Lacrimal gland budding requires PI3K-dependent suppression of EGF signaling

Abstract

The patterning of epithelial buds is determined by the underlying signaling network. Here, we study the cross-talk between phosphoinositide 3-kinase (PI3K) and Ras signaling during lacrimal gland budding morphogenesis. Our results show that PI3K is activated by both the p85-mediated insulin-like growth factor (IGF) and Ras-mediated fibroblast growth factor (FGF) signaling. On the other hand, PI3K also promotes extracellular signal-regulated kinase (ERK) signaling via a direct interaction with Ras. Both PI3K and ERK are upstream regulators of mammalian target of rapamycin (mTOR), and, together, they prevent expansion of epidermal growth factor (EGF) receptor expression from the lacrimal gland stalk to the bud region. We further show that this suppression of EGF signaling is necessary for induction of lacrimal gland buds. These results reveal that the interplay between PI3K, mitogen-activated protein kinase, and mTOR mediates the cross-talk among FGF, IGF, and EGF signaling in support of lacrimal gland development.

Fig. 1. PI3K signaling is required for lacrimal gland development.

(A) The lacrimal gland primordium arises as thickening of the conjunctival epithelium at mouse embryonic day 13.5 (E13.5). One day later, it elongates to form a bud followed by an extended stalk. At postnatal day 0 (P0), the lacrimal gland is composed of two glandular structures with extensive branches. iLG, intraorbital lacrimal gland; eLG, extraorbital lacrimal gland. (B to D) Lacrimal gland buds (arrows) were presented at E14.5 in control and p85^CKO mutants but not in p110^CKO embryos. H&E, hematoxylin and eosin. (E to J) Although the progenitor cell marker Sox9 was preserved in the lacrimal gland primordia, pAKT was reduced in p85^CKO and lost in p110^CKO mutants. (K to M) Compared to controls, p85^CKO and p110^CKO mutants exhibited fewer or even no pHH3⁺ cells, respectively. (N) Quantification of pHH3⁺ cells and pAKT staining. One-way analysis of variance (ANOVA): *P < 0.02 for p85^CKO versus control, **P < 0.05 for p85^CKO versus p110^CKO, #P < 0.0001 for p85^CKO versus control, and ##P = 0.0002 for p85^CKO versus p110^CKO. n = 5 for pHH3⁺ quantification of controls and n = 3 for all other genotypes.

Fig. 2. The p110α-Ras interaction is required for FGF to activate PI3K signaling.

(A) Schematic diagram of PI3K activation. The p110α catalytic subunit of PI3K can be stimulated by RTK via binding to p85, or it could be coupled to FGF signaling via interaction with Ras. (B) FGF2 treatment led to increased levels of pAKT in control but not in p110α^RBD/RBD MEF cells that carry mutations disrupting the p110α RBD domain. One-way ANOVA was used to test for statistical significance (n = 4 for data at 0′ and 5′ and n = 3 for data at 10′). N.S., not significant. (C and D) p110^RBD mutants still expressed Sox9 in lacrimal gland buds but exhibited reduced staining for both pAKT and pHH3, while TUNEL staining was increased. The lacrimal glands were much reduced in p110^RBD mutants at E15.5 and birth. (E) Quantification of pHH3 (n = 6)– and TUNEL (n = 3)–positive cells. Student’s t test was used to calculate statistical significance.

Fig. 3. p110 mutations disrupt PI3K signaling and expression of MAPK response genes.

(A) Segregation of E14.5 control, p110^RBD, and p110^CKO lacrimal gland transcriptomes shown by cluster analysis. (B) GO analysis showed that the differentially regulated genes in p110^CKO mutants were enriched in PI3K-regulated pathways. ECM, extracellular matrix. (C) GSEA revealed that IGF signaling was disrupted in p110^CKO mutants. FDR, false discovery rate. (D) As shown in volcano plot of the control versus the p110^CKO transcriptome, Igf1 and Igf2 were among up-regulated genes, and Fgf1, Etv4, Etv5, Dusp6, Six1, and Sox10 were significantly down-regulated. (E) Heatmap of differentially regulated genes implicated in IGF and MAPK pathways. (F to W) RNA in situ hybridization and immunostaining confirmed down-regulation of MAPK response genes in p110^RBD and p110^CKO mutants.

Fig. 4. PI3K activates MAPK via the p110α-Ras interaction.

