Our data uncovered several signaling pathways that may play important roles in lacrimal gland development and morphogenesis. From the large list of potential candidates, we chose to focus on one—the NOTCH signaling pathway—in order to directly evaluate its role in lacrimal gland development. We selected NOTCH because, as noted above, many genes of the NOTCH signaling pathway, including
Notch1,
Notch3,
Hes1,
Dlk1,
Jag1,
Jag2, and
Rbpj, were expressed at significantly higher levels in the embryonic lacrimal gland than in the adult lacrimal gland (
Figs. 11552–
3;
Supplementary Table S4). We began our study of the NOTCH pathway by treating embryonic lacrimal glands with DAPT, a nonselective inhibitor of NOTCH signaling. We cultured lacrimal glands from E15.5 mice in the presence of 10, 50, and 100 μM of DAPT. Untreated lacrimal glands or lacrimal glands cultured in the presence of Dimethyl Sulfoxide (DMSO) (stock solutions of DAPT were prepared in DMSO) were saved as controls. Three days after treatment, the glands were collected and morphometrically analyzed. We found that while DAPT treatment did not affect significantly the overall size of the cultured embryonic lacrimal glands, it did exert a strong effect on average lobule size and average number of lobules (
Fig. 5). DAPT treatment led to a reduction in the average size of lobules (93 ± 11 μm [DMSO], 66 ± 3 μm [10-μM DAPT], 60 ± 1 μm [50-μM DAPT], 73 ± 3 μm [100-μM DAPT]), but also to an increase in the average number of lobules (22 ± 2 [DMSO], 53 ± 2 [10-μM DAPT], 38 ± 4 [50-μM DAPT], 31 ± 1 [100-μM DAPT];
Fig. 5). We did not detect a difference between untreated and DMSO-treated lacrimal glands (
Fig. 5). To demonstrate direct effect of Notch signaling inactivation, we used Notch1
flox/flox mice. To this end, lacrimal glands isolated from Notch1
flox/flox mice and wild-type animals were treated with Ad-CMV-iCre, the adenovirus constitutively expressing Cre recombinase that catalyzes site-specific recombination of DNA between loxP sites. Untreated Notch1
flox/flox and wild-type lacrimal glands were saved as controls. Three days after Ad-CMV-iCre treatment, the glands were collected and morphometrically analyzed. In agreement with the DAPT data above, we found that Notch1 knockout in the embryonic lacrimal gland led to a reduction in the average size of lobules (90 ± 3 μm [wild-type untreated], 82 ± 1 μm [wild-type Ad-CMV-iCre treated], 81 ± 3 μm [Notch1
flox/flox untreated], 56 ± 2 μm [Notch1
flox/flox Ad-CMV-iCre treated]), but also to an increase in the average number of lobules (29 ± 3 [wild-type untreated], 37 ± 6 [wild-type Ad-CMV-iCre treated], 40 ± 2 [Notch1
flox/flox untreated], 51 ± 2 [Notch1
flox/flox Ad-CMV-iCre treated]), respectively (
Fig. 6). To verify that the observed effect was due to reduced Notch1 expression, two groups of Notch1
flox/flox untreated and Notch1
flox/flox Ad-CMV-iCre–treated lacrimal glands were used to test Notch1 expression by quantitative RT-PCR. We found that Notch1 expression was reduced in Ad-CMV-iCre–treated lacrimal glands (100 ± 9% [Notch1
flox/flox untreated] vs. 69 ± 3% [Notch1
flox/flox Ad-CMV-iCre treated],
P value = 0.02,
n = 5).