The<Emphasis Type="Italic"> Arabidopsis thaliana rlp</Emphasis> mutations revert the ectopic leaf blade formation conferred by activation tagging of the<Emphasis Type="Italic"> LEP</Emphasis> gene

Activation tagging of the gene LEAFY PETIOLE ( LEP) with a T-DNA construct induces ectopic leaf blade formation in Arabidopsis, which results in a leafy petiole phenotype. In addition, the number of rosette leaves produced prior to the onset of bolting is reduced, and the rate of leaf initiation is retarded by the activation tagged LEP gene. The ectopic leaf blade results from an invasion of the petiole region by the wild-type leaf blade. In order to isolate mutants that are specifically disturbed in the outgrowth of the leaf blade, second site mutagenesis was performed using ethane methanesulphonate (EMS) on a transgenic line that harbours the activation-tagged LEP gene and exhibits the leafy petiole phenotype. A collection of revertant for leafy petiole ( rlp) lines was isolated that form petiolated rosette leaves in the presence of the activated LEP gene, and could be classified into three groups. The class III rlp lines also display altered leaf development in a wild-type (non-transgenic) background, and are probably mutated in genes that affect shoot or leaf development. The rlp lines of classes I and II, which represent the majority of revertants, do not affect leaf blade outgrowth in a wild-type (non-transgenic) background. This indicates that LEP regulates a subset of the genes involved in the process of leaf blade outgrowth, and that genetic and/or functional redundancy in this process compensates for the loss of RLP function during the formation of the wild-type leaf blade. More detailed genetic and morphological analyses were performed on a selection of the rlp lines. Of these, the dominant rlp lines display complete reversion of (1) the leafy petiole phenotype, (2) the reduction in the number of rosette leaves and (3) the slower leaf initiation rate caused by the activation-tagged LEP gene. Therefore, these lines are potentially mutated in genes for interacting partners of LEP or in downstream regulatory genes. In contrast, the recessive rlp lines exhibit a specific reversion of the leafy petiole phenotype. Thus, these lines are most probably mutated in genes specific for the outgrowth of the leaf blade. Further functional analysis of the rlp mutations will contribute to the dissection of the complex pathways underlying leaf blade outgrowth.Communicated by G. Jürgens     

A preliminary investigation of the role of auxin and cytokinin in sylleptic branching of three hybrid poplar clones exhibiting contrasting degrees of sylleptic branching

Sylleptic branches grow out from lateral buds during the same growing season in which the buds are formed. This type of branching is present in poplar and in many tropical species. It results in the production of more branches, more leaves and expanded photosynthetic capacity and is thought to assist in increasing the overall growth and biomass of the tree at a young age. However, very little is known about the physiology of sylleptic branching in poplar, which is an extremely important source of fibre and fuel. In the present study of three hybrid poplar clones (11-11, 47-174 and 49-177) of Populus trichocarpa x P. deltoides exhibiting contrasting degrees of sylleptic branching, an analysis was carried out on parent shoot elongation and sylleptic branching, together with a preliminary comparison of the parent shoots' sensitivity to auxin (naphthaleneacetic acid) as a repressor of lateral bud outgrowth, and cytokinin (benzyladenine) as a promoter. Suggestive evidence was found for an inverse correlation between parent shoot sensitivity to auxin and the degree of sylleptic branching, as well as a partially positive correlation with respect to sensitivity to cytokinin. The present data are consistent with the hypothesis that auxin and cytokinin may play repressive and promotive roles, respectively, in the sylleptic branching of hybrid poplar.     

Neurite Outgrowth-Promoting Factors in Extracts of Denervated Chick Skeletal Muscle

1. An extract of denervated skeletal muscle contained activity for promotion of neurite outgrowth from telencephalic neurons, as well as that from neurons in the spinal cord. A factor responsible for the activity was characterized in cultures of dissociated neurons.2. The factor acted on neurons only when they were attached to the surface of culture dishes. Since treatments with proteases and lectins reduced the outgrowth-promoting activity, the factor was thought to be a glycoprotein.3. Among the monoclonal antibodies raised against the partially purified extract, five antibodies were found to inhibit the activity for spinal and telencephalic neurons. The most potent antibody, 4D2a, recognized mainly a 63-kD protein and other minor proteins in the extract. Although the 63-kD protein was confirmed to be chick serum albumin by analysis of amino acid sequence, the purified albumin exhibited no activity.4. From these observations, the factor was found to be a glycoprotein recognized by the neutralizing antibody as one of the minor components of the extract. This factor exhibits its activity in a substrate-bound form but not in a diffusible one.     

