Darrell J. Wiens

Neural crest cell motility in valproic acid

Neural crest cells (NCCs) exit the dorsal neural tube and migrate to sites where they form diverse tissues. Valproic acid (VPA) is an anticonvulsant drug that induces neural tube and related defects. Altered NCC migration and proliferation have been proposed as mechanisms of teratogenicity. We cultured neural tube segments from chick embryos in 0.75-3.0mM VPA. We used image analysis, proliferation assays, and fluorescence localization to investigate NCCs during VPA exposure. VPA inhibited attachment of explants and the number that produced migrating cells. VPA markedly decreased the proportion of cells migrating individually, promoting migration as epithelial sheets. VPA at 3mM decreased cellular spreading. Area and perimeter change per minute were reduced, but migration velocity was not. VPA at 2mM reduced proliferation 11% and 3mM arrested proliferation. Immunostaining of VPA-exposed explants revealed N-cadherin-positive cell boundaries within sheets, but independent NCCs did not stain. F-actin staining was reduced in independent NCCs. The data support a VPA mechanism involving interference with epithelial-mesenchymal transition.

Simulated Microgravity and Hypergravity Attenuate Heart Tissue Development in Explant Culture

Exposure to altered gravity may disturb the cytoskeleton-cell surface-extracellular matrix (ECM) interface of embryonic cells. Development of organs such as the heart depends on dynamic interactions across cell surfaces. Fibronectin (FN), for example, a glycoprotein that links the ECM to the cytoskeleton through integrin surface receptors, is required for normal heart development. Thus, altered gravity may perturb organogenesis. We cultured precardiac explants from chick embryos in a rotating bioreactor vessel to simulate microgravity (μG), or in a tissue culture centrifuge, for 18 h during heart development. Bioreactor μG did not alter external morphology of explants, but did significantly reduce the proportion that developed contractions. Immunostaining for FN of explant sections showed that it also significantly reduced the linear extent of staining present in basement membrane regions. Analysis of ultrastructure revealed a significant reduction in the number of desmosomes per unit area and other differences. Hypergravity dramatically abolished development of contractions and altered morphogenesis. The results indicate a probable sensitivity of cardiomyogenic development involving FN to altered gravity.

Influence of Folic Acid on Neural Connectivity during Dorsal Root Ganglion Neurogenesis

The vitamin folic acid (FA) is essential for DNA synthesis, repair and methylation, and for methionine synthesis. Although it is necessary for neural development, recent studies suggest a possible link between excess maternal supplemental FA intake and adverse interferences with single-carbon metabolism and neural development. Insufficient FA early in brain development can lead to failure of the neural tube closure, but the consequences of too much intake have not been fully investigated. Plasma FA concentrations can increase greatly with dietary supplementation. To model the development of neural connectivity, we cultured dorsal root ganglia (DRGs) taken from 8-day-old chick embryos in a range of pteroylmonoglutamate (PteGlu, synthetic supplemental FA) concentrations. DRGs were cultured for 36 h, fixed and immunostained to reveal the locations of neural networks with synaptic vesicles. We found a concentration-dependent relationship with significant reduction in neurite length in PteGlu concentrations from 0.25 to 20 μM. The average total of stained synaptic areas surrounding each cultured DRG was significantly reduced as well. To further characterize the effects, we carried out time-lapse imaging of growth cones at terminals of extending neurites. We found that PteGlu reduced the area-changing activity of the growth cone, hindering its exploratory capabilities, along with a tendency to inhibit overall advancement, thus altering the ability to extend and form synapses. Our results show that PteGlu at 250 nM and higher reduces neurite extension and synapse formation in a dose-dependent manner during neurogenesis, and that its effect is mediated through inhibition of growth cone motility.

Isotretinoin alters morphology, polarity, and motility of neural crest cells in culture

Migrating neural crest cells (NCCs) contribute to a diverse array of vertebrate head and neck structures. Retinoids are proven human and animal teratogens. To elucidate isotretinoins effects, cranial and trunk neural folds were microdissected from chick embryos and cultured. Image analysis and immunostaining were used to quantitate cell behavior. We found that a higher proportion of Stage 8, 9, and 10 treated NCCs were rounded and clustered. Medians and means for cell area, perimeter, and elongation index were lower for treated cells from Stage 9 and 10 embryos, but not from Stage 8. Cumulative medians and means for changes in area and perimeter, and cell migration were similarly lower. Thus interference with the transitory basal activity of the cytoskeleton that adjusts and determines cell-substratum adhesion, spreading, elongation, and migration may be the mechanism by which isotretinoin acts on NCCs in slightly older embryos.

Could folic acid influence growth cone motility during the development of neural connectivity

ABSTRACT Perinatal dietary supplementation, together with widespread fortification of grain-based foods with synthetic folic acid (FA) has resulted in rising concentrations of unmetabolized plasma FA in pregnant women. In a recently published study we reported on experiments in which we cultured dorsal root ganglia from chick embryos in a range of FA concentrations. We found that FA inhibited neurite extension, synaptogenesis, and growth cone motility. In this commentary we consider the possible mechanism further. The effect of FA is more likely to be on motility processes of growth cones with their exploratory filapodia than on neurotrophic stimulation. Receptors present in the filapodia membrane recognize and bind to environmental guidance cues. The presence of the NMDA receptor on filapodia, and the possible competition of FA with the neurotransmitter glutamate for binding to it, resulting in perturbation of growth cone guidance, are discussed. Whether excess FA exerts its inhibitory effects by such binding competition or via some other mechanism, further investigation is needed. Sufficient intake of folate from conception through the first month of human pregnancy is essential for neural tube closure. However, our results suggest that an upper limit for FA consumption after the first month should be considered.