The lacrimal glands are a pair of tear-producing exocrine glands located at the top, lateral side of each eye. These glands contain branched, tubular ducts that lead to secretory end units (acini), where tears are synthesized and transported to the surface of the eye. In mice, lacrimal gland development begins at embryonic day (E) 13.5 as an invaginating epithelial bud in the periocular mesenchyme. This initial epithelial tube undergoes branching morphogenesis to produce a functional gland by postnatal day (P) 14, corresponding to the time of eye opening (Makareova et al., 2000). The terminal ends of these branched tubes contain terminally differentiated acinar (excretory) cells, ductal cells, and myoepithelial cells (contractile epithelial cells) that together synthesize and secrete tears in response to neuronal stimulation. In the new Development article, "Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland," the authors investigated the cellular and molecular mechanisms behind the morphogenic maturation of the lacrimal gland (Farmer et al., 2017).
Single-cell sequencing reveals transcriptional diversity in the developing lacrimal gland
Employing single-cell mRNA sequencing, first author D’Juan Farmer and colleagues examined two key morphological time points in mouse lacrimal gland development: E 16, when initial epithelial branching is first apparent, and P 4, when differentiated structural features of acini and ducts are visible.
To perform these experiments, lacrimal glands from E 16 and P 4 mice were dissected and dissociated into single-cell suspensions. Dissociated cells were partitioned and processed on a 10x Chromium™ Controller with Chromium™ Single Cell 3’ reagents (v1) to generate barcoded cDNA libraries (one library was constructed for each ~20,000 cell sample; 2 samples at each timepoint). Libraries were sequenced on an Illumina® HiSeq® 2500.
To analyze this complex dataset, the authors first used the 10x Cell Ranger™ pipeline (v1.1 and 1.2 with default settings) to process the raw FASTQ files, filter barcodes, remove PCR duplicates, align sequences to the ensembl database and generate transcriptional profiles for each cell. The Cell Ranger outputs were further processed using Seurat (an R plug-in for single cell analysis) to perform principal component analysis and cell clustering (Satija et al 2015). Final results were displayed in tSNE plots to provide 2-dimensional representations of the multidimensional clustering data.
In figure 1 of Farmer et al., cell clustering patterns in the tSNE plots are overlaid with expression of canonical cell type marker genes. 6 different lacrimal cell populations are apparent at E 16 (Figure 1A), including 2 distinct mesenchymal cell clusters and a cluster of cells enriched for immune marker genes. At P 4, the tSNE plot shows 9 clusters, including 4 distinct mesenchymal cell clusters and 2 distinct immune cell clusters (macrophage/monocyte and mast/lymphocyte) (Figure 1B). In a closer view of the epithelial cell clusters (Figure 1 C), a distinct ductal cell cluster is apparent at E 16, and all three epithelial cell lineages (myoepithelial, ductal and acinary) are apparent at P 4.
To further define the genetic differentiation of the epithelial cell layer in lacrimal gland development, Farmer and colleagues performed qPCR using lacrimal glands dissected at seven time points spanning from late embryonic development (E 14) thru adulthood (16 weeks). The qPCR results confirmed the single-cell sequencing results at E 16 and P 4 and further revealed a highly dynamic pattern of acinar, ductal and myoepithelial gene expression throughout lacrimal gland differentiation (Figure 2 in the Farmer article).
Cell lineage studies
The authors performed lineage tracing experiments to identify possible progenitor cells that might replenish the differentiated epithelial cells in adult lacrimal glands. Using transgenic lines with fluorescent proteins (GFP or RFP) under the control of inducible, tissue-specific gene induction, they labeled individual myoepithelial or basal epithelial cells in mouse lacrimal glands and traced the animals for ~6 months before dissection. Dissected glands were processed for immunofluorescence and analyzed by confocal microscopy to determine what types of cells arose from the initial labeled cell. The results showed that myoepithelial cells can self-renew but do not give rise to any other epithelial cell type (Fig 6 D). In contrast, basal epithelial lineage tracing indicated that basal epithelial cells can give rise to ductal cells by asymmetrical cell division (Fig 7B). This represents the first evidence of progenitor cells in a healthy, adult lacrimal gland.
A model system for studying tubular morphogenesis
In their final set of experiments, the authors characterized the molecular and cellular patterning of lacrimal gland development in donated human fetal lacrimal glands. Using gene expression profiling and immunofluorescence, they show that many features of lacrimal gland maturation, including cell lineage relationships and transcriptional pathways, are conserved between human and mouse.
Having provided a detailed description of cell lineage, molecular differentiation and morphogenesis during lacrimal gland maturation, the authors have established a tractable, defined system for studying the process of branching tubulogenesis. Branching tubulogenesis is a key morphogenic process in multiple organs and tissues, including excretory glands, lungs and kidneys. Insights gained through the study of lacrimal gland development could thus have broader relevance by revealing essential aspects of tubular organ development.
Single-Cell RNA-seq as a tool for developmental biology
The results of the single-cell sequencing experiments described in this recent Development article revealed previously unidentified features of the lacrimal gland, including the presence of immune cells, molecular heterogeneity within mesenchymal cells and the early appearance of a myoepithelial lineage. All single-cell sequencing results in this paper were orthogonally validated by immunofluorescence and microscopy studies, which confirmed the presence of each cell lineage suggested by the single-cell sequencing analyses. Taken together, these results show that high-throughput, single-cell gene expression profiling is a reliable and viable technique for studying a dynamic developmental process at single-cell resolution.
The full article in Development can be found here.
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Farmer, D., Nathan, S., Finley, J., et al., "Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland," Development, June 2, 2017.
Makarenkova, H. P., Ito, M., Govindarajan, V., Faber, S. C., Sun, L., et al., "FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development," Development, 127 (2000): 2563–72.
Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. and Regev, A., "Spatial reconstruction of single-cell gene expression data," Nat. Biotechnol. 33 (2015): 495–502.