we find that homocysteine transiently activates MAP kinase (ERK2 isoform) in cultured VSMCs from chick embryos. Homocysteine activation of ERK2 is dose-dependent with an EC50 of approximately 500 nM and blocked by the MAP/Erk kinase (MEK) inhibitor PD98059. VSMC embryonic lineage is another determinant of homocysteine sensitivity. These findings demonstrate that homocysteine activates the MAP kinase signal transduction pathway and thus support the hypothesis that homocysteine may promote atherosclerosis by stimulation of growth promoting signal transduction pathways., (Copyright 1998 Academic Press.)

N-Methyl-D-aspartate (NMDA) receptors are a calcium-conducting class of excitatory amino acid receptors that are involved in neuronal development and migration. Certain well known teratogens (e.g. homocysteine, ethanol, and chloroform) that induce congenital neural tube and neural crest defects also have the capacity to act as NMDA receptor antagonists. We hypothesized that teratogenicity was a general property of NMDA receptor antagonists, and that high affinity NMDA receptor antagonists would induce neural tube and neural crest defects. Chicken embryos were given 5, 50, or 500 nmol/d of selected NMDA receptor antagonists for 3 consecutive days during the process of neural tube closure, beginning 4 h after the beginning of incubation. Selected NMDA receptor antagonists represented three classes of antagonists: ion channel blockers, glycine site antagonists, and glutamate site agonists and antagonists. All classes of NMDA receptor antagonists induced embryonic death and congenital defects of the neural crest and neural tube; however, the channel blockers were the most potent teratogens. Dextromethorphan at 500 nmol/embryo/d killed more than half the embryos and induced congenital defects in about one-eighth of the survivors; dextromethorphan was also highly lethal at 50 nmol/embryo/d. Glutamate site NMDA receptor agonists (NMDA and homoquinolinic acid) displayed weak toxicity relative to their known NMDA receptor potency. Taken together, these data indicate that NMDA receptor antagonists, particularly channel blockers, are potent teratogens in the chicken embryo model. Because dextromethorphan is a widely used nonprescription antitussive, its strong teratogeneticity using this model is particularly noteworthy.

Hyperhomocysteinemia has been recognized as an independent risk factor for cerebral, coronary, and peripheral atherosclerosis. To examine the contribution of homocysteine (H[cys]) in the pathogenesis of vascular diseases, we sought to determine whether the H[cys] effect on vascular smooth muscle (VSMC) proliferation is mediated by a specific receptor/transporter or is due to an interaction with growth factors or cytokines. We show that H[cys] induced c-fos and c-myb and increased DNA synthesis and cell proliferation 12-fold in neural crest-derived VSMC (N-VSMC). The H[cys] effect on N-VSMC proliferation is inhibited by Mk-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, a glutamate-gated calcium ion channel receptor, and CGS 19755, a competitive antagonist of NMDA-type glutamate receptor. H[cys] stimulates the synthesis of mass amounts of sn-1,2 diacylglycerol, and activates protein kinase C translocation from the nucleus and cytoplasm to cell membranes. Furthermore, protein kinase C inhibitors block the growth effect mediated by H[cys]. These findings indicate that H[cys]-mediated responses are coupled to diacylglycerol-dependent protein kinase C activation. Our results suggest that homocysteine activates a receptor/transporter-like factor in neural crest derived smooth muscle.

Previously, we demonstrated that avian vascular smooth muscle cells (VSMC) derived from embryonic abdominal and thoracic aorta grow differently in the presence of transforming growth factor beta (TGF-beta1) and platelet-derived growth factor (PDGF-BB) (Wrenn et al., In Vitro Cell. Dev. Biol. 29, 73-78, 1992). The thoracic VSMC (N-VSMC) are derived from neural crest, and therefore differentiate from ectoderm; the abdominal VSMC (M-VSMC) are derived from mesoderm. The present study was designed to identify factors that mediate the differential responses of the VSMC to TGF-beta1. We found that TGF-beta1 increased DNA synthesis by approximately sevenfold in N-VSMC. Levels of both alpha1 (I) procollagen and c-myb mRNAs were markedly induced in N-VSMC treated with TGF-beta1. Chimeric plasmids containing up to 3.5 kb of alpha1 (I) procollagen 5' flanking DNA were induced to equivalent levels as procollagen mRNA in N-VSMC. However, TGF-beta1 increased DNA synthesis by threefold in M-VSMC; there was no effect on alpha1 (I) procollagen expression, and c-myb was not expressed, as demonstrated by immunohistochemistry staining and RNA analyses. Antisense c-myb oligodeoxynucleotides blocked the TGF-beta1 induction of alpha1 (I) procollagen and the growth of N-VSMC. The increase in DNA synthesis by M- and N-VSMC was correlated with the secretion of PDGF-AA, and staurosporine and antibodies directed against PDGF-AA suppressed DNA synthesis. Our results demonstrate that TGF-beta1 activity and c-myb expression modulate the expression of alpha1 (I) collagen and cell proliferation in neural crest-derived smooth muscle. The regulation of these events by TGF-beta1 may be important during morphogenesis of blood vessels and vascular diseases.

