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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

Expression of tdT fluorescence in Lyve1CreERT2tdT pinna following low dose 4-OHT administration. Three weeks after 0.25 mg 4-OHT was administered to 6 Lyve1CreERT2tdT mice, the pinnas were harvested and labeled with antibodies to LYVE-1. Maximum intensity projection images obtained using confocal microscopy were used to determined the targeting specificity of the transgene. 2 images from each mouse pinna were analyzed. Using images similar to the image shown in a, tdT+ cells were quantified. Using images similar to that shown in b, it was determined whether the tdT+ cells were LYVE-1+ or LYVE-1– and whether these cells were a constituent of a lymphatic vessel. Most of the tdT+ cells were LYVE-1+ and were constituents of lymphatic vessels (93 %). Some of the tdT+ cells were LYVE-1+ or LYVE-1− and physically separated from a lymphatic vessel (7 %) (b,c). The histogram is data pooled from fields obtained from 6 similarly treated Lyve1CreERT2tdT mice. The size standards are 50 µm
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Fig3: Expression of tdT fluorescence in Lyve1CreERT2tdT pinna following low dose 4-OHT administration. Three weeks after 0.25 mg 4-OHT was administered to 6 Lyve1CreERT2tdT mice, the pinnas were harvested and labeled with antibodies to LYVE-1. Maximum intensity projection images obtained using confocal microscopy were used to determined the targeting specificity of the transgene. 2 images from each mouse pinna were analyzed. Using images similar to the image shown in a, tdT+ cells were quantified. Using images similar to that shown in b, it was determined whether the tdT+ cells were LYVE-1+ or LYVE-1– and whether these cells were a constituent of a lymphatic vessel. Most of the tdT+ cells were LYVE-1+ and were constituents of lymphatic vessels (93 %). Some of the tdT+ cells were LYVE-1+ or LYVE-1− and physically separated from a lymphatic vessel (7 %) (b,c). The histogram is data pooled from fields obtained from 6 similarly treated Lyve1CreERT2tdT mice. The size standards are 50 µm

Mentions: To track LEC clone fate, we developed low 4-OHT induction conditions such that tdT fluorescence was expressed in individual cells in the Lyve1CreERT2tdT pinna. As LYVE-1 is expressed in LECs and a population of macrophages [14], we expected both populations to express tdT following 4-OHT administration. Three weeks after 0.25 mg 4-OHT was administered to transiently active Cre in Lyve1CreERT2tdT mice, pinnas were harvested and labeled with antibodies to LYVE-1 to detect LECs. 93 % of the tdT+ cells were LYVE-1+ LECs integrated stochastically in cutaneous lymphatic vessels. The remaining 7 % were single tdT+ cells that were not constituents of a lymphatic vessel (Fig. 3a–c). This data showed that under the low 4-OHT conditions, 93 % of the tdT+ cells were LYVE-1+ LECs and 7 % were non-LEC, presumably macrophage or other single cells.Fig. 3


Invariant asymmetry renews the lymphatic vasculature during homeostasis.

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

Expression of tdT fluorescence in Lyve1CreERT2tdT pinna following low dose 4-OHT administration. Three weeks after 0.25 mg 4-OHT was administered to 6 Lyve1CreERT2tdT mice, the pinnas were harvested and labeled with antibodies to LYVE-1. Maximum intensity projection images obtained using confocal microscopy were used to determined the targeting specificity of the transgene. 2 images from each mouse pinna were analyzed. Using images similar to the image shown in a, tdT+ cells were quantified. Using images similar to that shown in b, it was determined whether the tdT+ cells were LYVE-1+ or LYVE-1– and whether these cells were a constituent of a lymphatic vessel. Most of the tdT+ cells were LYVE-1+ and were constituents of lymphatic vessels (93 %). Some of the tdT+ cells were LYVE-1+ or LYVE-1− and physically separated from a lymphatic vessel (7 %) (b,c). The histogram is data pooled from fields obtained from 6 similarly treated Lyve1CreERT2tdT mice. The size standards are 50 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Expression of tdT fluorescence in Lyve1CreERT2tdT pinna following low dose 4-OHT administration. Three weeks after 0.25 mg 4-OHT was administered to 6 Lyve1CreERT2tdT mice, the pinnas were harvested and labeled with antibodies to LYVE-1. Maximum intensity projection images obtained using confocal microscopy were used to determined the targeting specificity of the transgene. 2 images from each mouse pinna were analyzed. Using images similar to the image shown in a, tdT+ cells were quantified. Using images similar to that shown in b, it was determined whether the tdT+ cells were LYVE-1+ or LYVE-1– and whether these cells were a constituent of a lymphatic vessel. Most of the tdT+ cells were LYVE-1+ and were constituents of lymphatic vessels (93 %). Some of the tdT+ cells were LYVE-1+ or LYVE-1− and physically separated from a lymphatic vessel (7 %) (b,c). The histogram is data pooled from fields obtained from 6 similarly treated Lyve1CreERT2tdT mice. The size standards are 50 µm
Mentions: To track LEC clone fate, we developed low 4-OHT induction conditions such that tdT fluorescence was expressed in individual cells in the Lyve1CreERT2tdT pinna. As LYVE-1 is expressed in LECs and a population of macrophages [14], we expected both populations to express tdT following 4-OHT administration. Three weeks after 0.25 mg 4-OHT was administered to transiently active Cre in Lyve1CreERT2tdT mice, pinnas were harvested and labeled with antibodies to LYVE-1 to detect LECs. 93 % of the tdT+ cells were LYVE-1+ LECs integrated stochastically in cutaneous lymphatic vessels. The remaining 7 % were single tdT+ cells that were not constituents of a lymphatic vessel (Fig. 3a–c). This data showed that under the low 4-OHT conditions, 93 % of the tdT+ cells were LYVE-1+ LECs and 7 % were non-LEC, presumably macrophage or other single cells.Fig. 3

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