Background
The lymphatic vasculature is required for life and it regulates essential aspects of physiology and immunity during conditions of homeostasis and disease throughout life [
1,
2]. While it is well known that the lymphatic vasculature proliferates and remodels extensively to meet the demands of disease and wound repair conditions [
3,
4], there are no proposed conceptual self renewal models to explain how the lymphatic vasculature is maintained over the lifetime of an animal. To maintain homeostasis of any organ in the post-natal period, loss of cells must be balanced by a proliferation of newly generated cells. Given the importance of the lymphatic system, it seemed reasonable to consider the existence of a self-renewal program. We hypothesized that ‘new’ lymphatic endothelial cells (LECs) would replace LECs regularly to maintain the lymphatic vasculature. We considered mechanisms, such as proliferating neighboring LECs, or alternatively progenitor cells, as sources of the ‘new’ LECs. Although controversial, there are several lines of evidence that non-venous derived progenitor cells contribute to embryologic and pathologic lymphangiogenesis [
5‐
7].
To investigate this hypothesis, we used inducible cre-lox based genetic lableing and intravital microscopy approaches to directly visualize fluorescently labeled lymphatic vessels and individual LEC clones or small populations of LECs during conditions of homeostasis. Mice transgenic for the hormone regulated Cre recombinase driven by the LYVE-1 promoter (Lyve1CreERT2) were developed in our laboratory [
8]. The LYVE-1 promoter restricted Cre activity spatially to the LECs and a small population of LYVE-1
+ macrophages (discussed in greater detail below). The Cre-ERT2s generation construct is a fusion protein that is sensitive to low levels of tamoxifen and displays markedly less activation by endogenous estrogens [
9]. Lyve1CreERT2 mice were bred to mice transgenic for the tandem dimer tomato (tdT) fluorescence protein that contains an upstream stop codon flanked by lox P sites [
10], to produce Lyve1CreERT2
tdT mice. Cre activity was regulated temporally by administering 4-hydroxytamoxifen (4-OHT). By modifying the 4-OHT dose and schedule, we were able to induce tdT in nearly the entire cutaneous lymphatic vasculature or in individual LEC clones or small populations of LECs stochastically in Lyve1CreERT2
tdT mice. We adapted this labeling strategy from Mascre et al. [
11].
We used the Lyve1CreERT2
tdT mice as an in vivo platform for lineage tracing techniques. Lineage tracing is the marking and subsequent identification of all progeny from a founder or progenitor cell [
12]. One important principle of labeling cells by inductive genetic recombination is that the inductive agent is administered transiently rather than continuously at the start of the lineage tracing experiment. Transient Cre activation excises the stop codon in the reporter transgene such that these cells express the modified transgene and pass this modified constitutively expressed transgene to all progeny. This indelible labeling enables the detection and tracking of fluorescent founder cells and all progeny. We used this system to investigate the overall remodeling of the lymphatic vasculature and to quantify individual LEC behavior longitudinally over 11 months using intravital microscopy.
Methods
Mouse strains
All animal protocols were approved by Boys Town National Research Hospital Institutional Animal Care and Use Committee Institutional Review Board in accordance with NIH guidelines (Protocol #15–01). The development of the Lyve1CreERT2
tdT mouse strain has been described in detail [
8].
4-OHT induction protocols
In previous studies [
8], we modified the 4-OHT dose and administration schedule such that 1 mg 4-OHT suspended in sunflower oil administered to 8–10 week old male and female Lyve1CreERT2
tdT mice by intraperitoneal route on two consecutive days induced tdT expression in virtually all LECs. This 4-OHT dose and schedule was used for the high dose studies. 0.25 mg 4-OHT administered by intraperitoneal route induced tdT expression in small or clonal LEC populations. 0.25 mg 4-OHT dose was used for the low dose studies designed to follow clone or small LEC populations.
Microscope image acquisition
All images were acquired at ambient temperature: approximately 23 °C.