The biological basis or mechanism whereby folate supplementation protects against heart and neural tube defect is unknown. It has been hypothesized that the amino acid homocysteine may be the teratogenic agent, since serum homocysteine increases in folate depletion; however, this hypothesis has not been tested. In this study, avian embryos were treated directly with D,L-homocysteine or with L-homocysteine thiolactone, and a dose response was established. Of embryos treated with 50 microliters of the teratogenic dose (200 mM D,L-homocysteine or 100 mM L-homocysteine thiolactone) on incubation days 0, 1, and 2 and harvested at 53 h (stage 14), 27% showed neural tube defects. To determine the effect of the teratogenic dose on the process of heart septation, embryos were treated during incubation days 2, 3, and 4; then they were harvested at day 9 following the completion of septation. Of surviving embryos, 23% showed ventricular septal defects, and 11% showed neural tube defects. A high percentage of the day 9 embryos also showed a ventral closure defect. The teratogenic dose was shown to raise serum homocysteine to over 150 nmol/ml, compared with a normal level of about 10 nmol/ml. Folate supplementation kept the rise in serum homocysteine to approximately 45 nmol/ml, and prevented the teratogenic effect. These results support the hypothesis that homocysteine per se causes dysmorphogenesis of the heart and neural tube, as well as of the ventral wall.

Developmental studies have demonstrated that the vascular smooth muscle cells (VSMC) present within the elastic arteries are differentiated from two definitive origins, the neural crest and the mesoderm. Cells from these distinct progenitors differ in their ability to determine long-range spatial order of the extracellular matrix, in proliferative responses, and in the expression of critical proteins. The present study utilizes collagen gel contraction assays and the analysis of integrin receptor subunit expression to evaluate cell-matrix interactions. In the presence of serum and transforming growth factor-beta 1 (TGF) or TGF-beta 1 alone, VSMC isolated from the abdominal aorta (AA-VSMC) were found to contract collagen matrices to a significantly greater extent than VSMC from the thoracic aorta (TA-VSMC). However, in TA-VSMC, beta 1 integrin and gel contraction were stimulated only in the presence of serum factors. Metabolic labeling and immunoprecipitation of integrin subunits revealed that TGF-beta 1 induced beta 1 and alpha 5 integrin subunits in AA-VSMC four-and ninefold, respectively. AA-VSMC gel contraction stimulated by serum and TGF-beta 1 alone was inhibited with anti-beta 1 integrin antibody by 70 and 100%, respectively. However, the beta 1 integrin-specific antibody inhibited serum-induced TA-VSMC gel contraction by 25%. The data suggest that vascular smooth muscle cell ontogeny is an important determinant of cell function, phenotype, and response to growth factors such as TGF-beta 1.

The elastic matrix of the large arteries shows a high level of spatial order. However, the mechanisms by which such order is established and maintained are largely unknown. The embryonic development of the avian heart and great vessels provides an appropriate model to investigate these mechanisms. In control embryos, an elastic matrix with a high level of spatial order develops in the nascent great vessels. But after the normal vascular smooth muscle (VSM) progenitor cells in the great vessels are experimentally replaced by other VSM progenitor cells, the elastic extracellular matrix is congenitally disordered. The present study used this model to test the hypothesis that the proteoglycan decorin was involved in the establishment and maintenance of the normal three-dimensional spatial order of the vascular elastic matrix. The temporospatial expression of decorin was analysed during development of normal vessels and in experimental vessels with surrogate VSM. The results showed the following: (1) the expression of decorin was related in time and space to the establishment of large helical collagen type III fibers that are characteristic of the normal elastic extracellular matrix; (2) in the experimental extracellular matrix there were few helical fibers of collagen type III, but those that were present remained positive for decorin; and (3) in both control and experimental vessels, decorin associated with neither fibers of collagen type I nor fibers of collagen type III in any conformation other than the large helical fibers. These data indicate a previously unrecognized relationship between decorin and the spatial order of the physiologically significant helical fibers of collagen type III.

