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Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto-oncogene pp60c-src and high molecular weight neurofilament protein

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Abstract

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.

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Abbreviations

BCIP:

5-bromo-4-chloro-3-indolylphosphate p-toluidine salt

CNS:

central nervous system

DPBS:

Dulbecco's phosphate-buffered saline

GAM-AP:

goat anti-mouse IgG alkaline phosphatase conjugate

LB:

limb bud

NF:

high molecular weight neurofilament protein

NBT:

nitroblue tetrazolium chloride

SDS-PAGE:

polyacrylamide gel electrophoresis

PVDF:

polyvinylidene difluoride

RIPA:

radioimmunoprecipitation

TBS:

Tris-buffered saline

TTBS:

TBS with 0.05% Tween-20

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Authors and Affiliations

  1. Department of Environmental Health, University of Washington, Seattle, Washington

    Carol Sweeney, Zamyat Kirby & Elaine M. Faustman

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  1. Carol Sweeney
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  2. Zamyat Kirby
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  3. Elaine M. Faustman
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Presented in part at the 1989 and 1990 Teratology Society Meetings and the 1990 Society of Toxicology Meetings.

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Sweeney, C., Kirby, Z. & Faustman, E.M. Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto-oncogene pp60c-src and high molecular weight neurofilament protein. Cell Biol Toxicol 8, 113–128 (1992). https://doi.org/10.1007/BF00260562

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