The Keap1–Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Under homeostatic conditions, Keap1 interacts with Nrf2 and leads to its rapid proteasomal degradation, but when cells are exposed to oxidative stress/electrophiles, Keap1 senses them, resulting in an improper Keap1–Nrf2 interaction and Nrf2 stabilization. Keap1 is therefore considered both an “inhibitor” of and “stress sensor” for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), while there is only one in mammals, birds and reptiles. A phylogenetic analysis suggested that mammalian Keap1 is an ortholog of fish Keap1b, not Keap1a. In this study, we investigated the differences and similarities between Keap1a and Keap1b using zebrafish genetics. We generated zebrafish knockout lines of keap1a and keap1b . Homozygous mutants of both knockout lines were viable and fertile. In both mutant larvae, the basal expression of Nrf2 target genes and antioxidant activity were up-regulated in an Nrf2-dependent manner, suggesting that both Keap1a and Keap1b can function as Nrf2 inhibitors. We also analyzed the effects of the Nrf2 activator sulforaphane in these mutants and found that keap1a- , but not keap1b- , knockout larvae responded to sulforaphane, suggesting that the stress/chemical-sensing abilities of the two Keap1s are different.

. Significance: The scattering and polarization characteristics of various organs of in vivo wildtype zebrafish in three development stages were investigated using a non-destructive and label-free approach. The presented results showed a promising first step for the usability of Jones-matrix optical coherence tomography (JM-OCT) in zebrafish-based research. Aim: We aim to visualize and quantify the scatter and polarization signatures of various zebrafish organs for larvae, juvenile, and young adult animals in vivo in a non-invasive and label-free way. Approach: A custom-built polarization-sensitive JM-OCT setup in combination with a motorized translation stage was utilized to investigate live zebrafish. Depth-resolved scattering (intensity and attenuation coefficient) and polarization (birefringence and degree of polarization uniformity) properties were analyzed. OCT angiography (OCT-A) was utilized to investigate the vasculature label-free and non-destructively. Results: The scatter and polarization signatures of the zebrafish organs such as the eye, gills, and muscles were investigated. The attenuation coefficient and birefringence changes between 1- and 2-month-old animals were evaluated in selected organs. OCT-A revealed the vasculature of in vivo larvae and juvenile zebrafish in a label-free manner. Conclusions: JM-OCT offers a rapid, label-free, non-invasive, tissue specific, and three-dimensional imaging tool to investigate in vivo processes in zebrafish in various development stages.

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Breast cancer is a leading cause of death in female patients worldwide. Further research is needed to get a deeper insight into the mechanisms involved in the development of this devastating disease and to find new therapy strategies. The zebrafish is an established animal model, especially in the field of oncology, which has shown to be a promising candidate for pre-clinical research and precision-based medicine. To investigate cancer growth in vivo in zebrafish, one approach is to explore xenograft tumor models. In this article, we present the investigation of a juvenile xenograft zebrafish model using a Jones matrix optical coherence tomography (JM-OCT) prototype. Immunosuppressed wild-type fish at 1-month post-fertilization were injected with human breast cancer cells and control animals with phosphate buffered saline in the tail musculature. In a longitudinal study, the scatter, polarization, and vasculature changes over time were investigated and quantified in control versus tumor injected animals. A significant decrease in birefringence and an increase in scattering signal was detected in tumor injected zebrafish in comparison to the control once. This work shows the potential of JM-OCT as a non-invasive, label-free, three-dimensional, high-resolution, and tissue-specific imaging tool in pre-clinical cancer research based on juvenile zebrafish models.