Mesenchymal cells. Defining a mesenchymal progenitor niche at single-cell resolution

Abstract

Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here, we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication.

Figure 1. Marking cell clones in developing lung mesenchyme

A, Diagram of embryonic day E11.5 mouse lung (ventral view) showing airway epithelium (AE; white), mesenchyme (Me; light gray), mesothelium (Mt; dark gray), airway (ASM) and vascular (VSM) smooth muscle (red), and pulmonary arteries (PA) and vascular plexus (Pl) in blue. B, Labeled mesenchymal progenitor cell (green) in early lung (left), which at E11.5 (right) has generated a mixed lineage clone (green, dashed circle) with labeled ASM, mesothelial cells, and undifferentiated fibroblasts. C-F, Close-up of clones (pseudo-colored green) in indicated tissues of lungs of indicated ages. Clones were marked with MADM (C, E, F) or Rainbow (D) strategies and immunostained for E-cadherin (airway epithelium; blue), PECAM (vascular endothelium; blue in F) and smooth muscle α-actin (ASM and VSM; red). Cells in each clone are numbered. G, Structure of lung mesenchyme specific Cre transgene using lung mesenchyme-specific enhancer (LME) from Tbx4 locus and P_hsp68 minimal promoter to drive Cre recombinase expression. H-J, Cre mRNA in situ hybridization of lungs from Tbx4^LME-Cre transgenic mice of indicated ages. mRNA is robustly detected by E10.5 exclusively in lung mesenchyme (H), remains strong at E13.5 (I), and begins to downregulate by E14.5 (J). Similar results were obtained for Tbx4^LME-CreER (Figure S1). K, Tiled image of transverse section through E13.5 Tbx4^LME-Cre; R26R-EYFP lung immunostained for YFP lineage trace marker (white). Lineage-traced cells are confined to lung. NT, neural tube; Li, liver; St, stomach. L, Section through E13.5 Tbx4^LME-Cre; R26R-EYFP lung immunostained for YFP (green) and E-cadherin (magenta). Nearly all mesenchyme cells are lineage labeled (green). Lineage label is not detected in epithelium (magenta). M-O, Tbx4^LME-CreER; Rainbow embryos given limiting doses of tamoxifen (0.6 mg/dam) at E9.5 (M) or E10.5 (N, O) and analyzed at E13.5 by immunostaining for smooth muscle α-actin (SMA) and clone markers indicated. Clones are labeled by a single recombination reporter, either mCherry (M), mOrange (N) or Cerulean (O). Cells in each clone are numbered. Bars, 50 μm (C-F; H-J), 100 μm (L-O).

Figure 2. Cell dynamics of mesenchymal clones and structure of vascular smooth muscle clones

