TY - JOUR
T1 - Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridization
AU - Lawrence, Jeanne Bentley
AU - Taneja, Krishan
AU - Singer, Robert H.
N1 - Funding Information:
We thank Bruce Paterson, Robert Schwartz, Charles Ordahl, and Donald Cleveland for the generous gift of their actin probes and Jeff Robbins for the myosin heavy chain probe. Myosin antibodies were provided by John Coleman and actin antibodies by Jeannette Bu-linsky. We also thank Annette and John Coleman for help in the use of the microfluorimeter. We gratefully acknowledge the excellent technical assistance of Carol Villnave. This research was supported by HD18066 to R.H.S. and J.B.L. and an MDA Fellowship and grant to
PY - 1989/5
Y1 - 1989/5
N2 - The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and β isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for α-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, β-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.
AB - The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and β isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for α-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, β-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.
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U2 - 10.1016/0012-1606(89)90314-X
DO - 10.1016/0012-1606(89)90314-X
M3 - Article
C2 - 2651181
AN - SCOPUS:0024597064
SN - 0012-1606
VL - 133
SP - 235
EP - 246
JO - Developmental Biology
JF - Developmental Biology
IS - 1
ER -