Live imaging
Live imaging was performed on sedated Lyve1CreERTtdT mice. The pinna was depilated and placed between glass slides, and the mouse was positioned laterally. Images were obtained using a Leica MZ10F Fluo III microscope using a Leica Planap 1.0X objective and a Leica DFC310FX camera (acquisition software: LAS version 4.0.0.8777) or a Ziess Axio Zoom.V16 and a Zeiss Plan-NeofluarZ 1.0 × 0.25 na objective and a Zeiss AxioCam MRm camera (acquisition software: Zeiss Zen 2012, blue edition, version 1.1.1.0). To acquire the clonal and small population LEC data, we used low magnification light microscopy to identify the major blood vessels within the pinna. These large stable structures were used to develop a vascular map of the pinna. Using this information to provide image guidance and the identical power of magnification, we were able to visualize the same fields of interest using light and fluorescent microscopy.
Tissue staining and antibodies
To visualize lymphatic vessels within their microenvironment and study specific features that were identified during live imaging, pinnas were fixed in 1 % paraformaldehyde in PBS pH 7.4 and labeled as previously described using whole mount technique [
13]. Whole mount mouse cornea and pinna was stained with antibodies to LYVE-1 (11-034, AngioBio, Del Mar, CA), DAPI (Sigma–Aldrich) and the appropriate secondary antibody: using the fluorochromes Alexa488, (ThermoFisher Scientific) and DyLight488 (Jackson ImmunoResearch Laboratories, West Grove, PA). Fixed and labeled whole mounts were mounted in Vectashield H-1000 (Vector Laboratories, Burlingame, CA).
Epifluorescence microscopy
Epifluorescent images were acquired using a Zeiss Axio-Imager.A1 and an EC Plan-Neofluar 10 × 0.3 na objective and a Diagnostic Instruments SpotFlex model 15.2 64 Mp Shifting Pixel camera (acquisition software: SPOT windows version 4.6 or 5.1).
Confocal microscopy
Confocal images were acquired on either a Leica TCS SP8 MP (Creighton University Integrated Biomedical Imaging Facility) using either a HC PL Apochromat 20x 0.75 na objective or a HC PL Apochromat 40 × 1.3 na oil objective (acquisition software: Leica LAS AF version 3.2.1.9702, 12 bit) or a Zeiss AxioObserver LSM 710 (University of Nebraska Medical School Confocal Laser Scanning Microscope Core Facility) using either a Plan-Apochromat 20 × 0.8 na objective or an EC Plan-Neofluar 40 × 1.30 na oil objective (acquisition software: Zeiss Zen 2011).
Processing software
Channel separation and maximum intensity projections were done in the FIGI version of ImageJ (1.47v) or in the respective confocal acquisition software. Figures were prepared from original images in Adobe Photoshop.
Discussion
We tracked and quantified the behavior of individual LEC clones in vivo during homeostasis and showed heterogeneous LEC behavior that was consistent with an invariant asymmetry model of self renewal. This is the first report to explore the cellular mechanisms of lymphatic vessel self renewal.
The use of inductive genetic recombination and lineage tracing has generated significant advances in our understanding of the mechanisms that regulate epithelial [
11,
15‐
17] and tumor [
15,
18] cell and population dynamics during conditions of homeostasis and injury. Direct observation of labeled cells in vivo during lineage tracing facilitates the identification of founder or progenitor cells and the interpretation of cell behavior or fate over time [
12]. Thus, the direct observation techniques used here, allowed the identification of founding LECs and the analysis of individual LEC behavior. These direct observation techniques have limitations. For example, it is not possible to directly observe biologic events over time and simultaneously harvest tissue to obtain histologic ‘snapshot’ data of more classic indicators of proliferation without disrupting the experimental design.
