Stem cell-driven lymphatic remodeling coordinates tissue regeneration.

Lymphatic capillaries: A newly identified SC-niche component
Assessing vascular-SC spatial relationships was made possible by a recent clearing method that renders opaque tissue transparent while preserving cellular and subcellular tissue structures (17) (fig. S1A). Whole-mount immunofluorescence and 3D image reconstruction of skin exposed an array of dermal vessels, positive for panendothelial marker CD31, just below HF SGs. During telogen, large-diameter vessels closely approached keratin 24 (KRT24+) HFSCs within the lower bulge (fig. S1B and movie S1).
HFSC-associated vessels were not blood vessels (Endomucin+), but rather they were positive for both surface lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) and vascular endothelial growth factor tyrosine kinase receptor-3 (VEGFR3), establishing their lymphatic endothelial identity (Fig. 1, A and B; fig. S1, C to G; and movies S2 to S5). A similar association between lymphatic capillaries and the bulge was seen in human HFs, which spend most of their time in anagen (fig. S1H). We focused on mice, whose hair growth cycles are shorter and temporally choreographed. By studying telogen (Tel), it was clear that lymphatics were tightly associated with the bulge and, to a lesser extent, with progenitors (hair germ, HG) that are primed to undergo proliferation and fate commitment at the onset of tissue regeneration [anagen I (AnaI)] (1) (Fig. 1A and fig. S1I).
Lymphatic capillaries drain into collecting vessels, which differ molecularly and anatomically, as well as in terms of permissiveness to fluid and cell entry (18). Lymphatics associated with the HFSC niche were thin-walled and blind-ended lymphatic capillaries (VEGFR3+LYVE1+), whereas collecting vessels (VEGFR3+LYVE1neg) resided deeper within the dermis (Fig. 1, C and D, and fig. S1J). Moreover, relative to the posterior arrector pili muscle, capillaries were asymmetrically positioned anteriorly along each bulge (fig. S1K and movie S6), at sites where new SCs form during early anagen (3). During embryogenesis, connections between lymphatic capillaries and HFs also coincided with emergence of the quiescent bulge niche (16,19) (fig. S2 and movies S7 to S11).

Lymphatic capillaries maintain SC quiescence
To determine whether lymphatic integrity functions in HF quiescence, we perturbed the lymphatic vascular network. Using mice expressing CreER knocked into the Prox-1 locus (20) and harboring Rosa26-fl-stop-fl-YFP, we first activated lineage-tracing in telogen skin and confirmed by immunofluorescence and flow cytometry that Prox1-YFP+ cells were exclusively lymphatics (fig. S3, A to D). We then intercrossed a Cre-recombinase-inducible diphtheria toxin (DT) receptor line (iDTR) (21) with Prox1-CreER mice and induced iDTR expression during the extended second telogen.
A single DT intradermal injection induced lymphatic cell death and disrupted the network (Fig. 2A and fig. S3, E to H). This perturbation stimulated HFs to proliferate and enter anagen, irrespective of targeting lymphatics in first or second telogen or even early anagen (Fig. 2, B and C, and fig. S4). Anagen entry did not induce Prox1 expression in HFSCs, blood capillaries remained intact, and the skin immune cell milieu resembled normal telogen-to-anagen transition (22) (figs. S4 and S5).
To further assess whether precocious HFSC proliferation might arise directly from disrupting the lymphatic-SC niche network, we administered soluble VEGFR3 receptor intradermally during second telogen. Because VEGFR3 signaling is essential for lymphatic endothelial cell proliferation and survival, its interception causes lymphatic regression (23). Notably, precocious HF anagen entry was recapitulated with this model (Fig. 2D and fig. S6). Given that VEGFR3 (Flt4) was expressed by lymphatics and not HFSCs (fig. S4D), these data further underscored a role for lymphatic capillaries in HF regeneration.

