Abstract
Abstract: :
Purpose: Recent work has highlighted the importance of "signaling endosomes." These endosomes carry activated signaling molecules and appear to regulate signal transduction cascades in a spatial and temporal manner. Signaling by FGFs (MAP kinase pathway) and BMPs (SMAD pathway) is important in lens cell differentiation. We found that the molecules involved in these signal transduction pathways co–localize to endosomes in the developing mouse lens. Methods: Wild–type neonatal mouse lenses were fixed and sliced in 100 µm thick sections. Sections were permeabilized and stained with phosphorylation–specific antibodies to Raf (pRAF), pMEK, pERK, pSMAD1 and pSMAD2. The sections were co–stained with the endosomal markers, EEA–1 and Rab5b and viewed with a confocal microscope. Mice lacking Alk3 in the lens were generated by mating Alk3 floxed mice to Le–Cre mice, in which Cre recombinase is expressed in the lens. Results: All of the activated MAPK and SMAD signaling factors studied co–localized on early endosomes. The number of endosomes increased at the onset of fiber cell differentiation, corresponding to increased intensity of pRaf, pMEK, pERK and pSMAD immunostaining. There were also areas of activated Raf, MEK, and ERK staining that appeared endosomal, yet did not co–localize with EEA–1, suggesting that these signaling molecules may move out of early endosomes and into a different intracellular membrane compartment. Endosomal localization of pSMAD1 was markedly reduced in lenses from which the BMP receptor Alk3 had been deleted by homogous recombination, indicating that BMP signaling is important for endosomal localization of its downstream effectors. Conclusions: Signaling molecules in the MAPK and SMAD cascade co–localize to endosomes in the developing mouse lens. As in other systems, signaling endosomes will likely play an important role in the signal transduction network that regulates lens growth and development.
Keywords: signal transduction