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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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Functional assays of Hoxc11-lacZ expression in cell culture. (A/B) Explant cultures. (A) The proportion of β-gal-positve cells derived from explant cultures of lateral marginal veins and femoral arteries from 6 weeks old Hoxc11-lacZ mice is approximately 4.3% (± 1.92%) under culture conditions that include 20% fetal bovine serum; this increases to approximately 3-fold to 13.5% (± 5.34%) under serum-free conditions; P < 0.001. (B) Cell migration/scratch assay indicates a dramatic (71-fold) reduction in transgene expression in the group of migratory cells found in the scratch area, (8.6% ± 2.2 blue cells in normal (unwounded) area compared to 0.11% ± 0.46 in wounded area); difference of approximately 71-fold, P < 0.001.
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Figure 8: Functional assays of Hoxc11-lacZ expression in cell culture. (A/B) Explant cultures. (A) The proportion of β-gal-positve cells derived from explant cultures of lateral marginal veins and femoral arteries from 6 weeks old Hoxc11-lacZ mice is approximately 4.3% (± 1.92%) under culture conditions that include 20% fetal bovine serum; this increases to approximately 3-fold to 13.5% (± 5.34%) under serum-free conditions; P < 0.001. (B) Cell migration/scratch assay indicates a dramatic (71-fold) reduction in transgene expression in the group of migratory cells found in the scratch area, (8.6% ± 2.2 blue cells in normal (unwounded) area compared to 0.11% ± 0.46 in wounded area); difference of approximately 71-fold, P < 0.001.

Mentions: Continued Hoxc11-lacZ expression in only a subpopulation of cultured VSMCs raised the question whether these cells might be functionally distinct compared to β-gal-negative cells. This was addressed in a preliminary manner by performing two simple and straightforward in vitro assays that tested response to culture serum levels and migration behavior. The results show that the proportion of Hoxc11-lacZ-expressing cells increased approximately 3-fold to 13.5% under serum-free culture conditions compared to about 4.3% under conditions including 20% FBS (Fig. 8A). In scratch assays, the proportion of β-gal-positive cells was dramatically reduced (71-fold) among the migratory cells found in the scratch area compared to the cells residing in the undisturbed monolayer (Fig. 8B). These differential reporter gene expression patterns in cultured VSMCs undergoing phenotypic modulation suggest that Hoxc11 expression is associated with distinct VSMC phenotypes.


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)

Functional assays of Hoxc11-lacZ expression in cell culture. (A/B) Explant cultures. (A) The proportion of β-gal-positve cells derived from explant cultures of lateral marginal veins and femoral arteries from 6 weeks old Hoxc11-lacZ mice is approximately 4.3% (± 1.92%) under culture conditions that include 20% fetal bovine serum; this increases to approximately 3-fold to 13.5% (± 5.34%) under serum-free conditions; P < 0.001. (B) Cell migration/scratch assay indicates a dramatic (71-fold) reduction in transgene expression in the group of migratory cells found in the scratch area, (8.6% ± 2.2 blue cells in normal (unwounded) area compared to 0.11% ± 0.46 in wounded area); difference of approximately 71-fold, P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570687&req=5

Figure 8: Functional assays of Hoxc11-lacZ expression in cell culture. (A/B) Explant cultures. (A) The proportion of β-gal-positve cells derived from explant cultures of lateral marginal veins and femoral arteries from 6 weeks old Hoxc11-lacZ mice is approximately 4.3% (± 1.92%) under culture conditions that include 20% fetal bovine serum; this increases to approximately 3-fold to 13.5% (± 5.34%) under serum-free conditions; P < 0.001. (B) Cell migration/scratch assay indicates a dramatic (71-fold) reduction in transgene expression in the group of migratory cells found in the scratch area, (8.6% ± 2.2 blue cells in normal (unwounded) area compared to 0.11% ± 0.46 in wounded area); difference of approximately 71-fold, P < 0.001.
Mentions: Continued Hoxc11-lacZ expression in only a subpopulation of cultured VSMCs raised the question whether these cells might be functionally distinct compared to β-gal-negative cells. This was addressed in a preliminary manner by performing two simple and straightforward in vitro assays that tested response to culture serum levels and migration behavior. The results show that the proportion of Hoxc11-lacZ-expressing cells increased approximately 3-fold to 13.5% under serum-free culture conditions compared to about 4.3% under conditions including 20% FBS (Fig. 8A). In scratch assays, the proportion of β-gal-positive cells was dramatically reduced (71-fold) among the migratory cells found in the scratch area compared to the cells residing in the undisturbed monolayer (Fig. 8B). These differential reporter gene expression patterns in cultured VSMCs undergoing phenotypic modulation suggest that Hoxc11 expression is associated with distinct VSMC phenotypes.

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
Related in: MedlinePlus