Primary cultures as a model for studying ependymal functions: glycogen metabolism in ependymal cells.

Ependymal cells form a single-layered, ciliated epithelium at the interface between the cerebrospinal fluid and the brain parenchyma. Although their morphology has been studied in detail, ependymal functions remain largely speculative. We have established and characterized a previously described cell culture model to investigate ependymal glycogen metabolism. During growth in minimal medium lacking many non-essential amino acids including L-glutamate, but containing glucose at physiological concentration, the cells contained negligible amounts of glycogen (7+/-3 nmol glucosyl residues/mg protein) despite the presence of insulin. However, during a period of 24 h, the cells accumulated glycogen to very high levels after transferal to a medium containing insulin, glucose at a 5-fold higher concentration, and all proteinogenic amino acids except L-asparagine and L-serine (990+/-112 nmol glucosyl residues/mg protein). Omission of insulin resulted in a 50% reduction in glycogen accumulation. Upon glucose deprivation, glycogen was degraded with a half-life of 21 min. The ependymal primary cultures contained 80+/-5 mU glycogen phosphorylase (Pho)/mg protein and stained positively with antibodies raised against this enzyme. Astroglial cultures built up less glycogen and had less Pho activity under identical conditions. Ependymal glycogen was mobilized by noradrenaline and serotonin. Our results indicate that ependymal cells maintain glycogen as a functional energy store, subject to rapid turnover dependent on the availability of energy substrates and the presence of appropriate signal molecules. Thus ependymocytes appear to be active players in the multitude of processes resulting in normal brain function, and ependymal primary cultures are suggested as a suitable model for studying the role of ependymal cells in these processes.

The Toll/IL-1 receptor binding protein MyD88 is required for Xenopus axis formation.

The Toll/Dorsal pathway regulates dorsoventral axis formation in the Drosophila embryo. We had previously obtained evidence that a homologous pathway exists in Xenopus, however, its role during normal frog development had not been established. Here we report the cloning of Xenopus MyD88 (XMyD88), whose mammalian homologs are adaptor proteins linking Toll/IL-1 receptors and IRAK kinases. We show that in the frog embryo overexpression of a dominant-negative form of XMyD88 blocked Toll receptor activity, specifically inhibited axis formation and reduced expression of pivotal organizer genes. The observed stage-dependency of interference suggests a function for maternal XMyD88 soon after fertilization. We conclude that XMyD88 activity is required for normal Spemann organizer formation, implying an essential role for maternal Toll/IL-1 receptors in Xenopus axis formation.

Conserved Spätzle/Toll signaling in dorsoventral patterning of Xenopus embryos.

The Spätzle/Toll signaling pathway controls ventral axis formation in Drosophila by generating a gradient of nuclear Dorsal protein. Dorsal controls the downstream regulators dpp and sog, whose patterning functions are conserved between insects and vertebrates. Although there is no experimental evidence that the upstream events are conserved as well, we set out to ask if a vertebrate embryo can respond to maternal components of the fly Dorsal pathway. Here we demonstrate a dorsalizing activity for the heterologous Easter, Spätzle and Toll proteins in UV-ventralized Xenopus embryos, which is inhibited by a co-injected dominant Cactus variant. We conclude that the Dorsal signaling pathway is a component of the conserved dorsoventral (d/v) patterning system in bilateria.