The lymphatic-SC niche is dynamic during physiological regeneration
Shortly after hair cycle onset (AnaII to AnaIII) in normal skin, LYVE1+PROX1+VEGFR3+ lymphatic capillaries exhibited signs of remodeling around the SC niche (Fig. 3A and fig. S7A). Although capillaries still connected to underlying PROX1+VEGFR3+LYVE1neg collecting vessels and maintained cell numbers with little or no signs of apoptosis, they were dissociated from SC niches and dilated (Fig. 3, B to D; fig. S7, B and C; and movie S12). This dissociation was transient, and by AnaIV, lymphatics resumed their niche connection.
Given the well-established role of lymphatic vessels in controlling tissue fluid balance and macromolecule efflux (18,24), we also assessed their drainage during hair regeneration. After intradermal injections, both Evans blue dye clearance (25) and OVA-Ax488 drainage into brachial lymph nodes were significantly delayed during the narrow hair cycle window when lymphatics were dissociated (Fig. 3E). Morphologically, the dissociated capillaries displayed signs of reduced permeability (26, 27), with no major hair cycle-associated changes in blood vessel permeability (fig. S7, D and E). When fluid volume was artificially overloaded in skin, HFSCs precociously proliferated (fig. S7F), which is consistent with a role for transient lymphatic dilation in HF regeneration.
Although we cannot exclude the possibility that mechanical forces imposed by HF regeneration might influence SC-lymphatic dynamics, HFs were still growing downward after lymphatic-SC niche connections were restored. However, this timing did coincide with the return of bulge SCs to quiescence (4). Based on these considerations, lymphatic-SC connections seemed to be affecting HFSC behavior, but by mechanisms beyond HF downgrowth. Although outside the scope of this work, possibilities include draining proliferation stimuli (growth factors, metabolites, toxins) from the SC niche or, alternatively, producing inhibitory factors that keep HFSC self-renewal in check.

A lymphovascular switch at the onset of SC activation
When transplanted in vivo, cultured HFSCs can establish cycling HFs (1), suggesting that SCs participate in organizing their niche. This led us to speculate that HFSCs might be orchestrating lymphatic capillary connections. To address this possibility, we began by purifying and transcriptome-profiling HFSCs during (i) telogen, when SCs are quiescent and lymphatics are associated; and (ii) AnaII-III, when SCs are proliferative and lymphatics are dissociated (fig. S8A).
Established proangiogenic and lymphangiogenic factors, such as Vegfa, Vegfb, and Vegfc, showed little or no expression in either quiescent or proliferative bulge SCs and hence were unlikely to control bulge-lymphatic dynamics (Fig. 4A). Changes in stromal VEGFC expression and/or processing were also not detected during this time (fig. S8, B and C). Moreover, although a VEGFC gradient is known to elicit robust lymphatic VEGFR3-mediated sprouting, overt signs of enhanced sprouting were absent (fig. S7). Thus, SC-lymphatic dynamics appeared to be controlled by other factors.
A number of putative vascular regulators displayed expression patterns paralleling these dynamics. Most notably, Angptl7 was expressed in telogen bulge SCs, whereas Angptl4 and netrin-4 (Ntn4) were induced in AnaII-III bulge SCs concomitant with Angptl7 down-regulation (Fig. 4A). These factors appeared to be bulge-specific (Fig. 4B).
Focusing first on Angptl7, we find that prior RNA sequencing (RNA-seq) data (28) showed that Angptl7 was not even expressed in the primed HG cells of telogen or early anagen HFs (Fig. 4C and fig. S8D). To delineate the detailed temporal changes of Angptl7 within bulge SCs, we performed stage-specific single-cell RNA-seq. Three distinct temporal patterns emerged from t-distributed stochastic neighbor embedding (t-SNE) and unsupervised hierarchical clustering (Fig. 4D and fig. S8E). Using machine learning-based cell-cycle allocation analysis (29), it was concluded that proliferative cells resided in AnaII SCs, in agreement with our 5-ethynyl-2’-deoxyuridine (EdU) labeling studies. Additionally, in contrast to SC marker Sox9, which was maintained in bulge SCs at all stages, Angptl7 transcription in bulge SCs diminished shortly after anagen onset, even sooner than that of established quiescence regulator Nfatc1 (28). Moreover, the Angptl7 expression dip was transient, occurring only between AnaII and AnaIII (Fig. 4E).
Angptl7 dynamics were recapitulated at the chromatin level. As judged by chromatin immunoprecipitation sequencing (ChIP-seq) analyses of fluorescence-activated cell sorting (FACS)-purified HF cells (30), the Angptl7 chromatin state was active in telogen (H3K27ac+), poised in AnaIII (H3K27acnegH3K4me1+), and silent in AnaVI differentiated cells (H3K27acnegH3K4me1neg) (fig. S9A). Moreover, when either of the two Angptl7 locus-accessible chromatin elements (30) were used as enhancers to drive enhanced green fluorescent protein (eGFP) expression in vivo, reporters faithfully recapitulated Angptl7’s temporal dynamics in bulge expression during the hair cycle (Fig. 4F and fig. S9B). These results intimated that changes in SC-derived Angptl7 transcription are involved in reshaping the regenerative microenvironment of the niche.

