Functional analysis of pea aphid Noggin-like genes
Transforming growth factor (TGF) is one of seven key signalling pathways that determine the cell fate in metazoan organisms. Several extracellular antagonists are known to regulate the TGF signalling pathway. One of these antagonists is noggin, a cysteine knot family protein, well-known for its role in dorsal-ventral patterning in vertebrates. Earlier studies have shown that a conserved “Clip” domain at the N terminus of this protein is crucial for binding to and inhibiting BMPs. Recently, genome sequencing of arthropods has revealed the presence of Noggin-likes, orthologs of Noggins that lacks conserved Clip domain, in their genomes. Phylogenetic analysis showed that noggins and noggin-likes share a common ancestor with two developmental proteins in insects, Trunk and prothoracicotropic hormone (PTTH). Trunk and PTTH do not regulate BMP signalling; instead, they activate the Torso/MAPK pathway. Duncan et al., 2013 has suggested that Noggin-likes are likely to be ancestral genes of Trunk and PTTH and may regulate both signalling pathways. However, how similar cysteine knot proteins have coupled themselves in regulating different pathways is yet unknown. Insect Noggin-like genes may hold the answer to this question. In this study, we aimed to characterize molecular functions of pea aphid Noggin (ApNL1), Noggin-like gene (ApNL2) and PTTH of the arthropod pea aphid by exogenously expressing them during Xenopus and Drosophila dorsoventral (DV) patterning, and Drosophila terminal patterning to test their interactions with BMP/Dpp and MAPK signalling, respectively. Our studies have shown that ApNL1 behaves like canonical noggin, acting as a BMP antagonist in Xenopus and Dpp antagonist in Drosophila DV patterning. ApNL1 also mimics the activation of the MAPK pathway when overexpressed in Xenopus animal caps, indicating a dual function of this protein. Our studies have also shown that maternal germline expression of ApNL1 triggers cell death at stage 9 of oogenesis, and this system cannot be used to study Torso/MAPK activation of ApNL1 in Drosophila terminal patterning. In contrast, ApNL2 does not regulate BMP and Dpp signalling in Xenopus and Drosophila DV patterning. However, ApNL2 induced blood-like cells in Xenopus animal caps but failed to produce mesoderm markers in RT-PCR. Furthermore, maternal germline expression of ApNL2 in Trunk null mutant flies rescued Trunk mutant terminal phenotypes, implying that ApNL2 is a Torso/MAPK activator. ApPTTH, on the other hand, also rescued Trunk null mutant phenotypes when maternally expressed, indicating that ApPTTH is a ligand for the Torso/MAPK pathway. In addition, we have also shown that the Clip domain is essential for Dpp antagonism in Drosophila DV patterning but not for BMP antagonism in Xenopus DV patterning. Nevertheless, we also have shown that introducing the Clip domain into Drosophila Trunk, a cysteine knot protein, can introduce BMP antagonism activity. Overall, this research study has identified the molecular functions of arthropod pea aphid Noggin, Noggin-like gene and PTTH, and providing evidence that arthropod Noggin-likes are ancestral genes for Trunk/PTTH.
Advisor: Beck, Caroline; Dearden, Peter
Degree Name: Doctor of Philosophy
Degree Discipline: Zoology
Publisher: University of Otago
Keywords: Noggin; Noggin-like; TGF-beta; Torso; antagonists
Research Type: Thesis