Calorie Restriction Increases the Number of Competing Stem Cells and Decreases Mutation Retention in the Intestine.

Calorie restriction (CR) extends lifespan through several intracellular mechanisms, including increased DNA repair, leading to fewer DNA mutations that cause age-related pathologies. However, it remains unknown how CR acts on mutation retention at the tissue level. Here, we use Cre-mediated DNA recombination of the confetti reporter as proxy for neutral mutations and follow these mutations by intravital microscopy to identify how CR affects retention of mutations in the intestine. We find that CR leads to increased numbers of functional Lgr5+ stem cells that compete for niche occupancy, resulting in slower but stronger stem cell competition. Consequently, stem cells carrying neutral or Apc mutations encounter more wild-type competitors, thus increasing the chance that they get displaced from the niche to get lost over time. Thus, our data show that CR not only affects the acquisition of mutations but also leads to lower retention of mutations in the intestine.

Shp2-Mitogen-Activated Protein Kinase Signaling Drives Proliferation during Zebrafish Embryo Caudal Fin Fold Regeneration.

Regeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a-/- ptpn11b-/- zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb, indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a-/- ptpn11b-/- zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a-mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration.

Differential oxidation of protein-tyrosine phosphatases during zebrafish caudal fin regeneration.

Zebrafish have the capacity to regenerate lost tissues and organs. Amputation of the caudal fin results in a rapid, transient increase in H2O2 levels emanating from the wound margin, which is essential for regeneration, because quenching of reactive oxygen species blocks regeneration. Protein-tyrosine phosphatases (PTPs) have a central role in cell signalling and are susceptible to oxidation, which results in transient inactivation of their catalytic activity. We hypothesized that PTPs may become oxidized in response to amputation of the caudal fin. Using the oxidized PTP-specific (ox-PTP) antibody and liquid chromatography-mass spectrometry, we identified 33 PTPs in adult zebrafish fin clips of the total of 44 PTPs that can theoretically be detected based on sequence conservation. Of these 33 PTPs, 8 were significantly more oxidized 40 min after caudal fin amputation. Surprisingly, Shp2, one of the PTPs that were oxidized in response to caudal fin amputation, was required for caudal fin regeneration. In contrast, Rptpα, which was not oxidized upon amputation, was dispensable for caudal fin regeneration. Our results demonstrate that PTPs are differentially oxidized in response to caudal fin amputation and that there is a differential requirement for PTPs in regeneration.

Impaired caudal fin-fold regeneration in zebrafish deficient for the tumor suppressor Pten.

Zebrafish are able to completely regrow their caudal fin-folds after amputation. Following injury, wound healing occurs, followed by the formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. Here we show that, surprisingly, the phosphatase and tumor suppressor Pten, an antagonist of phosphoinositide-3-kinase (PI3K) signaling, is required for zebrafish caudal fin-fold regeneration. We found that homozygous knock-out mutant (ptena-/-ptenb-/- ) zebrafish embryos, lacking functional Pten, did not regenerate their caudal fin-folds. AKT phosphorylation was enhanced, which is consistent with the function of Pten. Reexpression of Pten, but not catalytically inactive mutant Pten-C124S, rescued regeneration, as did pharmacological inhibition of PI3K. Blastema formation, determined by in situ hybridization for the blastema marker junbb, appeared normal upon caudal fin-fold amputation of ptena-/-ptenb-/- zebrafish embryos. Whole-mount immunohistochemistry using specific markers indicated that proliferation was arrested in embryos lacking functional Pten, and that apoptosis was enhanced. Together, these results suggest a critical role for Pten by limiting PI3K signaling during the regenerative outgrowth phase of zebrafish caudal fin-fold regeneration.

Studying Protein-Tyrosine Phosphatases in Zebrafish.

Protein-tyrosine phosphatases (PTPs) are a large family of signal transduction regulators that have an essential role in normal development and physiology. Aberrant activation or inactivation of PTPs is at the basis of many human diseases. The zebrafish, Danio rerio, is being used extensively to model major aspects of development and disease as well as the mechanism of regeneration of limbs and vital organs, and most classical PTPs have been identified in zebrafish. Zebrafish is an excellent model system for biomedical research because the genome is sequenced, zebrafish produce a large number of offspring, the eggs develop outside the mother and are transparent, facilitating intravital imaging, and transgenesis and (site-directed) mutagenesis are feasible. Together, these traits make zebrafish amenable for the analysis of gene and protein function. In this chapter we cover three manipulations of zebrafish embryos that we have used to study the effects of PTPs in development, regeneration, and biochemistry. Microinjection at the one-cell stage is at the basis of many zebrafish experiments and is described first. This is followed by a description for measuring regeneration of the embryonic caudal fin, a powerful and robust physiological assay. Finally, the considerable but manageable troubleshooting of several complications associated with preparing zebrafish embryos for immunoblotting is explained. Overall, this chapter provides detailed protocols for manipulating zebrafish embryo samples with a compilation of tips collected through extensive experience from the zebrafish research community.