A SC-driven secretome switch is required for lymphatic remodeling
Angiopoietin-like (ANGPTL) proteins do not bind to classical angiopoietin receptors, and knowledge of these orphan ligands is still scant (31, 32). To address whether Angptl7 down-regulation functions in HFSC activation, we engineered mice to harbor an Angptl7 transgene that is selectively doxycycline-inducible in skin progenitors (33). We then induced at telogen and examined the consequences of maintaining ANGPTL7 through early stages of the newly entered hair cycle (fig. S10, A to E).
In contrast to the lymphatic-SC niche remodeling of normal HFs, lymphatic capillaries remained tightly associated and the bulge remained quiescent when Angptl7 was sustained (Fig. 5A). Consistent with a nonautonomous role for ANGPTL7 in controlling stemness activity, recombinant ANGPTL7 did not appreciably alter colony formation of HFSCs in vitro. Rather, concomitant with sustained lymphatic efflux and drainage capacity, entry into the hair cycle was markedly delayed (Fig. 5B and fig. S10, C to E). Because a role for ANGPTL7 in angiogenesis had been suggested, we looked for, but did not find, perturbations in blood vessel density and coverage in our Angptl7-induced skin (fig. S10E). ANGPTL7 overexpression also did not affect levels of CCBE1, a protein required for VEGFC-mediated lymphangiogenesis (fig. S10F).
Because the hair cycle was delayed upon sustaining Angptl7, we wondered whether ANGPTL7 dynamics might be important for other regenerative responses. We focused on hair plucking (waxing), as it mechanically perturbs the SC niche, generating a wound-like response (3,34). Plucking not only activated hair regeneration, as expected, it also restricted lymphatic drainage and disrupted lymphatic-SC associations (fig. S11, A and B). Moreover, when Angptl7 was sustained, wound-induced hair regeneration was abrogated; conversely, after skin injury in a normal setting, Angptl7 gene expression plummeted (fig. S11, C and D).
Turning to the flip side of the secretome switch, we engineered doxycycline-inducible versions of Angptl4. When induced in telogen, ANGPTL4 precociously disrupted lymphatic capillaries surrounding the bulge, reducing lymphatic drainage and activating HFSCs (Fig. 5, C and D, and fig. S12). Despite a purported role for ANGPTL4 in blood vasculature (35), endothelial vessel density was largely unchanged, both in AnaII-III of the normal hair cycle and in telogen of the Angptl4-induced hair cycle. Similar results were obtained with NTN4, previously implicated in endothelial cell biology (36–38) (fig. S12).
Our data suggested that the HFSC-derived secretome can influence lymphatic dynamics directly, which we tested by evaluating lymphatic tube formation in a 3D-Matrigel system. Formation of tubelike structures was enhanced with ANGPTL7 and impaired with ANGPTL4, supporting this hypothesis. Consistent with a nonautonomous role, SC-driven factors in vitro neither affected HFSC growth nor stimulated immune cell migration (fig. S13).
Although ANGPTL and NTN receptors are poorly studied, transcriptional landscaping of freshly isolated endothelial cells from skin revealed preferential expression of their putative receptors by the lymphatic vessels (fig. S14A). Telogen-associated lymphatic capillaries also preferentially expressed WNT inhibitors. As WNT signaling is critical for HFSC activation and hair cycling (30, 39), these correlations are suggestive of an additional layer of regulation by which lymphatic capillaries might coordinate SC regeneration.
Single-cell analyses of lymphatic capillary cells isolated and characterized from telogen and from anagen skins revealed similar transcriptomic patterns, with only 4 to 5% of mRNAs changed temporally by >2× (fig. S14B). Of these genes, the most notable changes appeared to be in lymphatic tube formation and fluid dynamics. Thus, if capillary growth factors participate in maintaining HFSC quiescence, their significance likely resides in the dynamic regulation of lymphatic-SC connections rather than their differential transcription during the hair cycle.

