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Invariant asymmetry renews the lymphatic vasculature during homeostasis.

Connor AL, Kelley PM, Tempero RM - J Transl Med (2016)

Bottom Line: Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period.The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis.These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosensory Genetics and Otolaryngology and Head and Neck Surgery, Boys Town National Research Hospital, 555 North 30th Street, Omaha, NE, 68131, USA.

ABSTRACT

Background: The lymphatic vasculature regulates tissue physiology and immunity throughout life. The self renewal mechanism that maintains the lymphatic vasculature during conditions of homeostasis is unknown. The purpose of this study was to investigate the cellular mechanism of lymphatic endothelial cell (LEC) self renewal and lymphatic vessel maintenance.

Methods: Inductive genetic techniques were used to label LECs with tandem dimer tomato (tdT) in adult mice. Two types of studies were performed, those with high dose inductive conditions to label nearly all the lymphatic vessels and studies with low dose inductive conditions to stochastically label individual clones or small populations of LECs. We coupled image guidance techniques and live fluorescence microscopy imaging with lineage tracing to track the fate of entire tdT(+) cutaneous lymphatic vessels or the behavior of individual or small populations of LECs over 11 months. We tracked the fate of 110 LEC clones and 80 small LEC populations (clusters of 2-7 cells) over 11 months and analyzed their behavior using quantitative techniques.

Results: The results of the high dose inductive studies showed that the lymphatic vessels remained tdT(+) over 11 months, suggesting passage and expression of the tdT transgene from LEC precursors to progenies, an intrinsic model of self- renewal. Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period. By following the behavior of labeled LEC clones or small populations of LECs individually over 11 months, we identified diverse LEC fates of proliferation, quiescence, and extinction. Quantitative analysis of this data revealed that the average lymphatic endothelial clone or small population remained stable in size despite diverse individual fates.

Conclusion: The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis. These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

No MeSH data available.


Related in: MedlinePlus

Live imaging methodology for capturing the same fields of interest overtime within the Lyve1CreERT2tdT pinna containing single tdT+ clones and small populations. A single 0.25 mg 4-OHT dose was administered to 4 Lyve1CreERT2tdT mice to induce tdT fluorescence stochastically in individual cells. Using low power brightfield microscopy, regions of interest within the pinna were identified based on the morphologic features of large blood vessels (a–c). An asterisk shows a blood vessel branch point as an example. Without adjusting the position of the pinna or the focus, the Texas Red filter was used to detect the endogenous tdT fluorescence (d–f). The magnified fields show examples of single tdT+ clones (arrow) and a tdT+ small population of 3 cells (arrowhead) (g–i). The tdT+ cells were visualized 20 (g), 25 (h), and 44 (i) days following labeling. This methodology was used to identify 110 tdT+ clones and 80 tdT+ small populations that were tracked over time. The size standard is 100 µm
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Fig4: Live imaging methodology for capturing the same fields of interest overtime within the Lyve1CreERT2tdT pinna containing single tdT+ clones and small populations. A single 0.25 mg 4-OHT dose was administered to 4 Lyve1CreERT2tdT mice to induce tdT fluorescence stochastically in individual cells. Using low power brightfield microscopy, regions of interest within the pinna were identified based on the morphologic features of large blood vessels (a–c). An asterisk shows a blood vessel branch point as an example. Without adjusting the position of the pinna or the focus, the Texas Red filter was used to detect the endogenous tdT fluorescence (d–f). The magnified fields show examples of single tdT+ clones (arrow) and a tdT+ small population of 3 cells (arrowhead) (g–i). The tdT+ cells were visualized 20 (g), 25 (h), and 44 (i) days following labeling. This methodology was used to identify 110 tdT+ clones and 80 tdT+ small populations that were tracked over time. The size standard is 100 µm

Mentions: Low dose 4-OHT was administered to a cohort of 4 Lyve1CreERT2tdT mice to induce tdT fluorescence in individual cells. We used guidance techniques based on blood vessel position and morphology visualized in the bright field to identify 3 regions in the pinna of sedated Lyve1CreERT2tdT mice. An example of one region is shown 20, 25, and 44 days post labeling (Fig. 4a–c). Without changing the position of the mouse, the texas red filter was used to visualize the tdT fluorescence in the pinna (Fig. 4d–f). These techniques were performed serially over the course of 323 days. In the images obtained using fluorescence microscopy single tdT+ cells and clusters of tdT+ cells were detected (Fig. 4g–i). We identified 110 tdT+ clones and 80 tdT+ small populations using these techniques and tracked the individual fate of these different populations over time.Fig. 4


Invariant asymmetry renews the lymphatic vasculature during homeostasis.