(A) In p110α^RBD/RBD MEF cells, the loss of the p110α-Ras interaction not only prevented FGF from inducing AKT phosphorylation but also down-regulated the levels of pERK and active Ras (Ras-GTP). One-way ANOVA test for statistical significance (n = 2). (B) Deletion of p110α and p110β in MEF cells disrupted IGF-induced AKT and ERK phosphorylation. One-way ANOVA test for statistical significance (n = 2). (C) pERK staining was down-regulated in p110^RBD glands and sharply reduced in p110^CKO mutants. Expression of the constitutively active Mek1^DD allele led to partial recovery of lacrimal gland budding and pERK staining in p110^CKO;Mek1^DD mutants. One-way ANOVA test (n = 5 for the control and n = 3 for the rest). (D) In comparison to control and Mek1^DD pups, p110^RBD mutants mostly displayed residual glands at birth, whereas p110^RBD;Mek1^DD animals showed more extensively branched glands. Similarly, lacrimal gland induction was observed in p110^CKO;Mek1^DD mutants but not in p110^CKO mutants.

Fig. 5. ERK cooperates with PI3K to control mTOR activity.

(A to L) pmTOR, p4EBP1, and pS6 were reduced in p85^CKO and p110^RBD lacrimal gland buds. In p110^CKO mutants, however, there was persistent pmTOR staining despite loss of p4EBP1 and pS6. (M to U) Although Lrp^CKO mutants lacked lacrimal gland buds, they retained pERK and pmTOR staining in the Sox9-positive progenitor cells. Erk^CKO mutants also preserved Sox9 expression, but the pERK staining was lost, and pmTOR was diminished. (V) In MEF cells treated with FGF2 (25 ng/ml), PI3K inhibitor LY294002 (LY) abolished induction of pAKT, but pERK and pmTOR were only partially reduced. MEK inhibitor U0126 (U) only abrogated pERK induction. In contrast, combined treatment of PI3K and MEK inhibitors (LY + U) abolished mTOR phosphorylation. One-way ANOVA test (n = 3).

Fig. 6. mTORC1 is required for lacrimal gland budding and ERK phosphorylation.

(A to L) Although deletion of the mTORC2 subunit Rictor did not affect lacrimal gland budding in Rictor^CKO embryos, loss of the mTORC1 subunit Raptor in Raptor^CKO mutants resulted in loss of pmTOR, p4EBP1, and pS6 in Sox9-expressing progenitor cells. (M to O) The expression of Sox10 was also lost in Raptor^CKO mutants. (P) The mTOR inhibitor Torin not only abolished FGF-induced phosphorylation of mTOR and AKT but also down-regulated the level of pERK. The statistical significance was calculated using one-way ANOVA test (n = 3 for pERK and n = 4 for pAKT). (Q to X) pERK staining was reduced in Raptor^CKO mutants, elevated in Mek1^DD, and partially recovered in Raptor^CKO;Mek1^DD embryos. As a result, although Raptor^CKO mutants lacked any lacrimal gland, residual lacrimal gland was observed in Raptor^CKO;Mek1^DD pups. (Y) Quantification of lacrimal gland phenotypes.

Fig. 7. Suppression of EGF signaling by PI3K and MAPK is necessary for lacrimal gland budding.

(A) Egfr expression obtained by RNA-seq analysis was elevated in p110^RBD and p110^CKO mutants. FPKM, Fragments Per Kilobase of transcript per Million. (B) In control lacrimal glands, Egfr was expressed in the stalk region but excluded from the bud. This distinction was lost in p110^RBD lacrimal glands. In p110^CKO, Fgfr2^CKO, Erk^CKO, and Raptor^CKO mutants, Egfr was uniformly expressed in the conjunctiva. (C) The explant assay was performed with the eye tissue dissected from E13.5 Le-Cre embryos that expressed GFP. After cultured on the floating membrane in the presence or absence of EGF, the eye rudiment was evaluated for formation of the GFP⁺ lacrimal gland bud. (D) In 2 days of culture, the lacrimal gland budding was inhibited by EGF (100 ng/ml) but not bovine serum albumin (BSA). (E) Quantification of lacrimal gland budding. Fisher’s exact test, P = 0.0006. (F) Model of PI3K-MAPK interaction in lacrimal gland development. The p110 subunit of PI3K is activated by both p85-mediated IGF signaling and Ras-mediated FGF signaling. Conversely, the p110α-Ras interaction also activates Ras to stimulate MAPK signaling, which cooperates with AKT to control mTOR activity. We propose that these signaling pathways may suppress Egfr expression (dashed lines), allowing the budding of the lacrimal gland from the conjunctiva.