A role for the lymphatic network in integrating SC-niche behavior across a tissue
Turning to the physiological relevance of the SC-lymphatic connection, we used our powerful in utero lentiviral delivery method to selectively and efficiently knock down Angptl7 in skin progenitors with one of two different short hairpin RNAs (shRNAs). When Scramble controls were in telogen, shAngptl7 HFs had already entered anagen. Full anagen follicles were interspersed with telogen ones, indicating hair cycle asynchrony (Fig. 6A and fig. S15A).
The failure of HFSCs to generate ANGPTL7 profoundly affected lymphatic biology. Lymphatics were discontinuous and abnormally dilated, and they displayed impaired drainage, as judged both functionally and morphologically (Fig. 6B and fig. S15B). This chronic lymphatic dysfunction was associated with HF hyperplasia and reduced bone morphogenetic protein (BMP)/pSMAD1/5/9 signaling, which are essential for maintaining SC quiescence (3) (Fig. 6, B and C; fig. S15C; and movie S13). Hyperplastic HFs were associated with the most highly dilated lymphatic capillaries, which maintained their identity but displayed reduced drainage (Fig. 6, B and D; fig. S15D; and movie S14). Blood vessel density was also increased at hyperplastic HFs (fig. S15E and movie S15), although this was unlikely to have driven lymphatic remodeling, given that SCs associated primarily with lymphatic capillaries in normal homeostasis.
The asynchrony across the hair coat did not appear to stem from variations in transgene integration, as this would have generated clonal patches of HFs at specific cycle stages, which we did not see. However, to unequivocally demonstrate that HF asynchrony arose from perturbations in the SC-lymphatic crosstalk, and not our lentiviral delivery method, we used Flt4Chy mice, which harbor a mutant Vegfr3 allele, creating VEGFR3 dimers with dysfunctional tyrosine kinase activity and dysfunctional lymphatics (40). Flt4Chy mice recapitulated the asynchrony of bulge-SC niches, accompanied with dysfunctional lymphatic vessels (Fig. 6E and fig. S15, F and G). Taken together, these data underscore the importance of lymphatic capillary dynamics, driven by SCs, in integrating SC-niche behavior across a tissue.

Lymphatic capillaries as a dynamic SC-niche newcomer that coordinates SC behavior
The niche microenvironments of quiescent SCs, such as those of bone marrow, muscle, and HFs, provide the input signals that keep these SCs in an undifferentiated, inactive state (41). Niche-SC interactions must be dynamic in order to mobilize SCs to regenerate tissues. Additionally, SCs have an intrinsic ability to communicate with their neighbors and reshape their niche (34).
Our study identifies lymphatic capillaries as dynamic SC-niche elements that integrate SC niches and synchronize SC behavior across the hair coat. We have learned that, to mobilize HFSCs for either normal regenerative demands or those induced by injury, these SCs undergo a secretome switch that triggers transient dissociation of lymphatics from the niche (Fig. 6F).
Although the possible ways by which lymphatics control HFSCs are numerous, and the communication circuits are likely complex, our genetic manipulations of the lymphatic-SC niche connection underscore its functional importance in balancing SC self-renewal and quiescence. Indeed, as soon as bulge SCs launch production of their committed proliferative progeny and self-renew, the lymphatic capillaries resume connections with their SC neighbors, and niche quiescence is restored. The process is a two-way street, because the dynamic remodeling of lymphatic capillaries is orchestrated by bulge SCs, whereas the lymphatics govern SC behavior.
Drainage of tissue interstitial fluids and macromolecules has been a subject of interest for decades. We have unearthed a need to balance cutaneous influx of fluids and macromolecules in controlling SC behavior. Future studies will be needed to dissect the impact of interstitial fluid composition, extracellular macromolecule dynamics, and immune cell efflux on stem cell biology. Additionally, short remodeling duration suggests that lymphatic dissociation from the SC niche may be deleterious, perhaps rendering SCs transiently vulnerable to toxins or increased fluid pressure.
Our discovery that lymphatics localize to both mouse and human HFSC niches suggests that the need to establish such connections is not only physiologically important but also evolutionarily conserved, raising the possibility that lymphatic capillaries may participate in other SC niches to meet their specialized regenerative demands. With our newfound understanding of skin SC-lymphatic interactions, it now merits addressing whether the stem cell exhaustion that accompanies wound-healing defects and hair loss in aging and in patients with lymphedema (24,42) might be rooted in decline of lymphatics and interstitial fluid draining. If such links exist, targeting lymphatic function could prove to be a promising preventative therapeutic target for hair loss and wound repair.

Supplementary Material
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