Connor AL, Kelley PM, Tempero RM - J Transl Med (2016)

Live imaging methodology for capturing the same fields of interest overtime within the Lyve1CreERT2tdT pinna containing single tdT+ clones and small populations. A single 0.25 mg 4-OHT dose was administered to 4 Lyve1CreERT2tdT mice to induce tdT fluorescence stochastically in individual cells. Using low power brightfield microscopy, regions of interest within the pinna were identified based on the morphologic features of large blood vessels (a–c). An asterisk shows a blood vessel branch point as an example. Without adjusting the position of the pinna or the focus, the Texas Red filter was used to detect the endogenous tdT fluorescence (d–f). The magnified fields show examples of single tdT+ clones (arrow) and a tdT+ small population of 3 cells (arrowhead) (g–i). The tdT+ cells were visualized 20 (g), 25 (h), and 44 (i) days following labeling. This methodology was used to identify 110 tdT+ clones and 80 tdT+ small populations that were tracked over time. The size standard is 100 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940917&req=5

Fig4: Live imaging methodology for capturing the same fields of interest overtime within the Lyve1CreERT2tdT pinna containing single tdT+ clones and small populations. A single 0.25 mg 4-OHT dose was administered to 4 Lyve1CreERT2tdT mice to induce tdT fluorescence stochastically in individual cells. Using low power brightfield microscopy, regions of interest within the pinna were identified based on the morphologic features of large blood vessels (a–c). An asterisk shows a blood vessel branch point as an example. Without adjusting the position of the pinna or the focus, the Texas Red filter was used to detect the endogenous tdT fluorescence (d–f). The magnified fields show examples of single tdT+ clones (arrow) and a tdT+ small population of 3 cells (arrowhead) (g–i). The tdT+ cells were visualized 20 (g), 25 (h), and 44 (i) days following labeling. This methodology was used to identify 110 tdT+ clones and 80 tdT+ small populations that were tracked over time. The size standard is 100 µm
Mentions: Low dose 4-OHT was administered to a cohort of 4 Lyve1CreERT2tdT mice to induce tdT fluorescence in individual cells. We used guidance techniques based on blood vessel position and morphology visualized in the bright field to identify 3 regions in the pinna of sedated Lyve1CreERT2tdT mice. An example of one region is shown 20, 25, and 44 days post labeling (Fig. 4a–c). Without changing the position of the mouse, the texas red filter was used to visualize the tdT fluorescence in the pinna (Fig. 4d–f). These techniques were performed serially over the course of 323 days. In the images obtained using fluorescence microscopy single tdT+ cells and clusters of tdT+ cells were detected (Fig. 4g–i). We identified 110 tdT+ clones and 80 tdT+ small populations using these techniques and tracked the individual fate of these different populations over time.Fig. 4

Bottom Line: Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period.The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis.These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosensory Genetics and Otolaryngology and Head and Neck Surgery, Boys Town National Research Hospital, 555 North 30th Street, Omaha, NE, 68131, USA.

ABSTRACT

Background: The lymphatic vasculature regulates tissue physiology and immunity throughout life. The self renewal mechanism that maintains the lymphatic vasculature during conditions of homeostasis is unknown. The purpose of this study was to investigate the cellular mechanism of lymphatic endothelial cell (LEC) self renewal and lymphatic vessel maintenance.

Methods: Inductive genetic techniques were used to label LECs with tandem dimer tomato (tdT) in adult mice. Two types of studies were performed, those with high dose inductive conditions to label nearly all the lymphatic vessels and studies with low dose inductive conditions to stochastically label individual clones or small populations of LECs. We coupled image guidance techniques and live fluorescence microscopy imaging with lineage tracing to track the fate of entire tdT(+) cutaneous lymphatic vessels or the behavior of individual or small populations of LECs over 11 months. We tracked the fate of 110 LEC clones and 80 small LEC populations (clusters of 2-7 cells) over 11 months and analyzed their behavior using quantitative techniques.

Results: The results of the high dose inductive studies showed that the lymphatic vessels remained tdT(+) over 11 months, suggesting passage and expression of the tdT transgene from LEC precursors to progenies, an intrinsic model of self- renewal. Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period. By following the behavior of labeled LEC clones or small populations of LECs individually over 11 months, we identified diverse LEC fates of proliferation, quiescence, and extinction. Quantitative analysis of this data revealed that the average lymphatic endothelial clone or small population remained stable in size despite diverse individual fates.

Conclusion: The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis. These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

No MeSH data available.


Related in: MedlinePlus