Comparison of embryonic samples (E18) vs. adult (34 weeks old) lacrimal gland transcriptome

As an element of the lacrimal apparatus, the lacrimal gland (LG) produces the aqueous part of the tear film, which protects the eye surface. Therefore, a defective LG can lead to serious eyesight impairment. Up to now, little is known about LG morphogenesis and subsequent maturation. In this study, we delineated elements of the cellular and molecular events involved in LG formation by using three epithelial markers, namely aSMA, Krt14 andKrt19. While aSMA marked a restricted epithelial population of the terminal end buds (TEBs) in the forming LG, Krt14 was found in the whole embryonic LG epithelial basal cell layer. Interestingly, Krt19 specifically labelled the presumptive ductal domain and subsequently, the luminal cell layer. By combining these markers, the Fucci reporter mouse strain and genetic fate mapping of the Krt14+ population, we demonstrated that LG epithelium expansion is fueled by a patterned cell proliferation, and to a lesser extent by epithelial reorganization and possible mesenchymal-to-epithelial transition. We pointed out that this epithelial reorganization, which is associated with apoptosis, regulated the lumen formation. Finally, we showed that the Notch signalling inhibition prevented the ductal identity from setting, and leads to a LG covered by ectopic TEBs. Taken together our results bring a deeper understanding on LG morphogenesis, epithelial domain identity, and organ expansion. 6 samples were analysed, 3 embryonic samples (biological triplicates, E18) and 3 adult samples (biological triplicates, 34wks old). Embryonic samples were composed of at least 10 LGs (5 individuals) pooled together. Samples were collected for RNA extraction and hybridization on Affymetrix microarrays (MTA1).作为泪器（lacrimal apparatus）的组成部分，泪腺（lacrimal gland, LG）可分泌泪膜的水相成分，从而保护眼表。因此，泪腺功能缺陷可导致严重的视力损害。截至目前，学界对泪腺的形态发生及后续成熟过程仍知之甚少。本研究通过使用三种上皮标志物——α平滑肌肌动蛋白（aSMA）、角蛋白14（Krt14）和角蛋白19（Krt19）——阐明了泪腺形成过程中的细胞与分子事件。其中，aSMA可标记发育中泪腺内终末芽（terminal end buds, TEBs）的特定上皮细胞群；Krt14则表达于整个胚胎泪腺上皮基底层。值得注意的是，Krt19可特异性标记预设的导管结构域，并随后标记腔细胞层。结合上述标志物、Fucci报告基因小鼠品系（Fucci reporter mouse strain）以及对Krt14阳性细胞群的遗传谱系示踪，我们证实泪腺上皮的扩增主要受模式化细胞增殖驱动，其次则依赖上皮重排及可能存在的间质-上皮转化（mesenchymal-to-epithelial transition, MET）。我们还发现，伴随细胞凋亡（apoptosis）的上皮重排可调控管腔形成。最后，我们证实抑制Notch信号通路会阻碍导管细胞身份的确立，并导致泪腺被异位终末芽覆盖。综上，本研究结果加深了我们对泪腺形态发生、上皮域身份确立及器官扩增过程的理解。本研究共分析6份样本：3份胚胎样本（生物学重复，胚胎第18天，E18）与3份成年样本（生物学重复，34周龄）。胚胎样本由至少10个泪腺（来自5只个体）混合制备。收集样本用于RNA提取及Affymetrix基因芯片（MTA1）杂交检测。