| dc.description.abstracteng | In early development, morphogen gradients orchestrate the establishment of body axes. Perturbations of such a fundamental process are often damaging to embryonal development, making it difficult to study the role of body patterning factors later during development or even in adult animals. The planarian flatworm Schmidtea mediterranea provides a valuable model for addressing this challenge: Its pluripotent stem cell population does not only allow it to regenerate and reestablish its body axes after injury but is also required for dynamic tissue turnover and body plan maintenance. Therefore, by introducing perturbations of body axis patterning by RNA-interference we can study the remodeling of the body plan and the functions of patterning factors in an adult animal. A specifically interesting body plan determinant is Bone morphogenetic protein (BMP), a member of the TGFβ signaling family, which has been shown to not only pattern the dorso-ventral axis in S. mediterranea, but also influences anterior-posterior and mediolateral patterning. It is expressed on the dorsal side in a mediolateral gradient with the highest expression on the midline and fading intensity towards the body edge. However, how this mediolateral bmp expression gradient is formed is poorly understood. Moreover, the spatial distribution of BMP signaling activity through the planarian body remains unstudied so far.
The overall goal of my thesis was to probe the mechanistic basis of BMP gradient formation in S. mediterranea. In the first part, I systematically annotated the TGFβ signaling pathway components including BMP in the S. mediterranea genome and reconstructed their phylogeny in the context of other metazoans. Next, I established a Western blotting assay for the quantification of BMP signaling activity in response to the knockdown of potential BMP signaling factors. Furthermore, I established a whole mount immunostaining assay for the activated BMP signaling transducer phospho-Smad1 to reveal the spatial patterns of BMP signaling activity. With these tools at hand, I was able to quantify BMP signaling activity and visualize its spatial distribution throughout the planarian body. In the last part of the thesis, I utilized these methods to gain understanding of the mechanisms that establish the mediolateral expression profile of bmp in S. mediterranea.
I found previously undescribed TGFβ ligands and identified candidates for putative regulators of BMP4 signaling activity in an RNAi screen. Furthermore, I was able to visualize the spatial distribution of BMP signaling activity in S. mediterranea wild type animals and found that BMP signaling is not only active in the sites of dorsal bmp4 expression, but also occurs in other tissues like the gut. Moreover, my findings indicate that BMP signaling is likely involved in gut branching. By assessing the expression pattern of bmp4 and the spatial distribution of BMP signaling activity, I found indications for autoregulation of BMP signaling in S. mediterranea, possibly by a patterning organizer on the midline. Moreover, the metalloproteinase Tolloid is possibly necessary for long range BMP signal distribution from the midline to the body edge and influences muscle development.
In summary, my work provides an overview of the TGFβ signaling components of S. mediterranea and their phylogenetic relation to components of other metazoans, new assays to assess BMP signaling activity in S. mediterranea, and insights into the mechanisms of the mediolateral distribution of BMP signaling activity. | de |