ABSTRACT
The Pax gene eyg is important for Drosophila eye development. eyg expression in the visual system changes dynamically during development. In this study, we found that the transcriptional regulation of eyg can be separated into four distinct temporal phases (E, L1, L2, and L3) and each is regulated by distinct cis-regulatory elements. Utilizing these enhancers for temporal and spatially specific manipulations, we addressed the regulation and function of eyg at different developmental stages. We found that Notch signaling is required and sufficient for eyg expression and this activity is restricted only to the L2 stage. We further showed that the function of eyg in eye development is required only at the second instar larval stage, while its function for head and antenna development can be provided at any time during embryo and larval development. Thus there is a temporal switch of the regulatory mechanism and function of eyg. We propose that eyg expression at L2 is induced and maintained by N signaling, and is turned off at L3 by a negative feedback loop involving the morphogenetic furrow.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/embryology , Eye/embryology , Gene Expression Regulation, Developmental/physiology , Regulatory Elements, Transcriptional/physiology , Animals , Immunohistochemistry , In Situ Hybridization , Receptors, Notch/metabolism , Signal Transduction/physiologyABSTRACT
Notch (N) signal is activated at the dorsoventral (DV) border of the Drosophila eye disc and is important for growth of the eye disc. In this study, we showed that the Pax protein Eyg is a major effector mediating the growth promotion function of N. eyg transcription is induced by N signaling occurring at the DV border. Like N, eyg controls growth of the eye disc. Loss of N signaling can be compensated by overexpressing eyg, whereas loss of the downstream eyg blocked the function of N signaling. In addition, we showed that N and eyg could induce expression of upd, which encodes the ligand for the Jak/STAT pathway and acts over long distance to promote cell proliferation. Loss of eyg or N can be compensated by overexpressing upd. These results suggest that upd is a major effector mediating the function of eyg and N. The functional link from N to eyg to upd explains how the localized Notch activation can achieve global growth control.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/growth & development , Eye/growth & development , Membrane Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Division/physiology , Gene Expression Regulation, Developmental/physiology , Receptors, Notch , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
It has been well established that hepatocyte growth factor (HGF) induces branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, focalization proteolysis, cell-cell interaction and differentiation. However, signaling pathways and proteins involved in HGF-induced tubulogenesis by MDCK cells have not been thoroughly studied. Because cell-matrix interactions play important roles in tubulogenesis, we analyzed whether HGF altered the expression of extracellular matrix receptor (alpha2, alpha3, beta1 and alphavbeta3 integrin). We found that among those proteins examined, alpha2beta1 integrin levels were enhanced by HGF. HGF-induced upregulation of alpha2beta1 integrin was mediated via upregulation of alpha2 integrin mRNA abundance. Cycloheximide blocked the HGF-induced increase in alpha2 integrin mRNA expression. To understand the signaling pathways leading to an HGF-induced increase in alpha2beta1 integrin levels, PD98059 (MEK1 inhibitor), LY294002 (PI3-kinase inhibitor), and GF109203X (PKC inhibitor) were used. We found that PD98059 blocked the HGF-induced increase in alpha2beta1 integrin expression. Furthermore, 5E8 (specific anti-alpha2beta1 integrin antibody) was employed to elucidate the potential role of HGF-induced upregulation of alpha2beta1 integrin in branching morphogenesis. 5E8 did not alter HGF-induced scattering effects but disrupted HGF-induced branching tubulogenesis in collagen gel via inhibition of cell-cell interactions and growth. Taken together, HGF upregulates alpha2beta1 integrin expression via an indirect pathway, the results of which contribute to the regulation of cell-cell interactions and cell growth during branching morphogenesis in collagen gel.