In the avian embryo, vascular smooth muscle cells (VSMC) in the aortic arch (elastic) arteries originate in the neural crest, whereas other VSMC develop from local mesoderm. These two lineages have been shown previously to be significantly different in the timing and expression of the smooth muscle phenotype and in their respective abilities to produce an orderly elastic matrix. Two differing kinds of VSMC also have been shown in mammals. In the experimental absence of neural crest (NC) in the avian embryo, the matrix is spatially disordered. The molecular basis of the difference between the normal NC-VSMC and the surrogate mesodermal (MDM)-VSMC has not previously been investigated. In this study the expression of vascular smooth muscle alpha-actin, tropoelastin, c-fos and c-jun were examined via immunoblotting, immunohistochemistry, Northern blot, and/or transcription run-on assays. Control avian VSMC of NC origin were compared with experimental MDM-derived VSMC that populate the cardiac outflow after surgical ablation of the NC. The results show that, when they are grown under identical conditions in vitro or freshly removed from an embryonic vessel, surrogate MDM-VSMC express about 10 times more alpha-actin and tropoelastin than the normal NC-VSMC; and MDM-VSMC express up to 15 times more c-jun, whereas c-fos was not different. These results show profound heterogeneity in the regulation of VSMC-specific genes that is based in the embryonic lineage of the cells.

Transforming growth factor-beta (TGF-beta), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-alpha exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-beta on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-beta increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-beta had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-beta potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-beta are heterogeneous and are functions of the embryonic lineage of the VSMC.

Previous studies of coronary artery ontogeny have stressed early development and therefore have dwelt mainly upon the origin of the endothelium of the nascent coronary artery stem. This study has analyzed the ontogeny of the vascular smooth muscle cells (VSMC) in the coronary arteries of the domestic chicken, by establishing the timing and deployment of smooth muscle alpha-actin (SMAA). Anti-SMAA was applied to sections of normal embryos, and to sections of experimental embryos that had undergone surgical ablation of the neural crest over somites 1-3. The results show an orderly symmetrical deployment of SMAA in control coronary arteries. SMAA was expressed significantly earlier in the coronary artery VSMC compared with those of the cardiac outflow vessels; this early expression may indicate a unique responsiveness to induction of the smooth muscle phenotype. The normal orderly development of coronary artery VSMC was dependent upon the presence of the neural crest, and therefore was disrupted in the experimental embryos whose neural crest was ablated.

The smooth muscle of the abdominal region of the chicken aorta derives from locally recruited mesenchyme (mesenchymal smooth muscle), whereas that of the thoracic region derives from the neural crest (ectomesenchymal smooth muscle). We hypothesized that this smooth muscle heterogeneity might affect important enzymatic functions of the vessel wall. Therefore, we measured angiotensin converting enzyme (ACE) activity in homogenates of chicken thoracic and abdominal aorta at different embryonic stages (days 10, 14, and 18 of gestation). ACE activity increased in both regions over the time of gestation (p less than 0.001 in both cases); the increase was steeper and ACE activity was higher in thoracic than in abdominal segments (p less than 0.001). Km values were similar (approximately 7 microM) at all times and between the two segments, whereas changes in Vmax values closely paralleled those in enzyme activity, indicating gestation-dependent increases in the amount of enzyme. Neural crest ablation at an early developmental stage resulted in an increase of ACE activity in thoracic homogenates (p less than 0.001), predictably leaving that in abdominal homogenates unaffected. Bovine pulmonary artery endothelial cell monolayers exposed to media conditioned with cultured mesenchymal or ectomesenchymal smooth muscle cells exhibited elevated ACE activity (46% and 83%, respectively, relative to control medium, with p less than 0.01 in both cases; p less than 0.05 between the two media). Increases in endothelial cell ACE activity corresponded to proportional increases in ACE protein determined by enzyme-linked immunosorbent assay (r = 0.99) and were interpreted as indicative of enhanced enzyme synthesis subsequent to exposure of endothelial cells to smooth muscle-conditioned media.(ABSTRACT TRUNCATED AT 250 WORDS)