Nerve Growth Factor-Induced Differentiation in Neuroblastoma Cells Expressing TrkA but Lacking p75NGFR

Abstract: Nerve growth factor (NGF) binds to two distinct cell surface receptors, TrkA, which is a receptor tyrosine kinase, and p75^NGFR, whose role in NGF-induced signal transduction remains unclear. We have found that human neuroblastoma IMR-32 cells express TrkA, but p75^NGFR expression was not detectable in these cells by northern blot analysis, immunoblotting, or chemical crosslinking experiments. Despite the lack of p75^NGFR expression, subnanomolar concentrations of recombinant human NGF induced neurite outgrowth, tyrosine phosphorylation, and immediate early gene expression in these cells. These results strongly suggest that NGF-induced neuronal differentiation in IMR-32 cells is initiated through TrkA in the absence of p75^NGFR. Thus, IMR-32 cells may provide a model for studying neurotrophic effects of NGF on adult striatal cholinergic neurons, which also lack p75^NGFR expression.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Neurite Outgrowth and Protein Phosphorylation in Chick Embryonic Sensory Ganglia Induced by a Brief Exposure to 12-O-Tetradecanoylphorbol 13-Acetate

Abstract: An exposure to 12- O -tetradecanoylphorbol 13-acetate (TPA) at 20 n M for as short as 30 min was sufficient to elicit neurite outgrowth from explanted chick embryonic sensory ganglia. Attachment of the ganglia to the collagencoated substratum during exposure to TPA was essential for subsequent neurite outgrowth. Pulse-labeling with [³⁵S]-methionine indicated no significant difference in protein synthesis between control and TPA-treated ganglia. In vitro phosphorylation assay revealed a prominent protein kinase C substrate with an apparent molecular mass of 66,000 dalton (66 kDa) in chick embryo ganglia extracts. Treatment of intact ganglia with TPA for 30 min also specifically stimulated the phosphorylation of the same protein. When staurosporine, a potent inhibitor of protein kinase C, was present during TPA treatment, both neurite outgrowth and the phosphorylation of the 66-kDa protein were blocked. Biochemical analysis of the phosphorylated 66-kDa protein indicated that (1) phosphorylation was only in serine residue, (2) the pI value was 4.5, (3) after V8 protease digestion, two phosphorylated peptide fragments, 6.0 and 7.5 kDa in size, were produced, and (4) it cross-reacted with an antiserum raised against a 66-kDa neurofilament subunit from rat spinal cord. These results suggest that early activation of protein kinase C and the phosphorylation of the 66-kDa protein may be involved in neuritogenesis.     

Amitriptyline inhibits neurite outgrowth in chick cerebral neurons: A possible mechanism

Previous studies showed that amitriptyline (AMI), a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants and inhibited adenylyl cyclase activity in cerebral membrane preparations. In the present study, we have investigated the possibility that AMI may have additional effects on cellular metabolism and signal transduction that underlie AMI-mediated inhibition of neurite outgrowth. In vitro AMI inhibited phospholipase C in a dose- and GTP-dependent manner in membranes from 8-day-old chick forebrain. Brain homogenates from 8-day-old chick embryos, treated in vivo for 6 days with AMI (20 μg/g/day), showed significant reductions in (1) phosphorylation of two polypeptides (49 and 105 kD), and (2) levels of three polypeptides (43, 53, and 92 kD). Western blots showed that the 43- and 53-kD polypeptides corresponded to actin and tubulin, respectively. Diolein and dilinolein, potent activators of protein kinase C, stimulated neurite outgrowth and reversed the inhibitory effects of AMI. Sphingosine, a protein kinase C inhibitor, significantly inhibited neurite outgrowth and eliminated the stimulatory effects of diolein and dilinolein on neurite outgrowth. These data suggest that AMI-mediated inhibition of neurite outgrowth involves multiple effects on cellular metabolism and signal transduction. A hypothesis consistent with our data is that AMI interferes in some manner with the action of G proteins in the signal transduction cascade. © 1993 John Wiley & Sons, Inc.     

Promoting and directing axon outgrowth

Establishment of appropriate neuronal connections during development and regeneration requires the extension of processes that must then grow in the correct direction, find and recognize their targets, and make synapses with them. During development, embryonic neurons gradually establish central and peripheral connections in an evolving cellular environment in which neurotrophic factors are provided by supporting and target cells that promote neuronal survival, differentiation, and process outgrowth. Some cells also release neurotropic factors that direct the outgrowth of neuronal processes toward their targets. Following development the neurotrophic requirements of some adult neurons change so that, although they respond to neurotrophic factors, they no longer require exogenous neurotrophins to survive or to extend processes. Within the central nervous system (CNS), the ability of neurons to extend processes is eventually lost because of a change in their cellular environment from outgrowth permissive to inhibitory. Thus, neuronal connections that are lost in the adult CNS are rarely reestablished. In contrast, the environment of the adult peripheral nervous system fosters process outgrowth and synapse formation. This article discusses the neurotrophic requirements of embryonic and adult neurons, as well as the importance of neurotropic factors in directing the outgrowth of regenerating adult axons.     

Neurite outgrowth from cultured CNS neurons is promoted by inhibitors of protein and RNA synthesis

We examined the effects of changes caused by the blocking of protein and RNA synthesis on neurite outgrowth from neurons of the central nervous system (CNS) in primary culture. Exposure to cycloheximide and actinomycin-D led to dramatic increases in the length of neurites in cultures of neurons from various rat or chick CNS regions. Inhibitor-induced neurite outgrowth was observed (1) from dopaminergic neurons in mixed cultures of the rat substantia nigra or (2) in pure cultures of rat and chick neurons grown on a polyornithine/laminin substratum. These results suggest that neurite outgrowth from CNS neurons is kept restricted, at least in culture, by the continuous production of a labile neurite-inhibiting protein intrinsic to the neurons, which rapidly decays following inhibition of protein or RNA synthesis. 1994 John Wiley & Sons, Inc.