Requirement of Mad for wingless signaling in Drosophila wing development
Signaling pathways play crucial role in pattern formation and cross talk between different pathways forms an important aspect of developmental biology. During early embryogenesis ,cells are exposed to various multitude of growth factors,which will be put together to generate a particular cell differentiation decision. Wingless/wnt and dpp/BMP pathways are widely studied for their roles in various aspects of developmental biology.Dpp is the Drosophila homolog of the vertebrate bone morphogenetic proteins (BMPs), which are members of the TGF-β superfamily.
The integration of these two vital signaling pathways leads to formation of anterior-posterior (A/P) and Dorso-Ventral (D/V). Previous work has shown that a gradient of Wnt is a major determinant of the (A–P) axis, with low levels causing head and high levels tail development,while BMP signals take care of D/V patterning in early embryo.
SMADs are a class of proteins that modulate the activity of BMP/transforming growth factor beta ligands and these SMADs form complexes, often with other SMAD’s, enter the nucleus, and serve as transcription factors. The SMADs are homologous to both the drosophila protein, mothers against decapentaplegic (MAD) and the Caenorhabditis elegans protein SMA.Hence they get their name ,which is a combination of both. SMADs are highly conserved across species, especially in the N terminal MH1 domain and the C terminal MH2 domain. The MH1 domain has DNA specific binding properties ,which will enable the protein to interact to specific sequence of DNA.
These smad’s can be phosphorylated by Bone Morphogenetic ProteinReceptors (BMPR),Mitogen Activated Protein Kinase (MAPK) and also by Glycogen Synthase Kinase 3 (GSK3). GSK-3’s homolog in the fruit fly Drosophila melanogaster is known as Shaggy . In Drosophila and the frog Xenopus laevis GSK-3 works in the Wnt signalling pathway to phosphorylate β-catenin/armadillo. After phosphorylation of armadillo, leads to ubiquitination and degradation by cellular proteases, preventing it from entering the nucleus and activating transcription factors. In the cell where wingless signaling is active, a protein called Disheveled is activated which further inhibits GSK-3 , allowing β-catenin to accumulate and effect transcription of Wnt target genes. GSK-3 also phosphorylates Ci in the Hedgehog (Hh) pathway, targeting it for proteolysis to an inactive form.
Therefore Smads offer the possibility of integrating three of the main signaling pathways – BMP,MAPK and Wnt – on a single molecule. The Drosophila genome contains a single BMP-Smad, called mothers against dpp (Mad) [13], which has a single canonical MAPK/Erk phosphorylation site and two GSK3 sites upstream of it. The fruit fly therefore offered an excellent system to investigate signaling integration.
Using this system Eddy de Robertis lab investigated that whether endogenous Mad was required for Wingless (Wg) signaling in Drosophila and results of the study were published recently online in Plos One.
For the study they generated specific antibodies against pMadGSK3 and pMadMAPK, and also Mad RNAi knockdown constructs in order to knowk down maternal or zygotic Mad mRNA.
Interestingly Mad RNAi clones eliminated the wing margin,which require proper wingless signalling from the D/V organizer in wing imaginal disc. Mad knockdown with RNAi inhibited the increases in senseless, optomotor blind, distalless and vestigial transcripts caused by Wg.Generally ,In the wing disc, Wg overexpression strongly increased senseless, distalless, optomotor blind, and vestigial transcripts, and co-expression of Mad RNAi inhibited this effect, without affecting Wg expression levels. All these results indicate role of MAD in wingless signalling. Another intersting result from the study led by Edward Eivers and Luis C. Fuentealba demonstrates a novel role for Mad during segment formation.
References and Image Credit :
Eivers E, Fuentealba LC, Sander V, Clemens JC, Hartnett L, et al. (2009) Mad Is Required for Wingless Signaling in Wing Development and Segment Patterning in Drosophila. PLoS ONE 4(8): e6543. doi:10.1371/journal.pone.0006543
Heldin CH, Miyazono K, ten Dijke P (December 1997). “TGF-beta signalling from cell membrane to nucleus through SMAD proteins”. Nature 390 (6659): 465–71. doi:10.1038/37284
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