Surgical ablation of the cardiac neural crest from the chicken embryo results in persistent truncus arteriosus (PTA) and a change in the elastic laminae of the great vessels, wherein elastin and the elastin microfibril show significant spatial disorder. The purpose of this study was to test the hypothesis that the interstitial collagens would also be disordered in the elastic laminae of chicken embryos with PTA. The birefringence characteristics of interstitial collagen were examined to evaluate spatial ordering. The results showed that collagen in the elastic laminae assumed an orderly configuration of well-defined fiber bundles in the great vessel walls of control embryos, whereas vessels from embryos with PTA lacked any distinct spatial order. Collagens type I and III were localized in the vessel walls. Type III collagen was the principal collagen of the elastic laminae, but was absent from the intima of all vessels. In the elastic laminae of vessels from control embryos, collagen type III showed well-defined fiber bundles whereas embryos with PTA had diffuse collagen type III in poorly defined laminae that were not separated by discrete layers of smooth muscle cells. Collagen type I was a minor component of the elastic laminae but formed robust pericellular fiber bundles throughout the media and intima. Collagen type I fibers appeared to be coarsened and less uniform in the vessels from embryos with PTA.

It is now well established that cells from the cardiac neural crest (CNC) are essential for normal conotruncal septation. The truncal septation complex consists of the aorticopulmonary (AP) septum and the myocardial sheath of the truncus. The principal role of the CNC cells during septation appears to be their differentiation into the elastogenic smooth muscle that forms the AP septum proper. The objective of this study was to integrate serial reconstruction and specific histochemical markers in order to provide a unified analysis of the relationships between the CNC and the other components of the truncal septation complex. The development of the septation complex was compared normal embryos vs. embryos from which the CNC had been surgically ablated. Embryos from each group were harvested after incubation periods of 4-8 days (Hamburger-Hamilton stages 23-34). Histochemical procedures were performed for positive identification of the elastic matrix and smooth muscle alpha-actin; the presence of these proteins was used as the criterion for "septal cells" and to define the boundaries of the septum. The results indicate that the shape, components, boundaries, and degree of organization of the septation complex may be different from previous descriptions. Furthermore, all of the components of the truncal septation complex are dysgenic in the absence of the CNC. Of special significance in the absence of CNC. Of special significance in the absence of CNC are: 1) the failure of the myocardial sheath to retract; 2) the apparently random distribution of surrogate ectomesenchyme; and 3) the impairment of truncal elastogenesis. These results indicate that the cells of neural crest origin interact with the surrounding mesenchyme during septation and that the entire septation complex depends upon the presence of the neural crest cells for normal development.

Previous studies have shown that the cells of the aorticopulmonary (AP) septum are similar to the smooth muscle cells of the mediae of the great vessels in their common origin from the cardiac neural crest and in their common expression of an elastic extracellular matrix. The purpose of this study was to test the cells of the AP septum for the presence of certain cytoplasmic proteins, especially smooth muscle alpha-actin (SMAA) whose presence is definitive of smooth muscle. A monoclonal antibody against SMAA was applied to normal chicken embryos at 3.5-8 days of incubation and to age-matched embryos from which the cardiac neural crest had been ablated surgically. Antibodies against the intermediate filaments desmin, cytokeratin, and vimentin also were applied. The results showed that the AP septal cells expressed SMAA during the process of septation, days 5-8; but when the cardiac neural crest was ablated and septation was defective, no cells in the conotruncal connective tissue expressed SMAA. None of the intermediate filament proteins were detected in the septum. These results indicate that the AP septal cells are smooth muscle and therefore may be hypothesized to have an active role in septation.

