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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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RT-PCR analysis of Hox gene expression in adult blood vessels. (A) Total RNA derived from hindlimb blood vessels (6 wk old FVB/NTac mice) as defined in the text was used for cDNA synthesis, and Hox-specific cDNA fragments were amplified by using primers specific for the AbdB-type Hoxc genes; this resulted in PCR products in the expected size range for Hoxc9 (323 bp), Hoxc10 (350 bp), and Hoxc11 (435 bp); PCR reactions with primers specific for the Hoxc11 and Hoxc10 paralogous genes (Hoxa11 and Hoxd11, as well as Hoxa10 and Hoxd10) resulted in amplification products in the expected size range for Hoxa11 (220 bp), Hoxd11 (131 bp), and Hoxd10 (286 bp), whereas no product was detected for Hoxa10; positive control reaction: Gapdh-specific primers; MW: molecular weight standards in base pairs (bp). (B) RT-PCR analysis of Hoxa3 and Hoxc11 expression in adult mouse (6 wk) blood vessel segments including aortic arch (AA), descending thoracic aorta (DA), and distal femoral artery (dFA); positive control reaction was performed with β-actin-specific primers.
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Figure 4: RT-PCR analysis of Hox gene expression in adult blood vessels. (A) Total RNA derived from hindlimb blood vessels (6 wk old FVB/NTac mice) as defined in the text was used for cDNA synthesis, and Hox-specific cDNA fragments were amplified by using primers specific for the AbdB-type Hoxc genes; this resulted in PCR products in the expected size range for Hoxc9 (323 bp), Hoxc10 (350 bp), and Hoxc11 (435 bp); PCR reactions with primers specific for the Hoxc11 and Hoxc10 paralogous genes (Hoxa11 and Hoxd11, as well as Hoxa10 and Hoxd10) resulted in amplification products in the expected size range for Hoxa11 (220 bp), Hoxd11 (131 bp), and Hoxd10 (286 bp), whereas no product was detected for Hoxa10; positive control reaction: Gapdh-specific primers; MW: molecular weight standards in base pairs (bp). (B) RT-PCR analysis of Hoxa3 and Hoxc11 expression in adult mouse (6 wk) blood vessel segments including aortic arch (AA), descending thoracic aorta (DA), and distal femoral artery (dFA); positive control reaction was performed with β-actin-specific primers.

Mentions: To determine whether the vascular Hoxc11- and Hoxa3-lacZ reporter gene expression reflected corresponding endogenous gene activities, we performed semi-quantitative reverse transcriptase (RT)-PCR analysis of total RNA isolated from defined vessel segments of 6 wk old mice. For a first set of reactions, we used RNA isolated from femoral artery and vein segments ranging from the mid-femoral region just proximal to the Hoxc11-lacZ expression boundary to the distal femoral artery branch point approximately at knee-level. In addition to examining this RNA sample for the presence of Hoxc11-specific transcripts, we determined whether there was evidence for expression of the remaining AbdB-type Hoxc genes (Hoxc9, Hoxc10, Hoxc12, and Hoxc13), as well as for the Hoxc11 and Hoxc10 paralogous genes (Hoxa11, Hoxd11, and Hoxa10, Hoxd10). The results indicate strong expression of Hoxc11 and Hoxc10, whereas Hoxc9 expression appeared less abundant, and expression of Hoxc12 and Hoxc13 was not detectable (Fig. 4A). Strong expression was also indicated for two of the paralogous genes, Hoxa11 and Hoxd10, whereas no RT-PCR amplification product was detected for Hoxa10, and the band corresponding to Hoxd11 was weak (Fig. 4A). These data suggest selective activity of AbdB-type Hoxc genes in major blood vessels of the mid-femoral hindlimb region. Specifically, the detection of Hoxc11-specific amplification products suggests that the vascular Hoxc11-lacZ reporter gene expression observed in this region reflects authentic Hoxc11 transcriptional activity. The detection of Hoxa11- and Hoxc9-specific amplification products is consistent with the isolation of HOXA11 and HOXC9 cDNAs from human smooth muscle cell cDNA libraries reported previously [18].


