Milne, C.A.; (2007) Analysis of neural development using ligand-trap transgenic lines. Doctoral thesis , University of London.

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Abstract

Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor-beta (TGF-beta) signalling protein superfamily. BMPs play important and diverse roles in cell-cell signalling, including establishing cell fate during the development of vertebrate embryos. Their activity is antagonised in vivo by a number of proteins such as noggin, which sequester BMP ligands, preventing them from binding to BMP receptors. This thesis describes studies to establish a binary genetic approach combined with a ligand trap system to manipulate BMP signalling in the frog embryo. This system has been used to investigate the roles of BMP signalling in dorso-ventral patterning of the forebrain Xenopus tropicalis. The binary system described utilises a variety of tissue- or region-specific gene promoters to drive expression of the GAL4 transcriptional activator. Such transgenic "driver" lines can be crossed with a "responder" line in which expression of a membrane-tethered fusion protein comprising human Noggin fused to GFP is regulated by a synthetic promoter responsive to GAL4 (UAS-flognog). Transient expression assays confirmed the effectiveness of the "responder" line, GAL4 transactivation of UAS-flognog resulted in the expression of Flognog and an expansion of neural progenitor tissue, indicated by the X-Sox3 marker. In a binary cross with the Otx2-gal4 driver line, targeted GAL4 transactivation lead to a decrease in phospho-Smad-1 staining in the anterior CNS and eye in a proportion of cross embryos. Such a cross resulted in embryos showing an open neural tube and alterations in both Pax6 (dorsal) and X-<U13 (ventral) forebrain markers, further indicating the efficacy of the binary, ligand-trap strategy. In order to achieve temporal control on the activity of the UAS-flognog responder line in the telencephalon, an inducible driver line comprising the Pax6 promoter driving hormone-inducible GalPR (an inducible chimeric GAL4) was created. In binary crosses with a UAS-gfp reporter line, GFP expression was detected in the forebrain, hindbrain and spinal cord only in the presence of the steroid hormone, RU486. Similarly, a second driver line, N-tubulin-GalPR yielded inducible GFP expression in the developing brain, spinal cord and lens tissue in the presence of RU486. In conclusion, these findings are evidence that the binary ligand trap approach is functional and can cause targeted knockdown of BMP signalling, resulting in alterations in neural development and patterning. Furthermore, using an inducible version of this approach, Flognog (or any other target gene) can be expressed in the telencephalon in a RU486-inducible manner.

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