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Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

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The distribution pattern of 1E12 was examined in primordial VSMCs within an intact embryonic vessel wall by whole-mount  immunolabeling. The stage 20 quail embryo shown in this figure was embedded in acrylamide, sectioned, and viewed by LSCM. (a) Low  magnification view of a projection of a series of 10 1-μm sagittal sections through the vessel wall. Primordial VSMCs are labeled with  1E12. At this early developmental stage, the VSMCs are loosely arrayed, with open spaces between the cells. The boxed area is magnified in c. (b) For purposes of orientation, a schematic view of the preparation is shown. The drawing depicts a side view of the aorta with  the anterior (A) to posterior (P) axis, and dorsal (D) to ventral (V) axis indicated. The area shaded in green is representative of the fluorescent image shown in a. (c) High magnification view of the boxed area in a and a series of 10 1-μm optical sections projected into one  image plane. A fortuitous optical section through the main axis of a cell (arrowhead) within the plane of the section shows that 1E12 labeling is concentrated at the distal tips of cellular projections, possible points of attachment. The cell marked with the box is magnified  further in e. (d) 1-μm optical section through the cell denoted by the arrowhead in c. 1E12 labeling is seen intracellularly in fine, short,  threadlike structures. (e) A high magnification view of the cell denoted by the box in c. Small 1E12-labeled clusters (arrowheads) are  similar to adhesive structures termed rosettes or podosomes. Bars: (a) 50 μm; (c and d) 10 μm; (e) 7.5 μm.
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Figure 3: The distribution pattern of 1E12 was examined in primordial VSMCs within an intact embryonic vessel wall by whole-mount immunolabeling. The stage 20 quail embryo shown in this figure was embedded in acrylamide, sectioned, and viewed by LSCM. (a) Low magnification view of a projection of a series of 10 1-μm sagittal sections through the vessel wall. Primordial VSMCs are labeled with 1E12. At this early developmental stage, the VSMCs are loosely arrayed, with open spaces between the cells. The boxed area is magnified in c. (b) For purposes of orientation, a schematic view of the preparation is shown. The drawing depicts a side view of the aorta with the anterior (A) to posterior (P) axis, and dorsal (D) to ventral (V) axis indicated. The area shaded in green is representative of the fluorescent image shown in a. (c) High magnification view of the boxed area in a and a series of 10 1-μm optical sections projected into one image plane. A fortuitous optical section through the main axis of a cell (arrowhead) within the plane of the section shows that 1E12 labeling is concentrated at the distal tips of cellular projections, possible points of attachment. The cell marked with the box is magnified further in e. (d) 1-μm optical section through the cell denoted by the arrowhead in c. 1E12 labeling is seen intracellularly in fine, short, threadlike structures. (e) A high magnification view of the cell denoted by the box in c. Small 1E12-labeled clusters (arrowheads) are similar to adhesive structures termed rosettes or podosomes. Bars: (a) 50 μm; (c and d) 10 μm; (e) 7.5 μm.

Mentions: To examine the expression of the 1E12 antigen in primordial vascular smooth muscle cells within an intact embryonic vessel wall (i.e., cells that had not been subjected to a tissue-culture environment or physically sectioned), we examined thick (200 μm), sagittal sections of acrylamideembedded, whole-mounted embryos by LSCM. The image in Fig. 3 a is a low magnification projection of 10 1-μm optical sections into one image plane. The tissue, viewed in the sagittal plane, contains undisturbed 1E12-positive cells embedded in the extracellular matrix (ECM) of the developing aortic wall (stage 20 quail embryo). For the purposes of orientation, we have provided a schematic representation of this section in Fig. 3 b.


Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

The distribution pattern of 1E12 was examined in primordial VSMCs within an intact embryonic vessel wall by whole-mount  immunolabeling. The stage 20 quail embryo shown in this figure was embedded in acrylamide, sectioned, and viewed by LSCM. (a) Low  magnification view of a projection of a series of 10 1-μm sagittal sections through the vessel wall. Primordial VSMCs are labeled with  1E12. At this early developmental stage, the VSMCs are loosely arrayed, with open spaces between the cells. The boxed area is magnified in c. (b) For purposes of orientation, a schematic view of the preparation is shown. The drawing depicts a side view of the aorta with  the anterior (A) to posterior (P) axis, and dorsal (D) to ventral (V) axis indicated. The area shaded in green is representative of the fluorescent image shown in a. (c) High magnification view of the boxed area in a and a series of 10 1-μm optical sections projected into one  image plane. A fortuitous optical section through the main axis of a cell (arrowhead) within the plane of the section shows that 1E12 labeling is concentrated at the distal tips of cellular projections, possible points of attachment. The cell marked with the box is magnified  further in e. (d) 1-μm optical section through the cell denoted by the arrowhead in c. 1E12 labeling is seen intracellularly in fine, short,  threadlike structures. (e) A high magnification view of the cell denoted by the box in c. Small 1E12-labeled clusters (arrowheads) are  similar to adhesive structures termed rosettes or podosomes. Bars: (a) 50 μm; (c and d) 10 μm; (e) 7.5 μm.
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Figure 3: The distribution pattern of 1E12 was examined in primordial VSMCs within an intact embryonic vessel wall by whole-mount immunolabeling. The stage 20 quail embryo shown in this figure was embedded in acrylamide, sectioned, and viewed by LSCM. (a) Low magnification view of a projection of a series of 10 1-μm sagittal sections through the vessel wall. Primordial VSMCs are labeled with 1E12. At this early developmental stage, the VSMCs are loosely arrayed, with open spaces between the cells. The boxed area is magnified in c. (b) For purposes of orientation, a schematic view of the preparation is shown. The drawing depicts a side view of the aorta with the anterior (A) to posterior (P) axis, and dorsal (D) to ventral (V) axis indicated. The area shaded in green is representative of the fluorescent image shown in a. (c) High magnification view of the boxed area in a and a series of 10 1-μm optical sections projected into one image plane. A fortuitous optical section through the main axis of a cell (arrowhead) within the plane of the section shows that 1E12 labeling is concentrated at the distal tips of cellular projections, possible points of attachment. The cell marked with the box is magnified further in e. (d) 1-μm optical section through the cell denoted by the arrowhead in c. 1E12 labeling is seen intracellularly in fine, short, threadlike structures. (e) A high magnification view of the cell denoted by the box in c. Small 1E12-labeled clusters (arrowheads) are similar to adhesive structures termed rosettes or podosomes. Bars: (a) 50 μm; (c and d) 10 μm; (e) 7.5 μm.
Mentions: To examine the expression of the 1E12 antigen in primordial vascular smooth muscle cells within an intact embryonic vessel wall (i.e., cells that had not been subjected to a tissue-culture environment or physically sectioned), we examined thick (200 μm), sagittal sections of acrylamideembedded, whole-mounted embryos by LSCM. The image in Fig. 3 a is a low magnification projection of 10 1-μm optical sections into one image plane. The tissue, viewed in the sagittal plane, contains undisturbed 1E12-positive cells embedded in the extracellular matrix (ECM) of the developing aortic wall (stage 20 quail embryo). For the purposes of orientation, we have provided a schematic representation of this section in Fig. 3 b.

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Show MeSH