Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland

Abstract

The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5⁺ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis.

Keywords: Development; Epithelia; Lacrimal gland; Single cell sequencing; Tubulogenesis.

Fig. 1.

Single-cell sequencing reveals the cellular composition, lineage diversity and epithelial lineage relationships in the developing lacrimal gland. (A,B) t-SNE projection of cells isolated from E16 (A) and P4 (B) lacrimal glands. Distinct clusters indicate unique cell populations (see labels). (C) Zoomed images of boxed region of epithelial compartments at E16 (A) and P4 (B), illustrating specific epithelial markers. Inset shows erythroid contamination (Hbba-2) within the myoepithelial compartment.

Fig. 2.

Gene expression analysis reveals dynamic expression of epithelial markers during lacrimal gland development. All samples were normalized to E14 lacrimal glands unless otherwise indicated. (A) qPCR analysis of acinar cell markers indicates early expression of Sox10, and gradual acquisition of Aqp5 and Mist1. Levels of Ltf, a marker of mature acini, increased steadily as mice aged. We found Lyz1 and Lyz2, previously considered to be produced by adult acinar cells, were expressed early but levels were reduced over time. (B) Krt19 (normalized to 8-week lacrimal glands) was robustly expressed early in lacrimal gland development but decreased at later stages, whereas Nkcc1 displayed variable and dynamic expression until adult stages. (C) Myoepithelial markers Krt5 and Krt14 (normalized to 16-week lacrimal glands) peaked at P1 and steadily decreased at later stages. All qPCR experiments were completed in biological triplicates (data are mean±s.e.m.).

Fig. 3.

Spatiotemporal analysis reveals dynamic patterns of acinar and duct markers during lacrimal gland development. (A-E) E16 to adult lacrimal glands were immunostained for acinar and ductal markers. SOX10 was readily detected in acinar cells by P1, but slowly declined at later stages of lacrimal gland development. (F-J) AQP5 expression increased at postnatal stages, was prominent in acinar cells before P4, and was apically restricted in acini and small ducts by P7. (K-O) MIST1 was expressed in some acinar cells by P4, and in most acinar cells by P7 and adulthood. (P-T) KRT19 marked ductal cells at all time points evaluated. (U-Y) NKCC1 was broadly expressed at E16, but switched between ductal and acinar compartments between P1 and P7. By adulthood, it was specifically expressed in ducts. Asterisks mark acini and arrowheads mark ducts. Scale bars: 100 μm.

Fig. 4.

SOX10 and MIST1 mark subpopulations of acinar cells in developing and adult acini. (A) SOX10 and MIST1 co-staining revealed the subpopulations of acinar cells at P4 and adult stages. Three types of epithelial cells were readily detected in acini, including SOX10⁺MIST1−, SOX10⁺MIST1⁺ and SOX10−MIST1⁺ cells. b,d are magnifications of boxed regions in a,c, respectively. Scale bars: 100 μm in a,c; 25 μm in b,d. (B) Quantification of the epithelial cells expressing SOX10 and MIST1 at P4 (n=3) and adult stages (n=3) (data are mean±s.e.m.).

Fig. 5.

AQP5 and NKCC1 are dynamically expressed in the epithelium during lacrimal gland development. (A) Immunolabeling for AQP5 and KRT19 in P7 (a,b) and adult (c-f) lacrimal glands showed that AQP5 was expressed in small ducts after P7. However, AQP5 was absent in large ducts (e). Scale bars: 100 μm in a,c,e; 25 μm in b,d,f. b, d and f are higher magnification images of boxed regions in a, c and e, respectively. (B) NKCC1 expression switched between duct and acinar cells from P1 to adult stages. At P1, NKCC1 expression overlapped with KRT19 (a), but less overlap was apparent at P4 and P7 (b,c). (C) Co-staining with MIST1 confirmed the expression of NKCC1 in acini at P4 (a), but an exclusive switch back to the MIST1⁻ ductal cells in adult lacrimal gland (b). Scale bar: 25 μm. Arrowheads indicate ducts and asterisks indicate acini.

Fig. 6.

Myoepithelial cells are present early in postnatal development but do not contribute to other epithelial lineages in the lacrimal gland. (A) Imaging of myoepithelial cell emergence. ACTA2 protein was not observed at P0 (a), but was detected by P3 (b) in rounded cells within the acini bordering the mesenchyme. Myoepithelial cells began to emerge from the columnar epithelium by P5 (c). By P7 (d), these cells began to radiate processes, assuming their stellate mature appearance by P10 (e), which was maintained in adult glands (f). Arrowheads mark processes. Scale bar: 25 μm. (B) KRT5 (a) and P63 (b) were expressed at P1 in cells of acini immediately adjacent to the mesenchyme. P63⁺ cells also expressed SOX10 (c). High-magnification image in d (from boxed region in c) confirmed co-expression of these two markers Scale bars: 100 μm in a-c; 25 μm in d. (C) By P4, SOX10⁺ cells at the border of acini co-expressed ACTA2 (a) and P63 (b). Likewise in adult lacrimal glands, ACTA2⁺ECAD⁺ myoepithelial cells remained SOX10⁺ (c) and P63⁺ (d). Arrowheads indicate positive cells. (D) Genetic lineage tracing using the Acta2 promoter indicated that myoepithelial cells do not contribute to acinar or ductal lineages. Recombination was induced by tamoxifen injection in adult Acta2^CreERT2;Rosa26^RFP mice, and cells were traced for 6 months. RFP⁺ cells co-expressed ACTA2 (a). b is a higher magnification image of the boxed area. Scale bars: 100 μm in a; 25 μm in b. Arrowhead indicates positive cells.

Fig. 7.

Basal cells are progenitors for ductal, but not acinar, cells. (A) Lineage tracing was performed using Krt5^CreERT2;Rosa26^mTmG with recombination induced at either P1 (a-c) or adult stages (d-f). In the absence of Cre, no GFP was detected (a,d). However, in young or adult mice, induction of Cre resulted in KRT14⁺/GFP⁺ basal cells and GFP⁺ ductal cells (b,c,e,f). Arrowheads indicate ducts. Scale bar: 25 μm. (B) GFP⁺ luminal ductal cells were adjacent to GFP⁺ basal cells but not unlabeled basal cells in partially lineage-traced ducts, suggesting luminal cells are derived from basal cells by asymmetric division. Arrowheads indicate luminal cells derived from asymmetric division. Scale bar: 25 μm.

Fig. 8.

Phenotypic and molecular features of developing human and murine lacrimal glands are highly conserved. (A) The human fetal lacrimal gland undergoes extensive epithelial branching between 16 weeks (a) and 23 weeks (b). Scale bar: 500 μm. (B) qPCR analysis showed human lacrimal glands express several lineage markers observed in the murine lacrimal gland. All qPCR experiments used three biological replicates (data are mean±s.e.m.). (C) Immunostaining revealed that AQP5, SOX10 and MIST1 localize to the acini of fetal human lacrimal gland. As in mouse tissue, AQP5 was enriched in the apical membrane of acini and small ducts (a). SOX10 and MIST1 were exclusively found in acinar cells (b,c). Unlike in the murine lacrimal gland, KRT5 was barely detectable in myoepithelial cells when compared with basal cells, whereas KRT14 protein levels were similar in both cell types (d-f). Arrowheads indicate ducts and asterisks indicate acini.