Core fucosylation is required for midline patterning during zebrafish development.

Complex carbohydrates represent one of the most polymorphic classes of macromolecules, but their functions during embryonic development remain poorly defined. Herein, we show that knockdown of FucT8, the fucosyltransferase responsible for adding an α1,6 fucosyl residue to the core region of N-linked oligosaccharides, results in defective midline patterning during zebrafish development. Reduced FucT8 expression leads to mild cyclopia, small forebrains, U-shaped somites, among other midline patterning defects. One of the principal FucT8 substrates was identified as Apolipoprotein B (ApoB), the major scaffold protein that is responsible for assembly and secretion of lipoprotein particles in vertebrates. In Drosophila, lipoprotein particles are thought to facilitate cell signaling by serving as a transport vehicle for lipid-modified cell signaling proteins, such as hedgehog. In this regard, knockdown of ApoB expression in zebrafish embryos leads to similar midline patterning defects as those seen in FucT8 morphant embryos. Furthermore, preliminary studies suggest that ApoB facilitates Sonic hedgehog signaling during zebrafish development, analogous to the function of lipoprotein particles during hedgehog signaling in Drosophila.

Regulation of muscle differentiation and survival by Acheron.

Acheron (Achn), a phylogenetically-conserved member of the Lupus antigen family of RNA binding proteins, was initially identified as a novel cell death-associated gene from the intersegmental muscles of the tobacco hawkmoth Manduca sexta. C(2)C(12) cells are a standard model for the study of myogenesis. When deprived of growth factors, these cells can be induced to: form multinucleated myotubes, arrest as quiescent satellite-like reserve cells, or undergo apoptosis. Achn expression is induced in myoblasts that form myotubes and acts upstream of the muscle specific transcription factor MyoD. Forced expression of ectopic Achn resulted in the formation of larger myotubes and massive reserve cell death relative to controls. Conversely, dominant-negative or antisense Achn blocked myotube formation following loss of growth factors, suggesting that Achn plays an essential, permissive role in myogenesis. Studies in zebrafish embryos support this hypothesis. Reduction of Achn with antisense morpholinos led to muscle fiber loss and an increase in the number of surviving cells in the somites, while ectopic Achn enhanced muscle fiber formation and reduced cell numbers. These results display a crucial evolutionarily conserved role for Achn in myogenesis and suggest that it plays key roles in the processes of differentiation and self-renewal.

belladonna/(Ihx2) is required for neural patterning and midline axon guidance in the zebrafish forebrain.

Some of the earliest axon pathways to form in the vertebrate forebrain are established as commissural and retinal axons cross the midline of the diencephalon and telencephalon. To better understand axon guidance in the forebrain, we characterized the zebrafish belladonna (bel) mutation, which disrupts commissural and retinal axon guidance in the forebrain. Using a positional cloning strategy, we determined that the bel locus encodes zebrafish Lhx2, a lim-homeodomain transcription factor expressed in the brain, eye and fin buds. We show that bel(Ihx2) function is required for patterning in the ventral forebrain and eye, and that loss of bel function leads to alterations in regulatory gene expression, perturbations in axon guidance factors, and the absence of an optic chiasm and forebrain commissures. Our analysis reveals new roles for Ihx2 in midline axon guidance, forebrain patterning and eye morphogenesis.

Inter-alu PCR detects high frequency of genetic alterations in glioma cells exposed to sub-lethal cisplatin.

Increased genomic instability contributes to higher frequency of secondary drug resistance and neoplastic progression in tumors as well as in cells exposed to sub-lethal concentrations of chemotherapeutic agents. We have used PCR based DNA fingerprinting techniques of randomly amplified polymorphic DNA (RAPD) and inter-alu PCR to study this phenomenon in the tumor genome. The choice of the primer, either random (for RAPD) or specific (inter-alu PCR) can determine the nature of alterations being assessed. We have compared the inter-alu PCR and RAPD profiles of U87MG glioblastoma cells exposed to sequentially increasing low doses of cisplatin for 24 passages to that of untreated controls. Inter-alu PCR, with 2 primers, demonstrated a number of alterations in the treated cells, in the form of loss / gain and changes in the intensity of bands. No changes were observed by RAPD analysis with 5 primers, however, indicating a preferential increase in the alu mediated recombination frequency in the treated cells (p = 1.866 x 10(-4)). The number of changes observed with respect to the corresponding leucocyte DNA in the inter-alu PCR profile of 26 primary tumors (Grade II = 13; Grade IV = 13), resected before chemotherapy, for the 2 inter-alu primers was very small. We present a novel application of the inter-alu PCR in detecting alterations in long term cultured cells at low dose exposure to a chemotherapeutic agent. Our results suggest that alu mediated recombination may be important in cells exposed to sub-lethal doses of cisplatin but not in the genesis of primary glioma.