Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning, and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily acts to repel axons from inappropriate targets, induces cytoskeletal rearrangements that results in growth cone collapse 1 . These effects require intra-axonal mRNA translation. Here we show that transcripts for RhoA, a small GTPase that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3'UTR. Sema3A induces intra-axonal translation of RhoA mRNA and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a novel mechanism for the regulation of RhoA signaling.Studies using Xenopus retinal axons demonstrated that cytoskeletal regulation of the growth cone by Sema3A requires intra-axonal, or "local", mRNA translation 2 . Sema3A treatment results in increased protein synthesis in growth cones as evidenced by metabolic labeling experiments and by phosphorylation of elongation factor 4E-BP1 2 . These effects occur within minutes of Sema3A application 2 . Furthermore, Sema3A-mediated growth cone collapse is blocked by ribosomal inhibitors. The mRNA translation that is required for Sema3A-mediated growth cone collapse occurs in the axon, as both Sema3A-induced collapse and inhibition of this collapse by ribosomal inhibitors is preserved in axons that are severed from their cell bodies 2 .To determine if intra-axonal mRNA translation is required for Sema3A signaling in mammalian neurons, we examined Sema3A-mediated growth cone collapse in embryonic rat dorsal root ganglia (DRG) explant cultures 3 -5 . To eliminate the possibility that the effects of Sema3A were mediated through somatic translation, axons were severed from their cell bodies 2 ( Supplementary Fig. 1a). Treatment of severed axons with Sema3A for 60 min resulted in an increase in collapsed growth cones from 17 ± 1.3% to 75 ± 2.8 % ( Supplementary Fig. 1b, c, g). This effect was blocked by pretreatment of axons with either cycloheximide or anisomycin ( Supplementary Fig. 1d-f), both of which are ribosomal inhibitors. Pretreatment of these cultures with rapamycin, an inhibitor of cap-dependent translation 6 , also blocked Sema3A-mediated growth cone collapse. Together, these data indicate that the requirement forCorrespondence and requests for materials should be addressed to S.R.J. (e-mail:srj2003@med.cornell.edu).. * These authors contributed equally to this work.Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Competing interests statementThe authors declare that they have no competing financial interests. Supplementary ...

During development of the nervous system, axons and growth cones contain mRNAs, such as β-actin, cofilin, and RhoA, that are locally translated in response to guidance cues. Intra-axonal translation of these mRNAs results in local morphological responses, however other functions of intra-axonal mRNA translation remain unknown. Here we show that axons of developing mammalian neurons contain mRNA encoding the cAMP-responsive element (CRE)-binding protein (CREB). CREB is translated within axons in response to NGF and is retrogradely trafficked to the cell body. In neurons that are selectively deficient in axonal CREB transcripts, increases in nuclear pCREB, CRE-mediated transcription, and neuronal survival elicited by axonal application of NGF are abolished, indicating a signalling function for axonally-synthesised CREB. These studies identify a signalling role for axonally-derived CREB, and indicate that signaldependent synthesis and retrograde trafficking of transcription factors enables specific transcriptional responses to signalling events at distal axons.An important mechanism by which the protein composition at specific subcellular sites is regulated is by the precise intracellular targeting and translation of certain mRNAs 1 . This mechanism particularly evident in axons of developing neurons, which contain mRNAs, ribosomes, and other translational machinery 2 . Most known axonal mRNAs encode proteins that are involved in cytoskeletal regulation, and are involved in mediating the response to guidance cues [3][4][5] . Roles for axonal mRNA translation in other processes have not been established.One critical function of axons during development is to detect signals in the extracellular environment and to transduce these signals into changes in gene expression in the nucleus [6][7][8][9] . In developing sympathetic and sensory axons, growth cones detect nerve growth factor (NGF) synthesised by target cells, resulting in the generation of a retrograde signal that is conveyed to the soma that promotes neuronal survival 10 .Here we describe a role for axonal mRNA translation in the regulation of neuronal survival and nuclear transcription elicited by axonal application of NGF. We find NGF triggers axonal protein synthesis, which is required for NGF-mediated retrograde survival. A cDNA library prepared from the axons of developing sensory neurons reveals that CREB mRNA is an axonally-localised transcript. We find that CREB is selectively translated in axons in response to NGF and retrogradely trafficked to the cell body. Furthermore, selective 3 Correspondence should be addressed to S.R.J. (srj2003@med.cornell.edu). NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2011 August 9. knockdown of axonal CREB mRNA reveals that axonally-synthesised CREB is required for NGF at axons to promote the accumulation of pCREB in the nucleus, transcription of a CRE-containing reporter gene, and neuronal survival. These data identify a role for axonally-synthesised CREB and identify a ...

ResearchFusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca 2+ , leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca 2+ oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCζ) has been identified in mice. Mouse PLCζ triggers Ca 2+ oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCζ homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCζ protein sequences indicates a shorter X-Y linker region in human PLCζ and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCζ elicits Ca 2+ oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCζ elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCζ from monkeys and mice. These results are consistent with the proposal that sperm PLCζ is the molecular trigger for egg activation during fertilization and that the role and activity of PLCζ is highly conserved across mammalian species.

Developing axons and growth cones contain "local" mRNAs that are translated in response to various extracellular signaling molecules and have roles in several processes during axonal development, including axonal pathfinding, orientation of axons in chemotactic gradients, and in the regulation of neurotransmitter release. The molecular mechanisms that regulate mRNA translation within axons and growth cones are unknown. Here we show that proteins involved in RNA interference (RNAi), including argonaute-3 and argonaute-4, Dicer, and the fragile X mental retardation protein, are found in developing axons and growth cones. These proteins assemble into functional RNA-induced silencing complexes as transfection of small interfering RNAs selectively into distal axons results in distal axon-specific mRNA knock-down, without reducing transcript levels in proximal axons or associated diffusion of small interfering RNA into proximal axons or cell bodies. RhoA mRNA is localized to axons and growth cones, and intra-axonal translation of RhoA is required for growth cone collapse elicited by Semaphorin 3A (Sema3A), an axonal guidance cue. Selective knock-down of axonal RhoA mRNA abolishes Sema3A-dependent growth cone collapse. Our results demonstrate functional and potent RNAi in axons and identify an approach to spatially regulate mRNA transcripts at a subcellular level in neurons.

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca2+ oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca2+ release. The ‘sperm factor’ hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca2+ transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLCζ, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLCζ removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLCζ content of a single sperm was sufficient to produce Ca2+ oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLCζ as the molecular trigger for development of a fertilised egg into an embryo.