The cells that form the aorticopulmonary septum in the avian embryo have been shown to be similar to the cells that form the walls of the great vessels in two ways: both are derived from the cardiac neural crest and both are able to synthesize an elastogenic matrix in the early embryo. Because of these similarities, and because ablation of the cardiac neural crest causes congenital defects of the outflow tract that are related to failure of proper septation, it was hypothesized that such an ablation also would cause the walls of the great vessels to be defective. The purpose of this study was to compare the elastic matrix in the mediae of the great vessels of normal embryos with those from which the cardiac neural crest had been ablated. The results show that the elastic matrix in the great vessels of the experimental embryos was impaired 1) in the rate of downstream propagation of the initiation of elastogenesis among younger embryos, incubation days 4-8 and 2) in the spatial configuration of the elastic matrix among the older embryos, incubation days 16-20. These results may provide a biological explanation for the elastin defect that affects the pulmonary artery of many patients with cyanotic congenital heart defects.

The purpose of this study was to measure the binding of concanavalin A (conA) in minute regions of tissue by labelling the conA with iron dextran; then by measuring the bound iron per site by a procedure which uses atomic absorption spectrophotometry, X-ray microanalysis, and image analysis. The resulting data for a given region gamma are entered into the formula: (Formula: see text). The resulting quantity "iron at gamma" is directly proportional to conA binding in that region. For this study, three regions of rat renal cortex were compared: distal tubules, collecting ducts and blood vessels; glomeruli; and proximal tubules. Regional iron concentrations were: Combined region (distal tubules, etc.), 0.147 +/- 0.107 microgram/mg tissue; glomeruli, 0.199 +/- 0.087 microgram/mg tissue; and proximal tubules, 1.711 +/- 0.303 microgram/mg tissue.

The onset of acid phosphatase activity was observed in neonatal rat substantia gelatinosa using the Gomori method. Although acid phosphatase activity was not present at birth it appeared during the first day postnatally. By six to ten days postnatally enzyme activity appeared to reach its adult level. The activity was quantified using atomic absorption spectrophotometry which showed that acid phosphatase activity reached its adult level by 6 days postnatally. Acid phosphatase in the substantia gelatinosa is fluoride resistant from its first appearance.

A direct Schiff reaction of elastic tissues has been known for many years, but the nature of the native aldehyde-rich components has not been clear. In this study, chicken, quail, and rat embryos and adult rat lung, aorta, and kidney were fixed in methacarn or in a formalin solution, embedded in paraffin, and sections of 8-10 micron obtained. Rehydrated sections were incubated for various periods in solutions of the enzymes chondroitinase ABC, clostripain, collagenase, elastase, heparatinase, hyaluronidase, subtilisin Carlsberg ("protease"), or trypsin, and in solutions of phosphomolybdic acid or sodium borohydride. After incubation, sections were placed, without prior oxidation, in Schiff's reagent, and were ultimately observed and photographed in transmitted light or with blue or green epifluorescence. A Schiff-positive substance was found, always and exclusively, in elastic tissues of the vasculature and lungs, which was hydrolyzed by the proteolytic enzymes to an extent that ranged from complete loss of Schiff reaction in minutes (trypsin) to no loss of Schiff reaction in 22 hr (clostripain). The Schiff-reactive protein preceded the time of appearance of elastin in the early embryos. We conclude that the aldehyde-rich protein responsible for this reaction is a harbinger of elastogenesis in vivo and speculate that it may represent the elastic microfibril or a component thereof.

Bone marrow nuclei fixed with modified Carnoy's, then stained with gallocyanin chromalum followed by air drying showed no difference in morphology when compared by means of scanning electron microscopy with similar nuclei prepared by critical point drying. Glutaraldehyde at pH 4.0 and 7.1, mercury-containing Zenker's fluid, and chromalum alone, all of which are considered to be nuclear protein cross-linking fixatives, failed to preserve the nuclear morphology as well as gallocyanin-chromalum or critical point prepared bone marro nuclei.

Dermopathy is a part of the diabetic syndrome that decreases the solubility of dermal collagen. In the present study, the PAS-reactive component of collagen has been analysed in diabetic and non-diabetic human dermis by photographic densitometry. The PAS-reaction was significantly lower in diabetics than non-diabetics, and age seemed to be of no consequence. The results are interpreted to indicate a decrease in collagen-associated sugar residues in diabetics.

