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Evidence for Hox-specified positional identities in adult vasculature.

Pruett ND, Visconti RP, Jacobs DF, Scholz D, McQuinn T, Sundberg JP, Awgulewitsch A - BMC Dev. Biol. (2008)

Bottom Line: These reporter gene patterns were validated as authentic indicators of endogenous gene expression by immunolabeling and PCR analysis.Furthermore, we show that persistent reporter gene expression in cultured cells derived from vessel explants facilitates in vitro characterization of phenotypic properties as exemplified by the differential response of Hoxc11-lacZ-positive versus-negative cells in migration assays and to serum.The data support a conceptual model of Hox-specified positional identities in adult blood vessels, which is of likely relevance for understanding the mechanisms underlying regional physiological diversities in the cardiovascular system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USA. pruettnd@musc.edu

ABSTRACT

Background: The concept of specifying positional information in the adult cardiovascular system is largely unexplored. While the Hox transcriptional regulators have to be viewed as excellent candidates for assuming such a role, little is known about their presumptive cardiovascular control functions and in vivo expression patterns.

Results: We demonstrate that conventional reporter gene analysis in transgenic mice is a useful approach for defining highly complex Hox expression patterns in the adult vascular network as exemplified by our lacZ reporter gene models for Hoxa3 and Hoxc11. These mice revealed expression in subsets of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) located in distinct regions of the vasculature that roughly correspond to the embryonic expression domains of the two genes. These reporter gene patterns were validated as authentic indicators of endogenous gene expression by immunolabeling and PCR analysis. Furthermore, we show that persistent reporter gene expression in cultured cells derived from vessel explants facilitates in vitro characterization of phenotypic properties as exemplified by the differential response of Hoxc11-lacZ-positive versus-negative cells in migration assays and to serum.

Conclusion: The data support a conceptual model of Hox-specified positional identities in adult blood vessels, which is of likely relevance for understanding the mechanisms underlying regional physiological diversities in the cardiovascular system. The data also demonstrate that conventional Hox reporter gene mice are useful tools for visualizing complex Hox expression patterns in the vascular network that might be unattainable otherwise. Finally, these mice are a resource for the isolation and phenotypic characterization of specific subpopulations of vascular cells marked by distinct Hox expression profiles.

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Hoxc11-lacZ expression is restricted to a subset of smooth muscle cells in vessel explant culture. (A) Cellular outgrowth of explanted femoral artery/lateral marginal vein vessel segments (~2 mm; central dark ovoid structure in panel A) from young adult Hoxc11-lacZ transgenic mice [TG(Hoxc11/lacZ)62D9Awg] is comprised of β-gal-positive (blue) and -negative cells (black arrows in panel A, inset). (B) X-Gal labeling of cultured, femoral artery SMCs of Hoxc11-lacZ reporter mice reveals β-gal-positive (blue) and β-gal-negative cells as indicated by grey and white arrows, respectively. (B') Subsequent to X-Gal labeling, the cells shown in panel B were immunolabeled with antibodies specific for SMC marker Acta2 (formerly known as SMαA) as indicated by green fluorescence; note that both β-gal-positive and -negative cells as marked by grey and white arrows, respectively (grey and white arrows correspond to the same cells in panels B and B'), are labeled by anti-Acta2; nuclei were labeled with Hoechst 33342 (blue). (C/C'/C") Double-labeling of explant-derived, cultured femoral artery SMCs of TG(Hoxc11/lacZ)62D9Awg mice with antibodies against Hoxc11 (red signal, panel C) and anti-β-gal (green signal, panel C') results in a merged yellow signal in panel C". (D/E) Double-immunolabeling of cultured mitotic femoral artery SMCs with antibodies against Hoxc11 (red signal) and two standard SMC markers, including Transgelin (Tagln, formerly known as Smooth muscle protein 22-alpha; green signal, panel D), and Acta2 (green signal, panel E); note, overlap between Hoxc11 and Tagln signals in panel D results in yellow fluorescence, whereas the lesser degree of overlap between Hoxc11 and Acta2 signals in panel E results in discernible Hoxc11 (red) signal in the peri-nuclear region. Nuclei (blue) were labeled with Hoechst 33342.
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Figure 7: Hoxc11-lacZ expression is restricted to a subset of smooth muscle cells in vessel explant culture. (A) Cellular outgrowth of explanted femoral artery/lateral marginal vein vessel segments (~2 mm; central dark ovoid structure in panel A) from young adult Hoxc11-lacZ transgenic mice [TG(Hoxc11/lacZ)62D9Awg] is comprised of β-gal-positive (blue) and -negative cells (black arrows in panel A, inset). (B) X-Gal labeling of cultured, femoral artery SMCs of Hoxc11-lacZ reporter mice reveals β-gal-positive (blue) and β-gal-negative cells as indicated by grey and white arrows, respectively. (B') Subsequent to X-Gal labeling, the cells shown in panel B were immunolabeled with antibodies specific for SMC marker Acta2 (formerly known as SMαA) as indicated by green fluorescence; note that both β-gal-positive and -negative cells as marked by grey and white arrows, respectively (grey and white arrows correspond to the same cells in panels B and B'), are labeled by anti-Acta2; nuclei were labeled with Hoechst 33342 (blue). (C/C'/C") Double-labeling of explant-derived, cultured femoral artery SMCs of TG(Hoxc11/lacZ)62D9Awg mice with antibodies against Hoxc11 (red signal, panel C) and anti-β-gal (green signal, panel C') results in a merged yellow signal in panel C". (D/E) Double-immunolabeling of cultured mitotic femoral artery SMCs with antibodies against Hoxc11 (red signal) and two standard SMC markers, including Transgelin (Tagln, formerly known as Smooth muscle protein 22-alpha; green signal, panel D), and Acta2 (green signal, panel E); note, overlap between Hoxc11 and Tagln signals in panel D results in yellow fluorescence, whereas the lesser degree of overlap between Hoxc11 and Acta2 signals in panel E results in discernible Hoxc11 (red) signal in the peri-nuclear region. Nuclei (blue) were labeled with Hoechst 33342.

