Heterologous protein expression of Hydra FGFs and siRNA approach to investigate FGF function
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Philipps-Universität Marburg
Abstract
The fibroblast growth factor receptor (FGFR) signaling pathway is essential during eumetazoan development. It regulates key aspects of cellular physiology such as proliferation, differentiation, and migration during patterning and morphogenesis. Hydra, a simple freshwater polyp at the base of eumetazoan evolution, provides an ideal model to investigate FGFR signaling on the basis of its astonishing regenerative capability and constantly active patterning processes in the adult animal. Two FGFRs (FGFRa and FGFRb) have been identified in this polyp, as well as one truncated FGFR. In addition, five potential ligands (Hv-FGF-a, Hv-FGF-b, Hv-FGF-c, Hv-FGF-e, and Hv-FGF-f) are known.
FGFRa has an essential function during asexual reproduction of Hydra for detachment of the vegetative bud and according to unpublished pilot experiments, it might have additional functions in cell migration and differentiation. Migration of i-cells is prevented upon local or systemic inhibition of FGFR signaling and siRNA mediated knockdown of hv-fgf-c reduces the number of neurons and increases the number of I-cells.
The function of the five potential ligands is currently unknown, but they show each a specific transription pattern during in situ hybridization. On the basis of their differential transcription and siRNA knockdown experiments, Hydra FGFR signaling might selectively guide already committed interstitial cells and/or provide differentiation signals. To confirm this model, it is important to gain more conclusive evidence than manipulations on the pharmacological and RNA levels can provide. To gain further insights into the functions of FGFR signaling in Hydra, the aim of this project was the expression and purification of recombinant Hydra FGF proteins to investigate their effects on cell migration and differentiation in functional bioassays in vivo.
This study reports the difficulty of expressing soluble Hydra FGFs in prokaryotic cells, whereas denatured protein was purified from inclusion bodies. However, refolding the denatured proteins to their native, functional conformation did not produce sufficient yields. Expression of Hydra FGFs in mammalian HEK293T cells is much more promising since they have the ability to add eukaryotic post-translational modifications that might be integral to protein function.
Further hints as to the function of Hv-FGF-c were investigated through siRNA mediated knockdown, suggesting a possible involvement of Hv-FGF-c for proper spacing of battery cells during cell migration in the tentacles. The knockdown resulted in tentacles with extended battery nodules containing an irregular pattern of nematocytes.
In addition, ten novel, predicted Hydra FGFs were identified and first insights into potential functions given through analysis of their transcription. Each of the ten new Hv-FGFs shows a specific transcription pattern. However, more data is needed to complete the picture.
FGFR signaling in Hydra remains an exciting field. This study provides the basis for further investigations into the function of the ligands and towards functional bioassays with recombinant proteins.
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This item has been published with the following license: In Copyright