The interconnections between stereocilia within individual tufts of auditory hair cells in the basilar papilla of the alligator lizard were examined with a transmission electron microscope. An elaborate array of fibers near the base of each stereocilium (where it tapers to anchor into the cuticular plate) connected it to each of its neighboring stereocilia. The tips of individual stereocilia, which were slightly larger in diameter than their shaft, contacted adjacent stereocilia. Fibers also connected the tip of the kinocilium to neighboring stereocilia in the first row. The remaining regions of the stereocilia were relatively free of connecting fibers. The integrity of these connecting fibers are likely to be important in maintaining the normal micromechanical tuning and mechanoelectric transduction in these auditory hair cells. The addition of 0.1% ruthenium red to the primary fixative enhanced the preservation of the connecting structures, implying the presence of glycosaminoglycans.

Ectomesenchyme derived from cardiac neural crest is critical to aorticopulmonary septation in the heart. However, any unique contribution of the cardiac ectomesenchyme to the extracellular matrix of the conotruncus has not been demonstrated previously. In this study the chronology and topography of soluble tropoelastin (STE) and the aldehyde-rich protein (ARP) of the elastic connective tissues have been examined in the chick embryo, stages 21-38, and in the quail-chick chimera, stages 24-35 (quail neural fold grafted onto a chick embryo). STE was located with immunofluorescence histochemistry, and ARP with Schiff's reagent. With these procedures prevenient sites of elastin synthesis are observed readily. The results show that the myocardium proper appears to have a role in the instigation of elastogenesis and in elastic fiber orientation; that the mesenchymal cells whose matrix contains elastic fibers are ectomesenchymal, of neural crest origin; and that elastin is deployed in an orderly proximal-distal sequence. It is hypothesized that elastogenesis is a critical event in aorticopulmonary septation.

The myometrical glycogen content of the uterine horns of cycling, castrated and castrated hormone-treated hamsters was basically unaffected by the presence of an intrauterine device (IUD). The study also indicates that visual evaluation of histochemical localization of glycogen is not adequate to determine minor differences in staining intensity. The "estrogen-like" effect or stimulatory effect of the IUD on uterine glycogen in the rat is not duplicated in the hamster.

Cells from the cardiac neural crest are essential for the normal development of both the heart and the great vessels. If cardiac neural crest is ablated surgically from Hamburger-Hamilton stage 9 chicken embryos, they will develop anomalies of both the heart and great vessels that are similar to anomalies that occur in humans. In the absence of cardiac neural crest, another area of neural ectoderm (nodose placode) provides replacement cells that are less competent than those of the neural crest. In this study, both the cardiac neural crest and the nodose placodes have been surgically ablated. A syndrome of unusual prevalence (47%) and severity was found among the survivors of this surgery, which was characterized by a large undivided aorta that arched dorsally without right or left deviation to become the dorsal aorta. There was no other tributary to the formation of the dorsal aorta. There were no ducti arteriosi, and the pulmonary arteries were both ectopic and hypoplastic. The brachiocephalic arteries were asymmetric and hypoplastic. The association of the aorta with the anlagen of the thyroid and thymus glands, as well as with the inferior ganglion of the vagus nerve, indicated that the solitary surviving aortic arch artery is that of arch III in this syndrome. These results establish a biological limit of the plasticity of the neural ectoderm and give a probable cellular basis for a lethal congenital septal defect.

The purpose of this study was to determine the organization of collagen in the wall of the human pulmonary alveolus. Samples of human lung obtained at surgery were processed for light and electron microscopy. Light microscopy confirmed the general findings of Orsos ('36): there were 3 common fibers called primary, secondary, tertiary in this study in order of their increasing size. Primary fibers (called "pericapillary" by Orsos) formed a continuous mesh in the alveolar wall and were often confluent within the intercapillary regions of the wall ("knötenpunkten," or nodes, Orsos). The tortuous secondary fibers ("circulatory fibers," Orsos) passed frequently across the thickness of the alveolar wall and were closely applied to capillary walls. Tertiary fibers ("respiratory fibers," Orsos) were continuous with the alveolar ostia and formed the supportive struts of the alveolar wall as they crossed the wall in a more direct course than the serpiginous secondary fibers. Electron microscopy (serial sections and stereo pairs) showed that the primary fibers inserted near the edge of an intercapillary region, where they were attached to the endothelial or epithelial basal lamina directly or by a smaller fiber or microfibril resembling the fibrous component of elastin or oxytalan. Primary fibers passed through a typical intercapillary region while describing a helix or a portion thereof. Secondary fibers were more coarse than primary, and both secondary and tertiary fibers resembled woven ropes.