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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

Little remodeling was detected in the lymphatic vessel network in the Lyve1CreERT2tdT pinna. The experimental strategy is illustrated in a. The vertical lines represent points in time when live imaging was performed. 4-OHT was administered to Lyve1CreERT2tdT mice to label LECs within the pinna. Live imaging epifluorescent microscopy of 2 sedated Lyve1CreERT2tdT mice was performed, first every 3 or 4 days, then with increasing intervals, at 3 low magnification fields within the right pinna to directly visualize the tdT+ lymphatic vessels. The morphology of the tdT+ lymphatic vessel network within the microenvironment was detectable and relatively stable over the course of 323 days post-labeling (panel B). The overall morphology of many of the lymphatic vessels was similar over the course of the study (b-inset). At the conclusion of this study, the pinnas were labeled with antibodies to LYVE-1 and evaluated using immunofluorescent microscopy techniques. Low power epifluorescent microscopy showed that most of the tdT+ cells were LYVE-1+ LECs comprising lymphatic vessels (c–e). The results are representative of 2 different Lyve1Cre-ERT2tdT mice. The size standards are 100 µm
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Fig2: Little remodeling was detected in the lymphatic vessel network in the Lyve1CreERT2tdT pinna. The experimental strategy is illustrated in a. The vertical lines represent points in time when live imaging was performed. 4-OHT was administered to Lyve1CreERT2tdT mice to label LECs within the pinna. Live imaging epifluorescent microscopy of 2 sedated Lyve1CreERT2tdT mice was performed, first every 3 or 4 days, then with increasing intervals, at 3 low magnification fields within the right pinna to directly visualize the tdT+ lymphatic vessels. The morphology of the tdT+ lymphatic vessel network within the microenvironment was detectable and relatively stable over the course of 323 days post-labeling (panel B). The overall morphology of many of the lymphatic vessels was similar over the course of the study (b-inset). At the conclusion of this study, the pinnas were labeled with antibodies to LYVE-1 and evaluated using immunofluorescent microscopy techniques. Low power epifluorescent microscopy showed that most of the tdT+ cells were LYVE-1+ LECs comprising lymphatic vessels (c–e). The results are representative of 2 different Lyve1Cre-ERT2tdT mice. The size standards are 100 µm

Mentions: To investigate the overall remodeling of the cutaneous lymphatic vasculature during homeostasis, high dose 4-OHT was administered to Lyve1CreERT2tdT mice (n = 2) to transiently activate Cre and induce tdT fluorescence in cutaneous LECs. In these same mice, live imaging was performed initially weekly and then monthly or every other month to visualize the cutaneous tdT+ lymphatic vessels within the pinna. The general design of this experiment is shown in Fig. 2a. In sedated Lyve1CreERT2tdT mice, we used guidance techniques based on the identification of prominent blood vessels and their branching structures to locate the microscopy fields of interest in bright field conditions. These relatively large blood vessels coursing within pinna were easy to identify and were stable over the duration of our studies Additional file 1: Figure S1. We captured the endogenous tdT fluorescence signal in three fields of the pinna in sedated Lyve1CreERT2tdT mice initially about every 7 days for 6 weeks. Over the first 6 weeks, we were unable to detect lymphatic vessel growth, regression, or significant changes in overall lymphatic vessel morphology. Based on the lack of detectable changes we increased the observation interval. The overall morphology of the tdT+ lymphatic vessel network within the cutaneous microenvironment appeared relatively stable over the course of 323 days. Many of the lymphatic vessels had similar morphology throughout the duration of the study (Fig. 2b panel and e-inset).Fig. 2


Invariant asymmetry renews the lymphatic vasculature during homeostasis.

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

Little remodeling was detected in the lymphatic vessel network in the Lyve1CreERT2tdT pinna. The experimental strategy is illustrated in a. The vertical lines represent points in time when live imaging was performed. 4-OHT was administered to Lyve1CreERT2tdT mice to label LECs within the pinna. Live imaging epifluorescent microscopy of 2 sedated Lyve1CreERT2tdT mice was performed, first every 3 or 4 days, then with increasing intervals, at 3 low magnification fields within the right pinna to directly visualize the tdT+ lymphatic vessels. The morphology of the tdT+ lymphatic vessel network within the microenvironment was detectable and relatively stable over the course of 323 days post-labeling (panel B). The overall morphology of many of the lymphatic vessels was similar over the course of the study (b-inset). At the conclusion of this study, the pinnas were labeled with antibodies to LYVE-1 and evaluated using immunofluorescent microscopy techniques. Low power epifluorescent microscopy showed that most of the tdT+ cells were LYVE-1+ LECs comprising lymphatic vessels (c–e). The results are representative of 2 different Lyve1Cre-ERT2tdT mice. The size standards are 100 µm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940917&req=5

Fig2: Little remodeling was detected in the lymphatic vessel network in the Lyve1CreERT2tdT pinna. The experimental strategy is illustrated in a. The vertical lines represent points in time when live imaging was performed. 4-OHT was administered to Lyve1CreERT2tdT mice to label LECs within the pinna. Live imaging epifluorescent microscopy of 2 sedated Lyve1CreERT2tdT mice was performed, first every 3 or 4 days, then with increasing intervals, at 3 low magnification fields within the right pinna to directly visualize the tdT+ lymphatic vessels. The morphology of the tdT+ lymphatic vessel network within the microenvironment was detectable and relatively stable over the course of 323 days post-labeling (panel B). The overall morphology of many of the lymphatic vessels was similar over the course of the study (b-inset). At the conclusion of this study, the pinnas were labeled with antibodies to LYVE-1 and evaluated using immunofluorescent microscopy techniques. Low power epifluorescent microscopy showed that most of the tdT+ cells were LYVE-1+ LECs comprising lymphatic vessels (c–e). The results are representative of 2 different Lyve1Cre-ERT2tdT mice. The size standards are 100 µm
Mentions: To investigate the overall remodeling of the cutaneous lymphatic vasculature during homeostasis, high dose 4-OHT was administered to Lyve1CreERT2tdT mice (n = 2) to transiently activate Cre and induce tdT fluorescence in cutaneous LECs. In these same mice, live imaging was performed initially weekly and then monthly or every other month to visualize the cutaneous tdT+ lymphatic vessels within the pinna. The general design of this experiment is shown in Fig. 2a. In sedated Lyve1CreERT2tdT mice, we used guidance techniques based on the identification of prominent blood vessels and their branching structures to locate the microscopy fields of interest in bright field conditions. These relatively large blood vessels coursing within pinna were easy to identify and were stable over the duration of our studies Additional file 1: Figure S1. We captured the endogenous tdT fluorescence signal in three fields of the pinna in sedated Lyve1CreERT2tdT mice initially about every 7 days for 6 weeks. Over the first 6 weeks, we were unable to detect lymphatic vessel growth, regression, or significant changes in overall lymphatic vessel morphology. Based on the lack of detectable changes we increased the observation interval. The overall morphology of the tdT+ lymphatic vessel network within the cutaneous microenvironment appeared relatively stable over the course of 323 days. Many of the lymphatic vessels had similar morphology throughout the duration of the study (Fig. 2b panel and e-inset).Fig. 2

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