MAP kinase in situ activation atlas during Drosophila embryogenesis

Abstract

Receptor tyrosine kinases (RTKs) and the signaling cascades that they trigger play central roles in diverse developmental processes. We describe the capacity to follow the active state of these signaling pathways in situ. This is achieved by monitoring, with a specific monoclonal antibody, the distribution of the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to the known RTKs. This specific detection has enabled us to determine the time of receptor activation, visualize gradients and boundaries of activation, and postulate the distribution of active ligands. Since the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the TEY motif, this approach is applicable to a wide spectrum of multicellular organisms.

A non-canonical Raf function is required for dorsal–ventral patterning during Drosophila embryogenesis

“…The EGFR signaling pathway has not previously been associated with ventral cell fates and previous studies have not detected phosphorylated ERK in ventral cells 8 , 81 . Raf 926 mutants do not make a ventral furrow preventing standard staining or in situ RNA hybridization from being informative.…”

“…Most importantly, this Raf function appears to be independent of the MAPK cascade, as there is no evidence of active MAPK in ventral cells. Studies looking at phosphorylated MAPK show activation at the anterior and posterior poles in early stages, lateral activity in early gastrulation and activation in the mesoderm 81 . Interaction of MAPK and Toll pathway has been observed at early stages through Capicua and WntD 96 , 97 .…”

Patient-associated mutations inDrosophilaAlk perturb neuronal differentiation and promote survival

“…Embryo staining was carried out after (Muller, 2008). For phospho-ERK staining we followed the protocol from (Gabay et al , 1997).…”

FGF Pyramus Has a Transmembrane Domain and Cell-Autonomous Function in Polarity

“…Immunostaining reveals that 1$2 mesoderm cells at the dorsal-most position are positive for di-phosphorylated Erk (dpERK) on each side of the migrating mesodermal collective ll in wild-type embryos at late stage 8 (Figures 5A and 5B) [25]. This pattern is completely lost when the pyr gene is deleted in entirety (i.e., Df(2R)pyr36; Figure 5C), demonstrating a requirement for pyr in Htl-dependent mitogen-activated protein kinase (MAPK) signaling activation, which is consistent with previous studies [8,9,26].…”