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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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Analysis of Hoxc11-lacZ reporter gene expression in hindlimb vasculature. (A) E12 embryo of the transgenic shows posteriorly restricted lacZ reporter gene expression (blue) in most of the hindlimb region and tailbud, as well as in the posterior zeugopodal forelimb region. (B) X-Gal staining of posterior trunk and hindlimbs of 0.5 d newborn Hoxc11-lacZ transgenic mouse after skinning reveals expression in skeletal muscles and blood vessels (yellow arrow). (C) Lateral view of stained hindlimb of 42 d Hoxc11-lacZ transgenic mouse shows conspicuous β-gal activity in sciatic artery. (D-F) Medial view of same hindlimb as shown in panel C is presented in panel E and reveals distally restricted expression in both arteries and veins; close-up views of upper and lower sections of femoral artery (red arrows) and vein (blue arrows) are shown in panels D and F, respectively. (G) Lateral view of stained hindlimb from 1 yr old Hoxc11-lacZ transgenic mouse shows persistent reporter gene expression in blood vessels, most prominently in sciatic artery. All mice were derived from transgenic line TG(Hoxc11-lacZ)62D9Awg; fl: forelimb; hl: hindlimb; tlb: tailbud.
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Figure 1: Analysis of Hoxc11-lacZ reporter gene expression in hindlimb vasculature. (A) E12 embryo of the transgenic shows posteriorly restricted lacZ reporter gene expression (blue) in most of the hindlimb region and tailbud, as well as in the posterior zeugopodal forelimb region. (B) X-Gal staining of posterior trunk and hindlimbs of 0.5 d newborn Hoxc11-lacZ transgenic mouse after skinning reveals expression in skeletal muscles and blood vessels (yellow arrow). (C) Lateral view of stained hindlimb of 42 d Hoxc11-lacZ transgenic mouse shows conspicuous β-gal activity in sciatic artery. (D-F) Medial view of same hindlimb as shown in panel C is presented in panel E and reveals distally restricted expression in both arteries and veins; close-up views of upper and lower sections of femoral artery (red arrows) and vein (blue arrows) are shown in panels D and F, respectively. (G) Lateral view of stained hindlimb from 1 yr old Hoxc11-lacZ transgenic mouse shows persistent reporter gene expression in blood vessels, most prominently in sciatic artery. All mice were derived from transgenic line TG(Hoxc11-lacZ)62D9Awg; fl: forelimb; hl: hindlimb; tlb: tailbud.

Mentions: We previously reported that transgenic mice (n = 4 founders) carrying a Hoxc11-lacZ reporter gene construct in which E.coli lacZ with SV40 RNA processing signals was fused in-frame to Hoxc11 exon 1 coding sequences exhibited a conspicuous and reproducible β-gal expression pattern in mid-gestation embryos at ≈ E12 [25]. This pattern (Fig. 1A) was consistent with the posteriorly restricted pattern of endogenous Hoxc11 expression in par-axial and hindlimb mesenchyme as determined by ISH [25,28,29]. Accordingly, we concluded that this Hoxc11-lacZ construct included most of the control elements required for establishing the native Hoxc11 expression pattern in mid-gestation embryos [25,28,29]. Here, we used one of these Hoxc11-lacZ transgenic lines as a tool for gaining insight into global aspects of Hoxc11 expression during fetal and postnatal development, as well as in adulthood. This revealed β-gal activity in the hindlimb vasculature that had previously escaped our attention. Examination of young adult transgenic mice showed lacZ expression (X-Gal labeling) in all major blood vessels of the hindlimb, including arteries and veins (Fig. 1D–F) with strongest expression at the level of the zeugopod and lower stylopod (femur). In several vessels, including the femoral artery, X-Gal labeling was rather uniform in the lower limb and became progressively mosaic in upper femoral limb regions near the expression boundary (Fig. 1E, F). However, vessels showing mosaic β-gal expression patterns could also be seen next to vessels that appeared to be uniformly stained at a given proximal-distal level of the zeugopod. Persistent vascular expression was observed in animals of 1 year of age (Fig. 1G), the latest stage examined.


