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

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.

miR-205 is a critical regulator of lacrimal gland development.

The tear film protects the terrestrial animal's ocular surface and the lacrimal gland provides important aqueous secretions necessary for its maintenance. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an important gene for lacrimal gland development. Mice lacking miR-205 fail to properly develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal gland development. Specifically, more than half of knockout lacrimal glands never initiated, suggesting a critical role of miR-205 at the earliest stages of lacrimal gland development. RNA-seq analysis uncovered several up-regulated miR-205 targets that may interfere with signaling to impair lacrimal gland initiation. Supporting this data, combinatorial epistatic deletion of Fgf10, the driver of lacrimal gland initiation, and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways related to Fgf10 in order to regulate glandular development. These data show that a single microRNA is a key regulator for early lacrimal gland development in mice and highlights the important role of microRNAs during organogenesis.

Salivary gland development and disease.

Mammalian salivary glands synthesize and secrete saliva via a vast interconnected network of epithelial tubes attached to secretory end units. The extensive morphogenesis required to establish this organ is dependent on interactions between multiple cell types (epithelial, mesenchymal, endothelial, and neuronal) and the engagement of a wide range of signaling pathways. Here we describe critical regulators of salivary gland development and discuss how mutations in these impact human organogenesis. In particular, we explore the genetic contribution of growth factor pathways, nerve-derived factors and extracellular matrix molecules to salivary gland formation in mice and humans.

Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye.

BACKGROUND: Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, we have been analyzing R7 photoreceptor neuron development in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). We were able to remove PcG genes specifically from post-mitotic R1/R6/R7 precursors, allowing us to probe these genes' roles in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. RESULTS: Here, we show that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. We find that this fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, we show that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. CONCLUSIONS: We conclude that cells can use PcG genes specifically to maintain a subset of their binary fate choices.

Manipulating the murine lacrimal gland.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

Parasympathetic innervation regulates tubulogenesis in the developing salivary gland.

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.

Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development.

Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. We show that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. We find that the earlier defect coincides with a failure to localize synaptic vesicles, suggesting that it reflects a failure in presynaptic assembly. We then analyze the relationship between syd-1 and Liprin-α in R7s. We find that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: we show that Liprin-α promotes Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. We show that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. We therefore conclude that the two defects are caused by distinct molecular mechanisms. We find that Trio overexpression rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity.