Differential functions of Pyr and Ths

As described above Pyr and Ths support mesoderm formation by signalling through the FGFR Heartless. While Pyr and Ths have very similar function not all of these functions rely on Pyr and Ths in equal measure (Kadam et al. 2009; Klingseisen et al. 2009).

98 While this is depicted in their differential expression pattern, the differential function of these two signalling factors seems not to rely on the expression pattern alone. More likely Pyr and Ths, although most likely derived from an ancient gene duplication (Gryzik and Müller 2004; Stathopoulos 2004), have evolved to serve different functions.

Pyr is more essential for the migration of the mesoderm and differentiation of pericardial cells and the dorsal muscles, whereas these tissues are hardly affected in ths mutant embryos (Klingseisen et al. 2009; Tulin and Stathopoulos 2010). In contrary, mesoderm spreading relies on both Pyr and Ths (Kadam et al. 2009; Klingseisen et al.

2009). To understand how Pyr and Ths exactly play these divergent roles, even though they share the same receptor and have a mostly overlapping expression pattern, it is tempting to speculate that there are additional mechanisms that regulate signalling of the two FGF ligands. Processing of Pyr and Ths by different proteases would represent a possible regulatory mechanism to achieve differential signalling of the two factors through the same receptor. The identification of the cleaving proteases would represent valuable insight into to the mechanism through which differential signalling of Pyr and Ths is achieved.

The experimental data provided by this study is encouraging the hypothesis that Pyr and Ths are cleaved by different proteases. Mapping of the cutting sites in Pyr and Ths shows that Pyr and Ths are cleaved in different positions of the protein. Pyr is cleaved in the C-terminal half of the protein, resulting in an N-terminal fragment of approximately 60kDa. Ths on the other hand is cleaved much further N-terminal, straight after the FGF domain, resulting in a small N-terminal fragment of around 20kDa. In silico analysis of the cutting sites and their surrounding area showed no similarities within the amino acid sequence that would reveal the nature of the protease. However analysis of the amino acid sequence and subsequent experimental verification revealed that charged residues at the Pyr cutting site are crucial for its cleavage.

Additionally secretion is also prevented by the removal of the charged amino acids at the presumptive cleavage site of Pyr, suggesting a connection between proteolytic processing and secretion of Pyr. Cleavage of Pyr might be a requirement for secretion of Pyr. Another possible connection would be the processing during secretion, which would reveal the localization of the protease in question.

Furthermore, analysis of a construct with a deletion of the prospective cleavage site showed that deletion of the Pyr cutting site did not prevent cleavage in cell culture and even resulted in the same smaller N-terminal fragment that was observed in the unmodified Pyr. A possible explanation for this behaviour of the Pyr deletion construct would be that the responsible protease does not cleave at a specific amino acid

99 sequence but rather at a certain position in the folded Pyr protein. Concluding from these findings, the protease cleaving Pyr is most likely depending on charged residues.

Subsequent initiation of cleavage at a relative position between the amino acids 293 and 310 is presumably independent of a defined cleavage motif.

Additionally the Western blot data is suggesting that Pyr and Ths are cleaved in different subcellular locations. The Pyr fragment was detectable both in the supernatant and within the cell lysate suggesting intracellular cleaving. Ths however is found cleaved only within the supernatant indicating extracellular cleavage or alternatively cleavage during a late stage of its secretion.

Taken together the data is indicating that Pyr and Ths are cleaved by different proteases, which might comprise different regulatory mechanisms to achieve the temporal and spatial divergent signalling of the two FGF8-homologues Pyr and Ths.

This theory is further strengthened by the study of truncated Pyr and Ths constructs, which has shown that cleavage of Pyr and Ths is most likely associated with increased activity of the proteins (Tulin and Stathopoulos 2010). Therefore, processing seems to be essential for the activation of Ths and Pyr. Additionally the requirement of two individual proteases could explain the temporal and spatial divergence of Pyr and Ths signalling during embryonic development simply by distinct expression patterns of the two proteases.

To confirm this hypothesis the proteases cleaving Pyr and Ths have to be identified.

One attempt that will be followed in future is based on the knowledge that cutting takes place both in S2- and Kc-cell lines and in early embryos. Comparison of the proteases that are expressed based on Chip-data in all mentioned cases led to al list of candidate proteases. This list of candidates was shortened by excluding components of the proteasom and characterized proteases involved in apoptosis and autophagy.

Furthermore, some of the remaining proteases like the Rhomboid family could be excluded based on the cleavage preference within membranes thereby only cleaving transmembrane domains (Lee et al. 2001; Urban et al. 2001). Additionally literature research for proteases that promotes cleavage extracellular or within the secretion pathway can possibly narrow the group of candidates. Preliminary testing of some of the candidates using RNAi driven knock down using maternal Gal4 and the novel TRIP lines (Dietzl et al., 2007; Ni et al., 2008) is ongoing. An alternative way would be the knock down of the candidate proteases in cell culture using Kc-cells that take up RNA molecules without transfection.

100 The remaining list of candidates will be ordered as mutant or RNAi lines if available and subsequently tested for their influence on Pyr and Ths in early embryos. However, since Pyr has shown to have a greater influence on embryonic development and seems to have the ability to partially substitute for Ths (Kadam et al. 2009; Klingseisen et al.

2009) this test might be specific for the protease cleaving Pyr.

Following the identification of the protease or proteases cleaving Pyr and Ths study of lines mutant for the protease and rescue experiments with truncated constructs can be used to confirm the necessity of the protease for the mesoderm formation and the activity of the truncated proteins.

101