Mentions: Blood vessel explants (segments of ≈ 2 mm length) of the lateral marginal vein and the distal femoral artery of 6 wk old Hoxc11-lacZ mice were cultured to determine whether reporter gene activity was retained in outgrowing cells. Initial outgrowth was observed after ≈ 4 days under standard conditions (M199 medium supplemented with 20% FBS and pen-strep glutamine; see Materials and Methods), and X-Gal staining after 11 days of culture consistently showed β-gal activity in a fraction of the outgrowing cells (Fig. 7A, B), while control explants from corresponding regions of non-transgenic mice did not produce β-gal-positive cells (not shown). Co-labeling with antibodies directed against a common marker for smooth muscle cells (SMCs), i.e. Acta2 (previously known as SMαA) showed that the β-gal-positive cells expressed Acta2, whereas not all of the Acta2-expressing cells were β-gal-positive (compare panels B and B' in Fig. 7). Using Hoxc11- and β-gal-specific antibodies (see Materials and Methods for preparation and testing of Hoxc11-specific antibodies) in double-immunolabeling studies, we determined that these β-gal-positive cells do express endogenous Hoxc11 (Fig. 7C,C',C"). Furthermore, using the Hoxc11 antibodies in double-immunolabeling assays with Acta2 antibodies, as well as antibodies against a second SMC marker, Transgelin (Tagln; previously known as SM22) showed co-labeling in the perinuclear region of individual cultured VSMCs from the femoral artery of TG(Hoxc11/lacZ)62D9Awg mice in both cases (Fig. 7D,E).


Evidence for Hox-specified positional identities in adult vasculature.

Pruett ND, Visconti RP, Jacobs DF, Scholz D, McQuinn T, Sundberg JP, Awgulewitsch A - BMC Dev. Biol. (2008)