A, Scatter plots of each Tbx4^LME-CreER; Rainbow mesenchyme clone (Table 1) showing percent differentiated cells (cells expressing airway smooth muscle, vascular smooth muscle, mesothelial, or cartilage markers) in each clone as function of clone size (number of cells in clone). Clones harvested at E13.5 (dots) have < 200 cells and range from undifferentiated to fully differentiated, whereas clones harvested at E14.5 (squares) show the E13.5 pattern or are large (> 200 cells) and mostly undifferentiated clones (dashed box), indicating emergence of highly proliferative fibroblast population soon after E13.5. Clone size is greater (P = 9 × 10⁻⁷) and degree of differentiation less (P = 9 × 10⁻⁵) for clones harvested at E14.5 compared to E13.5. B, Violin plots of clones as above showing clone size (red dot, mean) as function of cell type complexity (number of mesenchymal cell types). n, number of clones analyzed. C, Ratio of cell number in E13.5 lungs of "twinspot" mesenchyme clones (n = 53 clones; Table S1) generated by HPRT-Cre; MADM, which differentially labels the two daughter cells of the original recombined cell with GFP or DsRed. Ratios were binned in increments of 0.5 (black bars) and shown with data from 10,000 simulated twinspots (gray bars; clone frequency values divided by 250) assuming a Poisson distribution in cell number. Note overrepresentation of clones with strong asymmetries (ratios > 5) relative to clones in Poisson simulation (P = 3 × 10⁻¹⁰¹ for all ratios >5, P = 1 × 10⁻¹⁸ with solo twinspots excluded). D, E, Projections of confocal z-stacks of twinspot clones 22 (D) and 47 (E) as above immunostained for GFP (clone mark, green), DsRed (sibling cell clone mark, magenta) and for E-cadherin (epithelium, blue), SMA (smooth muscle, red) and in D for PECAM (endothelium, also in blue). Values at bottom left show cell numbers in each twinspot; note asymmetry (D) and outright loss of one twinspot (E). No pattern was observed between loss of a twinspot and the clone’s location in the lung or position along a branch. F, Mesenchyme clones generated as above showing how arrangement of labeled cells in clones varies widely, from tight clusters (green-circled clone) to amorphous clouds of cells (yellow-circled clone, and clone in panel D). A slight bias was noted for clones arranged along the length of a branch, as in the yellow-circled clone, but no correlation with specific lobes or branches formed by a particular mode was apparent. G, H, Clones including VSM cells. In schematics (G', H'), cells in each clone are numbered. Note similar arrangement of cells, with undifferentiated mesenchyme cells (green in schematics) adjacent to SMA-expressing VSM cells (green with black dot in schematics). I, Model of vascular smooth muscle recruitment. Progenitors (gray) in mesenchyme directly surrounding endothelial cells (blue) are recruited to form VSM of artery wall (red). Bars, 50 μm (C, D, F), 10 μm (G-H).

Figure 3. Clonal analysis reveals airway smooth muscle progenitor niche, dynamics, and lineage boundaries

A, Airway branches of whole mount E13.5 mouse lung immunostained for E-cadherin (epithelium, blue) and smooth muscle α-actin (smooth muscle, red). SMA-positive airway smooth muscle (ASM) fibers cover airway stalks but are excluded from tips. Arrowheads, distal boundary of smooth muscle fibers. B-D, ASM-containing clones in E13.5 lungs immunostained for tissue markers as in A and for GFP clone marker (green). Cells in clone are numbered. In schematics (B’-D’), clone cells are green and those that are SMA-positive are marked with black dots. ASM clones fall into three classes: those with undifferentiated mesenchyme at distal tip plus elongating but SMA-negative cells around tip epithelium (B, B’); those with elongated but SMA-negative cells plus SMA-positive ASM cells (C, C’); and those with all cells SMA-positive (D, D’). Examples shown were chosen to illustrate the morphologies and distribution of clone cells; differences in cell number between clones are arbitrary. E, A large clone (outlined) with extensive labeling of mesenchyme along trachea and right primary airway stalk at E11.5. E’, Close up of boxed region. Note lack of recruitment into ASM layer from labeled mesenchyme at branch side, with lineage boundary marked by dotted line. F, Three step model of ASM formation. Fate of one progenitor (green) is highlighted. Proliferating progenitors are located in mesenchyme at branch tip (progenitor zone), initiate differentiation through interaction with epithelium (recruitment zone), and mature along airway stalk (maturation zone) as branch extends. Bars, 100 μm (A, E), 50 μm (B-D), 10 μm (E’).

Figure 4. Tissue grafts and lineage tracing show ASM progenitors are located in tip mesenchyme and form anew with new domain branches

