Darft part 1 2nd report

Abstract

During the development of Drosophila Melanogaster embryo, the product of Decapentaplegic (Dpp) gene plays an essential role in the final organization of dorsal-ventral pattern. It is a homolog of Bone Morphogenetic Proteins 2 and 4 (BMP 2/4) and acts as an extracellular morphogen to form a gradient with the effect of some other regulators including short gastrulation (Sog), twisted gastrulation (Tsg), Dcg1, Viking (Vkg) and Tolloid (Tld). Among these Dpp regulators, Sog and Tsg are products of zygotic genes, and Vkg and Dcg1 belong to the type IV collagens. In our project, we checked the interaction between Tsg, Vkg, Sog and Dpp by GST pull down assay to confirm the result from other researches, and tried to divide the Sog and Dpp proteins into smaller fragments in order to find out the region where the interaction occurs. We also performed in situ hybridization on Dpp and Dcg1 mutated embryos and wild type embryos to compare the expression patterns of Race and U-shaped (Ush) genes which are related to the expression of Dpp. And we found that there are differences between the wild type and mutated embryos.

Introduction

As kind of insect, Drosophila Melanogaster undergoes germ layer differentiation named gastrulation and forms endoderm, mesoderm and ectoderm during embryo development. This differentiation process rises from the different spatial positions of the cells in the embryo and is mostly controlled by morphogens. (Ashe and Briscoe, 2006)
During the early stages of embryo Drosophila Melanogaster development, the product of spätzle gene acts as a morphogen to establish the dorsal-ventral pattern (Morisato and Anderson, 1994). Then, the control is taken away by other morphogens. In previous studies, it has been reported that two homologs of Bone Morphogenetic Proteins 2 and 4 (BMP 2/4), Decapentaplegic (Dpp) and Screw (Scw) which are members of TGF-β superfamily, are the control factors for the formation of dorsoal ectoderm in Drosophila Melanogaster. Further researches indicated that Dpp plays the main role since all dorsal cells assume ventral lateral fates and the mutant phenotype of the zygotically required genes appears the most severely with the lack of Dpp (Arora and Nusslein-volhard, 1992; Ferguson and Anderson, 1992a). Experiments also showed that after the dorsal-ventral (DV) axes is patterned, the effect of the morphogenetic Dpp depends on the local concentration level. In detail, high Dpp level leads to the fate of being amnioserosa whilst lower levels make the cell become dorsal ectoderm tissue (Shimmi and O'Connor, 2003). The Dpp/Scw functions by binding to a receptor complex which is supposed to be composed of type I and type II transmembrane serine/threonine kinases (Hogan, 1996; Nakayama et al., 2000). It has been elucidated that in Drosophila, the type I receptor Thick veins (Tkv) interact with the Dpp molecule and the type II receptor Punt (Put) to form a heteromeric complex. The active Put within this complex phosphorylate the Tkv consequently and thus activates the associated type I kinases which will phosphorylate the cytoplasmic protein Mothers against Dpp (Mad). The Mad which belongs to the Smad superfamily then function inside of the cell and lead to the differentiation and in this way the signal of Dpp is transduced. Moreover, in the signal transduction of Scw, it seems that another type I receptor Saxophone (Sax) takes the place of Tkv in Dpp signal transduction. However experiment also showed that high level of Tkv is able to compensate the loss of Sax, and indicates that these two morphogens shares a similar intracellular pathway. (Affolter et al., 2001)
Based on the knowledge above, the regulation of dorsal ectoderm and amnioserosa formation by BMP signalling pathway is suggested to be achieved by the gradient distribution of Dpp. This hypothesis is confirmed by the visualization of Dpp distribution in wild type and mutant Drosophila embryos (Ferguson and Wang, 2005; Shimmi et al., 2005). The work done by the Shimmi group also revealed the fact that the Dpp and Scw may function effectively by forming a Dpp-Scw heterodimer which is able to induce a 10 to 100 fold phosphorylated Mad accumulation than Dpp or Scw homodimer in the cells exposed to these materials (Shimmi et al., 2005).
An interesting and indispensable phenomenon of the gradient of Dpp is that it is not smoothly distributed. It has been shown that Dpp distribution has a peak level at the 8-10 dorsalmost cells which will later form the amnioserosa, and there is a sharp fall of Dpp level at the edge of the dorsalmost cells where cells on the different sides of the confine receive high and very low BMP signal (Shimmi and O'Connor, 2003). This step gradient ensures the normal development of amnioserosa and dorsal epidermis.
According to the model of morphogen distribution which concerns the Dpp/Scw heterodimer alone, this phenomenon is unable to be explained. In the classical model, specified cell produces the morphogen and the morphogen diffuses to the tissue around either via extracellular or intracellular pathway. Thus a morphogen concentration gradient which peaks at the secreting cell and smoothly deduced along with the increasing distance to the secreting cell (Ashe and Briscoe, 2006). In this way, the formation of this step gradient of Dpp should be created by another mechanism with some other factors.
So far we have already known that Dpp signalling activity is regulated by the products of two zygotic genes short gastrulation (Sog) and twisted gastrulation (Tsg), two type IV collagens Viking (Vkg) and Dcg1, and at last a protease Tolloid (Tld) (Ashe, 2005). It is known that Sog gene is a homologue of Xenopus Chordin (Chd) gene which produces antagonist of BMPs both structural and functional. (FERGUSON and ANDERSON, 1992b; Piccolo et al., 1996). And later studies have discovered that Tsg is also a secreted protein which acts as extracellular antagonists of BMP (Chang et al., 2001; Ross et al., 2001). These works gave the evidence that Sog and Tsg bind to BMP directly and inhibit the interaction between BMP and its receptor (Tkv and Put) to form a Tsg-Sog-Dpp-Scw complex in order to block the signal transduction (Ashe, 2005). However, this inhibition will not destroy the BMP itself, and the formed complex can be divided into the original single molecular as well.



References

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