We observed minimal lymphatic vessel remodeling over the length of the study. This was surprising, as we anticipated detecting more dynamic lymphatic vessel remodeling within the cutaneous microenvironment. The persistence of the tdT label in the LECs prompted us to consider several mechanisms of self renewal. This finding raised the question of whether all of the founding tdT
+ LECs persisted throughout the entire study. We considered this to be unlikely. Alternatively, we considered an intrinsic mechanism of self renewal, such that some of the founding tdT
+ LECs proliferated and passed the modified tdT transgene to LEC progeny. Recently, a non-venous origin of the dermal lymphatic vasculature was described as a mechanism of lymphatic vessel development in the mouse lumbar and thoracic regions during embryogenesis [
6]. We considered whether a similar extrinsic mechanism would self renew the lymphatic vasculature in the post-natal period. The replacement of tdT
+ LECS with an unlabeled LEC progenitor, would predictably result in a loss of tdT
+ LECs and the accumulation of tdT
− lymphatic vessels over time. We did not observe such findings.
We explored the possibility of an intrinsic self renewal mechanism by tracking and quantifying individual tdT
+ LEC behavior. By modifying the induction scheme, we were able to label single cell clones and small populations of LECs with tdT. Using the blood vasculature as an internal guide, we were able to reproducibly capture serial images using fluorescent microscopy. We tracked the fate of these populations over 11 months and investigated clone fate by applying quantitative analysis to study the behavior of these populations. We visualized the loss of detection, the persistence, and the expansion of single tdT
+ cells and small tdT
+ populations that remained as assemblages changing slowly over the course of months. Endothelial cell shuffling could explain these observations; however, we do not favor this interpretation. Endothelial cell rearrangement has been suggested as a highly dynamic cellular mechanism of angiogenesis (cells moving in real time). This model is based upon results of studies conducted primarily in vitro or ex vivo [
19,
20]. The results of recently published work showed that LECs do not rearrange or shuffle in vivo during corneal lymphangiogenesis [
8]. In addition, we found it difficult to reconcile the shuffling model with the results presented here. For example, the spatial position of most of the tdT
+ cells was highly conserved over time (Fig.
4; Additional file
1: Figure S1) and 30 % of the tdT
+ quiescence LEC clones were detected in the exact same tissue position over 11 months. We acknowledge that lymphatic endothelial rearrangement may occur slowly at a rate that could not be detected experimentally.
Quantitative analysis of clonal fate data has provided new insights into the homeostatic mechanisms of cycling or proliferative tissue such as the mammalian intestine and epidermis [
21,
22]; however, far less is known about other mammalian organ systems. In part, these types of studies are changing the concepts of stem cells and mechanisms that regulate tissue maintenance. The ability to track the fate of proliferating cells in vivo with genetic lineage tracing has lead to a rebirth of the concepts that govern the basic principles that establish the proliferative hierarchy and capacity in adult tissue. Multiple lines of evidence have revealed a population asymmetry in tissue with high proliferative demands in which the balance of proliferation and differentiation is regulated at the level of the stem cell population [
16,
23]. The hallmark of such population asymmetry is that the average clone size increases and clonal heterogeneity diminishes over time [
21,
23]. There are examples of invariant asymmetry self renewal where one stem cell gives rise to a stem cell and a differentiated cell in invertebrates [
24] and satellite muscle cells [
25], resulting in a mosaic of ‘clonal units’ responsible for tissue homeostasis. The hallmarks of invariant asymmetry are a stable clone size and a fraction of surviving clones over time. We identified both of these findings in the lymphatic vasculature during homeostasis.
Here, we show that the fates of individual LECs are diverse; however, the population is balanced. Quantitative analysis revealed a plateau of the LEC clone survival and the constant average LEC clone size of 1. One limitation of this study is that the targeting transgene labeled LECs and macrophages. Approximately 7 % of the tdT
+ cells were macrophage (Fig.
3). Because of technical reasons, largely tissue orientation, it was not possible to orient and positively identify single antibody stained cells within the pinna. This made it difficult to prove conclusively that the tracked tdT
+ cells were LECs. Although the macrophages comprised 7 % of the tdT cells, our interpretation of the data remains unchanged.
Authors’ contributions
ALC participated in the design of the study, collected the data, and contributed in manuscript preparation. PMK engineered the Lyve1Cre-ERT mice. RMT conceived the study and is responsible for the overall design and manuscript preparation. All authors read and approved the final manuscript.