A, Graft strategy. Mesenchyme excised from stalk or tip of ZsGreen-expressing donor E11.5 lung were grafted into tip of an unlabeled littermate host lung, and grafted cells (green) were traced in culture. B, Quantification of progenitor cell migration from the graft in tip-to-tip (T>T) or stalk-to-tip (S>T) mesenchyme grafts, measuring the distance from the graft margin to the furthest invading graft cell, along the shortest inferred migration path. n, number of grafts. Note robust invasion of tip-derived cells (all grafts showed > 100 μm migration) relative to stalk-derived cells (11% showed > 100 μm migration). C, C’, Stereomicroscope image showing recipient lung four days after tip-to-tip micrograft (C), and single confocal optical slice of grafted region (C’). Note in C’ that tip cells have migrated away from lung margin (dotted line) along the airways (dashed lines) and some graft cells (arrowheads) are expressing ASM differentiation marker smooth muscle actin (red). D, Stalk-to-tip mesenchyme graft. Grafted cells fail to leave graft site, and do not migrate along airways or express smooth muscle actin (D’). E, Strategy of lineage trace of smooth muscle of parent branch of L.M1, a late-forming domain branch. Budding L.M1 (highlighted in insets) is first medial domain branch formed along left primary airway; it buds through existing ASM layer of parent branch and forms its own ASM layer. Saturating tamoxifen (3 mg) was administered at E11.5 to SM-MHC (smooth muscle myosin heavy chain)-CreER; mTmG to label differentiated ASM of parent branch and early forming lateral branches (L.L1 – L.L3) with heritable GFP expression (green bar on timeline, with tamoxifen assumed active for 24 hours) before emergence of L.M1 bud at E12.5. Lungs were analyzed at E14.5 after L.M1 has budded and formed its ASM (“new” ASM, black bar in inset). F, Confocal z-stack projections (ventral view) showing close-up of boxed region (F’) of SMMHC-CreER;mTmG E14.5 embryo treated as above and immunostained for lineage trace of smooth muscle of parent branch (GFP, green) and all airway smooth muscle (SMA, red). Note ASM of L.M1 daughter branch is not lineage labeled (border highlighted with dashed line in F’ merge) indicating a new progenitor pool formed ahead of new branch that is lineally independent of existing ASM. There is no cell mixing across smooth muscle lineage boundary (dashed line), with exception of two lineage-labeled cells (arrowheads in F') in ASM of L.M1. Dots, lineage labeled non-ASM cells. Bars, 50 μm.

Figure 5. Effect of Wnt bead implants and transgenic activation of Wnt pathway show that Wnt signaling primes ASM progenitors

A-C, Stereoscope images of stalk-to-tip mesenchyme grafts performed as in Figure 4D, except also implanted with control (A) or ligand-soaked (B, C) agarose beads as indicated. Asterisk, position of bead. A'-C' show single confocal slices of graft region after immunostaining for GFP (graft marker, green), E-cadherin (epithelium, blue), and SMA (ASM, red). Graft cells exposed to PBS- or FGF10-soaked beads showed little or no migration along airways (A', B'), whereas those exposed to WNT1-soaked beads (C') migrated along airways where some (arrowheads) differentiated to ASM (separate channels shown in Figure S5E). D, Quantification of graft cell invasion in untreated grafts (reproduced from Figure 4B), and following exposure to ligand-soaked beads and Wnt inhibitors, as indicated. Note that WNT1 beads (column 5) induce stalk mesenchyme invasion at levels comparable to that seen in tip-to-tip grafts (column 1), and the effect is reduced in the presence of 100 ng/ml Wnt inhibitors DKK1 or SFRP1 (column 6). E-H, Right middle lobes of E12.5 control (E) and Tbx4^LME-Cre;βcat^ex3/+ lungs that express stabilized β-catenin to activate canonical Wnt pathway throughout mesenchyme (F-H) immunostained for E-cadherin (epithelium, green), SMA (smooth muscle, red), and PECAM (endothelium, blue). Wnt pathway activation results in disorganized smooth muscle around airways (F) and ectopic wrapping of vascular plexus by cells continuous with ASM (arrowheads in G). (H) Single confocal slice of branch as in G showing that the ectopic smooth muscle wrapping the plexus (arrowhead) expresses vascular smooth muscle marker NG2 (magenta). Bars, 50 μm (A-E), 25 μm (F, G).

Figure 6. Diverse mechanisms generate mesenchymal cell derivatives

(A) Single labeled mesenchyme cells (green) proliferate (clonal expansion) and daughter cells disperse to seed progenitor "niches" that generate stromal and support cells by different mechanisms. (B) ASM (airway smooth muscle) progenitors are recruited to branch stalks from a niche distal to the branch tip. VSM (vascular smooth muscle) progenitors are recruited by local condensation of mesenchyme immediately surrounding the vessel. Mesothelium forms by self-duplication and limited spreading of progenitors. Black, airway epithelium; grey, mesenchyme; dark gray, mesothelium; red, smooth muscle; blue, endothelium.