Protein tyrosine phosphatases in zebrafish development and regeneration

Abstract

Protein tyrosine phosphatases (PTPs) have a central role in cell signaling, by dephosphorylating other proteins. PTPs are highly susceptible to oxidation, due to the low pKa of their active sites. Their reactive cysteine gets oxidized, which leads to the temporary inactivation of the PTP. It has been shown in many different species that a burst of Reactive Oxygen Species (ROS) is produced upon injury. This burst of ROS plays an important role in wound healing and regeneration. We hypothesized that the ROS could start a signaling cascade by oxidizing PTPs. Previously, eight PTPs were identified that were specifically oxidized in response to amputation of the tail fin of adult zebrafish. One of these PTPs, Shp2a, was shown to be essential for regeneration. In chapter 2 we describe how we generated zebrafish knockouts of 6 PTPs that were shown previously to be oxidized upon amputation of the caudal fin and their paralogs. We show that these PTPs were not essential for regeneration. In addition, we did not recapitulate all the phenotypes we expected. We hypothesize that this is due to genetic compensation. In chapter 3 we describe our investigation of zebrafish with a knockout of ptpn4a. We show that the majority of the mutant zebrafish died at the juvenile stage and suffered from epileptic seizures. This corresponds to human patients who suffer from intellectual disability and epileptic seizures due to nonsense mutations in PTPN4. We show that a minority of the mutant zebrafish compensated for the loss of Ptpn4a and survived, but also that a separate knockout line lost its compensation ability over time and showed enhanced lethality at the juvenile stage. In chapter 4 we describe our investigation of zebrafish with a knockout of ptpn6. We show that these mutant zebrafish displayed fully penetrant mortality at the larval stage and died from lethal hyperinflammation. This corresponds to the phenotype of the motheaten mice lacking SHP1 and human patients suffering from neutrophilic dermatoses and emphysema. We show that loss of Shp1 led to a reduced number of emerging HSPCs, increased HSPC proliferation, an increased number of macrophages and a decreased number of neutrophils. In addition, we show that loss of Shp1 affected the behavior of neutrophils and macrophages and led to reduced directional migration upon wounding. In chapter 5 we describe how we investigated the role of oxidation of Shp2a during regeneration, by creating a fusion protein consisting of Shp2a linked with Catalase. Due to the close vicinity, the Catalase protected Shp2a from oxidation. We show that by inhibiting oxidation of Shp2a, we inhibited regeneration. This indicates that transient oxidation of Shp2a is an essential step in the signaling cascade that results in regeneration. This research puts an end to the apparent paradox that Shp2a is oxidized during regeneration, but that catalytic activity of Shp2a is essential for regeneration to proceed. We hypothesize that transient oxidation of Shp2a leads to a reduction of a negative feedback loop, resulting in increased signaling activity once Shp2a is reduced again.