Hoxc11-lacZ expression is restricted to a subset of smooth muscle cells in vessel explant culture. (A) Cellular outgrowth of explanted femoral artery/lateral marginal vein vessel segments (~2 mm; central dark ovoid structure in panel A) from young adult Hoxc11-lacZ transgenic mice [TG(Hoxc11/lacZ)62D9Awg] is comprised of β-gal-positive (blue) and -negative cells (black arrows in panel A, inset). (B) X-Gal labeling of cultured, femoral artery SMCs of Hoxc11-lacZ reporter mice reveals β-gal-positive (blue) and β-gal-negative cells as indicated by grey and white arrows, respectively. (B') Subsequent to X-Gal labeling, the cells shown in panel B were immunolabeled with antibodies specific for SMC marker Acta2 (formerly known as SMαA) as indicated by green fluorescence; note that both β-gal-positive and -negative cells as marked by grey and white arrows, respectively (grey and white arrows correspond to the same cells in panels B and B'), are labeled by anti-Acta2; nuclei were labeled with Hoechst 33342 (blue). (C/C'/C") Double-labeling of explant-derived, cultured femoral artery SMCs of TG(Hoxc11/lacZ)62D9Awg mice with antibodies against Hoxc11 (red signal, panel C) and anti-β-gal (green signal, panel C') results in a merged yellow signal in panel C". (D/E) Double-immunolabeling of cultured mitotic femoral artery SMCs with antibodies against Hoxc11 (red signal) and two standard SMC markers, including Transgelin (Tagln, formerly known as Smooth muscle protein 22-alpha; green signal, panel D), and Acta2 (green signal, panel E); note, overlap between Hoxc11 and Tagln signals in panel D results in yellow fluorescence, whereas the lesser degree of overlap between Hoxc11 and Acta2 signals in panel E results in discernible Hoxc11 (red) signal in the peri-nuclear region. Nuclei (blue) were labeled with Hoechst 33342.
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Figure 7: Hoxc11-lacZ expression is restricted to a subset of smooth muscle cells in vessel explant culture. (A) Cellular outgrowth of explanted femoral artery/lateral marginal vein vessel segments (~2 mm; central dark ovoid structure in panel A) from young adult Hoxc11-lacZ transgenic mice [TG(Hoxc11/lacZ)62D9Awg] is comprised of β-gal-positive (blue) and -negative cells (black arrows in panel A, inset). (B) X-Gal labeling of cultured, femoral artery SMCs of Hoxc11-lacZ reporter mice reveals β-gal-positive (blue) and β-gal-negative cells as indicated by grey and white arrows, respectively. (B') Subsequent to X-Gal labeling, the cells shown in panel B were immunolabeled with antibodies specific for SMC marker Acta2 (formerly known as SMαA) as indicated by green fluorescence; note that both β-gal-positive and -negative cells as marked by grey and white arrows, respectively (grey and white arrows correspond to the same cells in panels B and B'), are labeled by anti-Acta2; nuclei were labeled with Hoechst 33342 (blue). (C/C'/C") Double-labeling of explant-derived, cultured femoral artery SMCs of TG(Hoxc11/lacZ)62D9Awg mice with antibodies against Hoxc11 (red signal, panel C) and anti-β-gal (green signal, panel C') results in a merged yellow signal in panel C". (D/E) Double-immunolabeling of cultured mitotic femoral artery SMCs with antibodies against Hoxc11 (red signal) and two standard SMC markers, including Transgelin (Tagln, formerly known as Smooth muscle protein 22-alpha; green signal, panel D), and Acta2 (green signal, panel E); note, overlap between Hoxc11 and Tagln signals in panel D results in yellow fluorescence, whereas the lesser degree of overlap between Hoxc11 and Acta2 signals in panel E results in discernible Hoxc11 (red) signal in the peri-nuclear region. Nuclei (blue) were labeled with Hoechst 33342.
Mentions: Blood vessel explants (segments of ≈ 2 mm length) of the lateral marginal vein and the distal femoral artery of 6 wk old Hoxc11-lacZ mice were cultured to determine whether reporter gene activity was retained in outgrowing cells. Initial outgrowth was observed after ≈ 4 days under standard conditions (M199 medium supplemented with 20% FBS and pen-strep glutamine; see Materials and Methods), and X-Gal staining after 11 days of culture consistently showed β-gal activity in a fraction of the outgrowing cells (Fig. 7A, B), while control explants from corresponding regions of non-transgenic mice did not produce β-gal-positive cells (not shown). Co-labeling with antibodies directed against a common marker for smooth muscle cells (SMCs), i.e. Acta2 (previously known as SMαA) showed that the β-gal-positive cells expressed Acta2, whereas not all of the Acta2-expressing cells were β-gal-positive (compare panels B and B' in Fig. 7). Using Hoxc11- and β-gal-specific antibodies (see Materials and Methods for preparation and testing of Hoxc11-specific antibodies) in double-immunolabeling studies, we determined that these β-gal-positive cells do express endogenous Hoxc11 (Fig. 7C,C',C"). Furthermore, using the Hoxc11 antibodies in double-immunolabeling assays with Acta2 antibodies, as well as antibodies against a second SMC marker, Transgelin (Tagln; previously known as SM22) showed co-labeling in the perinuclear region of individual cultured VSMCs from the femoral artery of TG(Hoxc11/lacZ)62D9Awg mice in both cases (Fig. 7D,E).

Bottom Line: These reporter gene patterns were validated as authentic indicators of endogenous gene expression by immunolabeling and PCR analysis.Furthermore, we show that persistent reporter gene expression in cultured cells derived from vessel explants facilitates in vitro characterization of phenotypic properties as exemplified by the differential response of Hoxc11-lacZ-positive versus-negative cells in migration assays and to serum.The data support a conceptual model of Hox-specified positional identities in adult blood vessels, which is of likely relevance for understanding the mechanisms underlying regional physiological diversities in the cardiovascular system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USA. pruettnd@musc.edu

ABSTRACT

Background: The concept of specifying positional information in the adult cardiovascular system is largely unexplored. While the Hox transcriptional regulators have to be viewed as excellent candidates for assuming such a role, little is known about their presumptive cardiovascular control functions and in vivo expression patterns.

Results: We demonstrate that conventional reporter gene analysis in transgenic mice is a useful approach for defining highly complex Hox expression patterns in the adult vascular network as exemplified by our lacZ reporter gene models for Hoxa3 and Hoxc11. These mice revealed expression in subsets of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) located in distinct regions of the vasculature that roughly correspond to the embryonic expression domains of the two genes. These reporter gene patterns were validated as authentic indicators of endogenous gene expression by immunolabeling and PCR analysis. Furthermore, we show that persistent reporter gene expression in cultured cells derived from vessel explants facilitates in vitro characterization of phenotypic properties as exemplified by the differential response of Hoxc11-lacZ-positive versus-negative cells in migration assays and to serum.

Conclusion: The data support a conceptual model of Hox-specified positional identities in adult blood vessels, which is of likely relevance for understanding the mechanisms underlying regional physiological diversities in the cardiovascular system. The data also demonstrate that conventional Hox reporter gene mice are useful tools for visualizing complex Hox expression patterns in the vascular network that might be unattainable otherwise. Finally, these mice are a resource for the isolation and phenotypic characterization of specific subpopulations of vascular cells marked by distinct Hox expression profiles.

Show MeSH