title: In vitro growth conditions and development affect differential distributions of RNA in axonal growth cones and shafts of cultured rat hippocampal neurons.
abstract: Local synthesis of proteins in the axons participates in axonogenesis and axon guidance to establish appropriate synaptic connections and confer plasticity. To study the transcripts present in the growth cones and axonal shafts of cultured rat hippocampal neurons, two chip devices, differing in their abilities to support axonal growth and branching, are designed and employed here to isolate large quantities of axonal materials. Cone-, shaft- and axon-residing transcripts with amounts higher than that of a somatodendritic transcript, Actg1 (γ-actin), are selected and classified. Since the chips are optically transparent, distribution of transcripts over axons can be studied by fluorescence in situ hybridization. Three transcripts, Cadm1 (cell adhesion molecule 1), Nefl (neurofilament light polypeptide), and Cfl1 (non-muscle cofilin) are confirmed to be preferentially localized to the growth cones, while Pfn2 (profilin2) is preferentially localized to the shafts of those axons growing on the chip that restricts axonal growth. The different growing conditions of axons on chips and on conventional coverslips do not affect the cone-preferred localization of Cadm1 and shaft-preferred localization of Pfn2, but affect the distributions of Nefl and Cfl1 over the axons at 14th day in vitro. Furthermore, the distributions of Cadm1 and Nefl over the axons growing on conventional coverslips undergo changes during in vitro development. Our results suggest a dynamic nature of the mechanisms regulating the distributions of transcripts in axonal substructures in a manner dependent upon both growth conditions and neuronal maturation.
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
authors.forename: Yi-Yun
authors.initials: YY
authors.lastname: Wang
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
authors.forename: Huei-Ing
authors.initials: HI
authors.lastname: Wu
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
authors.forename: Wei-Lun
authors.initials: WL
authors.lastname: Hsu
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
authors.forename: Hui-Wen
authors.initials: HW
authors.lastname: Chung
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
authors.forename: Pei-Hung
authors.initials: PH
authors.lastname: Yang
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC; Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 300 30013, Taiwan, ROC. Electronic address: ycchang@life.nthu.edu.tw.
authors.forename: Yen-Chung
authors.initials: YC
authors.lastname: Chang
authors.affiliation: Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 300 30013, Taiwan, ROC; Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC. Electronic address: wychow@life.nthu.edu.tw.
authors.forename: Wei-Yuan
authors.initials: WY
authors.lastname: Chow
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000367:Age Factors; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D051338:Cofilin 1 / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian; D005260:Female; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism; D020439:Growth Cones / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology*; D017404:In Situ Hybridization, Fluorescence; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D011247:Pregnancy; D051304:Profilins / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000199:Actins; D015815:Cell Adhesion Molecules; C495812:Cfl1 protein, rat; D051338:Cofilin 1; D015693:Gene Products, nef; D008565:Membrane Proteins; D051304:Profilins; D012337:RNA, Ribosomal, 18S
keywords: Axonal RNA; Axonal growth; Axonal shaft; Growth cone