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)

Analysis of Hoxc11-lacZ reporter gene expression in hindlimb vasculature. (A) E12 embryo of the transgenic shows posteriorly restricted lacZ reporter gene expression (blue) in most of the hindlimb region and tailbud, as well as in the posterior zeugopodal forelimb region. (B) X-Gal staining of posterior trunk and hindlimbs of 0.5 d newborn Hoxc11-lacZ transgenic mouse after skinning reveals expression in skeletal muscles and blood vessels (yellow arrow). (C) Lateral view of stained hindlimb of 42 d Hoxc11-lacZ transgenic mouse shows conspicuous β-gal activity in sciatic artery. (D-F) Medial view of same hindlimb as shown in panel C is presented in panel E and reveals distally restricted expression in both arteries and veins; close-up views of upper and lower sections of femoral artery (red arrows) and vein (blue arrows) are shown in panels D and F, respectively. (G) Lateral view of stained hindlimb from 1 yr old Hoxc11-lacZ transgenic mouse shows persistent reporter gene expression in blood vessels, most prominently in sciatic artery. All mice were derived from transgenic line TG(Hoxc11-lacZ)62D9Awg; fl: forelimb; hl: hindlimb; tlb: tailbud.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Analysis of Hoxc11-lacZ reporter gene expression in hindlimb vasculature. (A) E12 embryo of the transgenic shows posteriorly restricted lacZ reporter gene expression (blue) in most of the hindlimb region and tailbud, as well as in the posterior zeugopodal forelimb region. (B) X-Gal staining of posterior trunk and hindlimbs of 0.5 d newborn Hoxc11-lacZ transgenic mouse after skinning reveals expression in skeletal muscles and blood vessels (yellow arrow). (C) Lateral view of stained hindlimb of 42 d Hoxc11-lacZ transgenic mouse shows conspicuous β-gal activity in sciatic artery. (D-F) Medial view of same hindlimb as shown in panel C is presented in panel E and reveals distally restricted expression in both arteries and veins; close-up views of upper and lower sections of femoral artery (red arrows) and vein (blue arrows) are shown in panels D and F, respectively. (G) Lateral view of stained hindlimb from 1 yr old Hoxc11-lacZ transgenic mouse shows persistent reporter gene expression in blood vessels, most prominently in sciatic artery. All mice were derived from transgenic line TG(Hoxc11-lacZ)62D9Awg; fl: forelimb; hl: hindlimb; tlb: tailbud.
Mentions: We previously reported that transgenic mice (n = 4 founders) carrying a Hoxc11-lacZ reporter gene construct in which E.coli lacZ with SV40 RNA processing signals was fused in-frame to Hoxc11 exon 1 coding sequences exhibited a conspicuous and reproducible β-gal expression pattern in mid-gestation embryos at ≈ E12 [25]. This pattern (Fig. 1A) was consistent with the posteriorly restricted pattern of endogenous Hoxc11 expression in par-axial and hindlimb mesenchyme as determined by ISH [25,28,29]. Accordingly, we concluded that this Hoxc11-lacZ construct included most of the control elements required for establishing the native Hoxc11 expression pattern in mid-gestation embryos [25,28,29]. Here, we used one of these Hoxc11-lacZ transgenic lines as a tool for gaining insight into global aspects of Hoxc11 expression during fetal and postnatal development, as well as in adulthood. This revealed β-gal activity in the hindlimb vasculature that had previously escaped our attention. Examination of young adult transgenic mice showed lacZ expression (X-Gal labeling) in all major blood vessels of the hindlimb, including arteries and veins (Fig. 1D–F) with strongest expression at the level of the zeugopod and lower stylopod (femur). In several vessels, including the femoral artery, X-Gal labeling was rather uniform in the lower limb and became progressively mosaic in upper femoral limb regions near the expression boundary (Fig. 1E, F). However, vessels showing mosaic β-gal expression patterns could also be seen next to vessels that appeared to be uniformly stained at a given proximal-distal level of the zeugopod. Persistent vascular expression was observed in animals of 1 year of age (Fig. 1G), the latest stage examined.

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