ABSTRACT
We have previously demonstrated that the growth of peripheral nervous system axons is strongly attracted towards limb buds and skin explants in vitro. Here, we show that directed axonal growth towards skin explants of Xenopus laevis in matrigel is associated with expression of matrix metalloproteinase (MMP)-18 and also other MMPs, and that this long-range neurotropic activity is inhibited by the broad-spectrum MMP inhibitors BB-94 and GM6001. We also show that forced expression of MMP-18 in COS-7 cell aggregates enhances axonal growth from Xenopus dorsal root ganglia explants. Nidogen is the target of MMPs released by cultured skin in matrigel, whereas other components remain intact. Our results suggest a novel link between MMP activity and extracellular matrix breakdown in the control of axonal growth.
Subject(s)
Axons/physiology , Matrix Metalloproteinases/metabolism , Neurogenesis/physiology , Skin/innervation , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Coculture Techniques , Microscopy, Fluorescence , Polymerase Chain Reaction , RNA, Messenger/analysis , XenopusABSTRACT
Gene ACIAD1637 from Acinetobacter baylyi ADP1 encodes a 182 amino acid putative antibiotic resistance protein. The structure of this protein (termed acepita) has been solved in space group P(2) to 2.35 A resolution. Acepita belongs to the GCN5-related N-acetyltransferase (GNAT) family, and contains the four sequence motifs conserved among family members. The structure of acepita is compared with that of pita, its homologue from Pseudomonas aeruginosa. Acepita has a similar substrate profile to pita and performs a similar function.
Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , Acinetobacter/enzymology , Catalytic Domain/physiology , Acetyltransferases/genetics , Acinetobacter/metabolism , Amino Acid Sequence , Crystallization , Kinetics , Molecular Sequence Data , Molecular Structure , Mutation/genetics , Sequence Alignment , Substrate Specificity/physiologyABSTRACT
Axonal regeneration is enhanced by the prior ;conditioning' of peripheral nerve lesions. Here we show that Xenopus dorsal root ganglia (DRG) with attached peripheral nerves (PN-DRG) can be conditioned in vitro, thereafter showing enhanced neurotrophin-induced axonal growth similar to preparations conditioned by axotomy in vivo. Actinomycin D inhibits axonal outgrowth from freshly dissected PN-DRG, but not from conditioned preparations. Synthesis of mRNAs that encode proteins necessary for axonal elongation might therefore occur during the conditioning period, a suggestion that was confirmed by oligonucleotide microarray analysis. Culturing PN-DRG in a compartmentalized system showed that inhibition of protein synthesis (but not RNA synthesis) in the distal nerve impaired the conditioning response, suggesting that changes in gene expression in cultured DRG depend on the synthesis and retrograde transport of protein(s) in peripheral nerves. The culture system was also used to demonstrate retrograde axonal transport of several proteins, including thioredoxin (Trx). Cyclopentenone prostaglandins, which react with Trx, blocked the in vitro conditioning effect, whereas inhibition of other signalling pathways thought to be involved in axonal regeneration did not. This suggests that Trx and/or other targets of these electrophilic prostaglandins regulate axonal regeneration. Consistent with this hypothesis, morpholino-induced suppression of Trx expression in dissociated DRG neurons was associated with reduced neurite outgrowth.
Subject(s)
Axons/physiology , Prostaglandin D2/analogs & derivatives , Prostaglandins A/pharmacology , Animals , Axons/drug effects , Cells, Cultured , Dactinomycin/pharmacology , Ganglia, Spinal/drug effects , Nerve Regeneration/drug effects , Nerve Regeneration/physiology , Peripheral Nerves/drug effects , Peripheral Nerves/metabolism , Prostaglandin D2/pharmacology , RNA, Messenger/metabolism , Thioredoxins/metabolism , XenopusABSTRACT
Gastrulation movements in Xenopus laevis are becoming increasingly well characterised, however the molecular mechanisms involved are less clear. Active migration of the leading edge mesendoderm across the fibronectin-coated blastocoel roof is necessary for further development of tissues such as head mesoderm, heart, blood and liver. The zinc finger transcription factors GATA4 and GATA6 are expressed in this migratory tissue during gastrulation, but their role here is unknown. This study further characterises the expression of GATA4 and 6 during gastrulation, and investigates their function in migratory behaviour. Gain-of-function experiments with these GATA factors induce cell spreading, polarisation and migration in non-motile presumptive ectoderm cells. Expression of a dominant-interfering form of GATA6, which inhibits transactivation of GATA targets, severely impairs the ability of dorsal leading edge mesendoderm to spread and translocate on fibronectin. Mosaic inhibition of GATA activity indicates that GATA factors function cell autonomously to induce cell spreading and movement in dorsal mesendoderm. Knockdown of specific GATA factors using anti-sense morpholinos indicates that GATA4 and GATA6 both contribute to dorsal mesendoderm migration in vitro. GATA4 and GATA6 are known to be involved in cell-specification of mesoderm and endoderm-derived tissues, but this is the first description of an additional role for these factors in cell migration.