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)

RT-PCR analysis of Hox gene expression in adult blood vessels. (A) Total RNA derived from hindlimb blood vessels (6 wk old FVB/NTac mice) as defined in the text was used for cDNA synthesis, and Hox-specific cDNA fragments were amplified by using primers specific for the AbdB-type Hoxc genes; this resulted in PCR products in the expected size range for Hoxc9 (323 bp), Hoxc10 (350 bp), and Hoxc11 (435 bp); PCR reactions with primers specific for the Hoxc11 and Hoxc10 paralogous genes (Hoxa11 and Hoxd11, as well as Hoxa10 and Hoxd10) resulted in amplification products in the expected size range for Hoxa11 (220 bp), Hoxd11 (131 bp), and Hoxd10 (286 bp), whereas no product was detected for Hoxa10; positive control reaction: Gapdh-specific primers; MW: molecular weight standards in base pairs (bp). (B) RT-PCR analysis of Hoxa3 and Hoxc11 expression in adult mouse (6 wk) blood vessel segments including aortic arch (AA), descending thoracic aorta (DA), and distal femoral artery (dFA); positive control reaction was performed with β-actin-specific primers.
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Figure 4: RT-PCR analysis of Hox gene expression in adult blood vessels. (A) Total RNA derived from hindlimb blood vessels (6 wk old FVB/NTac mice) as defined in the text was used for cDNA synthesis, and Hox-specific cDNA fragments were amplified by using primers specific for the AbdB-type Hoxc genes; this resulted in PCR products in the expected size range for Hoxc9 (323 bp), Hoxc10 (350 bp), and Hoxc11 (435 bp); PCR reactions with primers specific for the Hoxc11 and Hoxc10 paralogous genes (Hoxa11 and Hoxd11, as well as Hoxa10 and Hoxd10) resulted in amplification products in the expected size range for Hoxa11 (220 bp), Hoxd11 (131 bp), and Hoxd10 (286 bp), whereas no product was detected for Hoxa10; positive control reaction: Gapdh-specific primers; MW: molecular weight standards in base pairs (bp). (B) RT-PCR analysis of Hoxa3 and Hoxc11 expression in adult mouse (6 wk) blood vessel segments including aortic arch (AA), descending thoracic aorta (DA), and distal femoral artery (dFA); positive control reaction was performed with β-actin-specific primers.
Mentions: To determine whether the vascular Hoxc11- and Hoxa3-lacZ reporter gene expression reflected corresponding endogenous gene activities, we performed semi-quantitative reverse transcriptase (RT)-PCR analysis of total RNA isolated from defined vessel segments of 6 wk old mice. For a first set of reactions, we used RNA isolated from femoral artery and vein segments ranging from the mid-femoral region just proximal to the Hoxc11-lacZ expression boundary to the distal femoral artery branch point approximately at knee-level. In addition to examining this RNA sample for the presence of Hoxc11-specific transcripts, we determined whether there was evidence for expression of the remaining AbdB-type Hoxc genes (Hoxc9, Hoxc10, Hoxc12, and Hoxc13), as well as for the Hoxc11 and Hoxc10 paralogous genes (Hoxa11, Hoxd11, and Hoxa10, Hoxd10). The results indicate strong expression of Hoxc11 and Hoxc10, whereas Hoxc9 expression appeared less abundant, and expression of Hoxc12 and Hoxc13 was not detectable (Fig. 4A). Strong expression was also indicated for two of the paralogous genes, Hoxa11 and Hoxd10, whereas no RT-PCR amplification product was detected for Hoxa10, and the band corresponding to Hoxd11 was weak (Fig. 4A). These data suggest selective activity of AbdB-type Hoxc genes in major blood vessels of the mid-femoral hindlimb region. Specifically, the detection of Hoxc11-specific amplification products suggests that the vascular Hoxc11-lacZ reporter gene expression observed in this region reflects authentic Hoxc11 transcriptional activity. The detection of Hoxa11- and Hoxc9-specific amplification products is consistent with the isolation of HOXA11 and HOXC9 cDNAs from human smooth muscle cell cDNA libraries reported previously [18].

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