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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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Validation of endogenous Hoxa3 expression boundary in adult carotid artery of FVB/NTac mouse. (A) Diagram of right carotid segment indicating reporter gene expression domain (blue shading) with anterior boundary at level of lingual artery (la) as observed in Hoxa3-lacZ transgenic mouse (see Fig. 2E for comparison); yellow arrow points to carotid bifurcation into internal (ica) and external carotid arteries (eca), respectively; sections corresponding to levels L1 and L2 as indicated in the cartoon were used for detection of Hoxa3 and Acta2 by immunofluorescence as shown in the three upper (B,B'B") and lower (C,C',C") panels to the right. Both L1 and L2 sections were incubated with Hoxa3-specific and Cy3-conjugated Acta2-specific antibodies; using Cy5-conjugated secondary antibodies, red-fluoresent-labeled Hoxa3 protein was detected only in section L2 (panel C'), whereas green-fluorescent-labeled Acta2 expressed in VSMCs was detected in both L1 and L2 sections (panels B" and C"); accordingly, multichannel composite micrographs (panels B and C) for detecting red (Cy5), green (Cy3) and blue (Hoechst 33342 for nuclear labeling; this is not shown individually) indicate co-localization of Hoxa3 and Acta2 (yellow) in VSMCs only in section L2 (panel C). fa: facial artery; oa: occipital artery.
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Figure 5: Validation of endogenous Hoxa3 expression boundary in adult carotid artery of FVB/NTac mouse. (A) Diagram of right carotid segment indicating reporter gene expression domain (blue shading) with anterior boundary at level of lingual artery (la) as observed in Hoxa3-lacZ transgenic mouse (see Fig. 2E for comparison); yellow arrow points to carotid bifurcation into internal (ica) and external carotid arteries (eca), respectively; sections corresponding to levels L1 and L2 as indicated in the cartoon were used for detection of Hoxa3 and Acta2 by immunofluorescence as shown in the three upper (B,B'B") and lower (C,C',C") panels to the right. Both L1 and L2 sections were incubated with Hoxa3-specific and Cy3-conjugated Acta2-specific antibodies; using Cy5-conjugated secondary antibodies, red-fluoresent-labeled Hoxa3 protein was detected only in section L2 (panel C'), whereas green-fluorescent-labeled Acta2 expressed in VSMCs was detected in both L1 and L2 sections (panels B" and C"); accordingly, multichannel composite micrographs (panels B and C) for detecting red (Cy5), green (Cy3) and blue (Hoechst 33342 for nuclear labeling; this is not shown individually) indicate co-localization of Hoxa3 and Acta2 (yellow) in VSMCs only in section L2 (panel C). fa: facial artery; oa: occipital artery.

Mentions: Evidence that the anterior Hoxa3-lacZ boundary in carotid arteries (Fig. 2E) reflects the endogenous Hoxa3 pattern in these vessels was provided at the protein level by immunolabeling studies with Hoxa3-specific antibodies that detected Hoxa3 protein exclusively in sections of carotid vessel segments located posterior of this boundary (Fig. 5). Furthermore, while Hoxa3-lacZ expression in the endothelial layer was difficult to discern by X-Gal staining as exemplified in Figure 6A, endogenous Hoxa3 protein expression in both ECs and VSMCs of the aortic arch was confirmed by immunolabeling (Fig. 6B,C). Combined, these data strongly suggest that Hoxa3-lacZ mimics the authentic Hoxa3 expression pattern in adult blood vessels. The Hoxa3-lacZ patterns correlate remarkably well with the broad spectrum of vascular defects previously observed in Hoxa3 mice, which included abnormalities of the carotid system, thinning of the aorta and enlargement of major veins [4,6]. Lack of information about the Hoxa3 vascular expression pattern, however, largely precluded interpretation of these data with regard to potential underlying mechanisms.


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)

Validation of endogenous Hoxa3 expression boundary in adult carotid artery of FVB/NTac mouse. (A) Diagram of right carotid segment indicating reporter gene expression domain (blue shading) with anterior boundary at level of lingual artery (la) as observed in Hoxa3-lacZ transgenic mouse (see Fig. 2E for comparison); yellow arrow points to carotid bifurcation into internal (ica) and external carotid arteries (eca), respectively; sections corresponding to levels L1 and L2 as indicated in the cartoon were used for detection of Hoxa3 and Acta2 by immunofluorescence as shown in the three upper (B,B'B") and lower (C,C',C") panels to the right. Both L1 and L2 sections were incubated with Hoxa3-specific and Cy3-conjugated Acta2-specific antibodies; using Cy5-conjugated secondary antibodies, red-fluoresent-labeled Hoxa3 protein was detected only in section L2 (panel C'), whereas green-fluorescent-labeled Acta2 expressed in VSMCs was detected in both L1 and L2 sections (panels B" and C"); accordingly, multichannel composite micrographs (panels B and C) for detecting red (Cy5), green (Cy3) and blue (Hoechst 33342 for nuclear labeling; this is not shown individually) indicate co-localization of Hoxa3 and Acta2 (yellow) in VSMCs only in section L2 (panel C). fa: facial artery; oa: occipital artery.
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Figure 5: Validation of endogenous Hoxa3 expression boundary in adult carotid artery of FVB/NTac mouse. (A) Diagram of right carotid segment indicating reporter gene expression domain (blue shading) with anterior boundary at level of lingual artery (la) as observed in Hoxa3-lacZ transgenic mouse (see Fig. 2E for comparison); yellow arrow points to carotid bifurcation into internal (ica) and external carotid arteries (eca), respectively; sections corresponding to levels L1 and L2 as indicated in the cartoon were used for detection of Hoxa3 and Acta2 by immunofluorescence as shown in the three upper (B,B'B") and lower (C,C',C") panels to the right. Both L1 and L2 sections were incubated with Hoxa3-specific and Cy3-conjugated Acta2-specific antibodies; using Cy5-conjugated secondary antibodies, red-fluoresent-labeled Hoxa3 protein was detected only in section L2 (panel C'), whereas green-fluorescent-labeled Acta2 expressed in VSMCs was detected in both L1 and L2 sections (panels B" and C"); accordingly, multichannel composite micrographs (panels B and C) for detecting red (Cy5), green (Cy3) and blue (Hoechst 33342 for nuclear labeling; this is not shown individually) indicate co-localization of Hoxa3 and Acta2 (yellow) in VSMCs only in section L2 (panel C). fa: facial artery; oa: occipital artery.
Mentions: Evidence that the anterior Hoxa3-lacZ boundary in carotid arteries (Fig. 2E) reflects the endogenous Hoxa3 pattern in these vessels was provided at the protein level by immunolabeling studies with Hoxa3-specific antibodies that detected Hoxa3 protein exclusively in sections of carotid vessel segments located posterior of this boundary (Fig. 5). Furthermore, while Hoxa3-lacZ expression in the endothelial layer was difficult to discern by X-Gal staining as exemplified in Figure 6A, endogenous Hoxa3 protein expression in both ECs and VSMCs of the aortic arch was confirmed by immunolabeling (Fig. 6B,C). Combined, these data strongly suggest that Hoxa3-lacZ mimics the authentic Hoxa3 expression pattern in adult blood vessels. The Hoxa3-lacZ patterns correlate remarkably well with the broad spectrum of vascular defects previously observed in Hoxa3 mice, which included abnormalities of the carotid system, thinning of the aorta and enlargement of major veins [4,6]. Lack of information about the Hoxa3 vascular expression pattern, however, largely precluded interpretation of these data with regard to potential underlying mechanisms.

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