A screen for regeneration-associated silencer regulatory elements in zebrafish.

Regeneration involves gene expression changes explained in part by context-dependent recruitment of transcriptional activators to distal enhancers. Silencers that engage repressive transcriptional complexes are less studied than enhancers and more technically challenging to validate, but they potentially have profound biological importance for regeneration. Here, we identified candidate silencers through a screening process that examined the ability of DNA sequences to limit injury-induced gene expression in larval zebrafish after fin amputation. A short sequence (s1) on chromosome 5 near several genes that reduce expression during adult fin regeneration could suppress promoter activity in stable transgenic lines and diminish nearby gene expression in knockin lines. High-resolution analysis of chromatin organization identified physical associations of s1 with gene promoters occurring preferentially during fin regeneration, and genomic deletion of s1 elevated the expression of these genes after fin amputation. Our study provides methods to identify "tissue regeneration silencer elements" (TRSEs) with the potential to reduce unnecessary or deleterious gene expression during regeneration.

Heterogeneous fates and dynamic rearrangement of regenerative epidermis-derived cells during zebrafish fin regeneration.

The regenerative epidermis (RE) is a specialized tissue that plays an essential role in tissue regeneration. However, the fate of the RE during and after regeneration is unknown. In this study, we performed Cre-loxP-mediated cell fate tracking and revealed the fates of a major population of the RE cells that express fibronectin 1b (fn1b) during zebrafish fin regeneration. Our study showed that these RE cells are mainly recruited from the inter-ray epidermis, and that they follow heterogeneous cell fates. Early recruited cells contribute to initial wound healing and soon disappear by apoptosis, while the later recruited cells contribute to the regenerated epidermis. Intriguingly, many of these cells are also expelled from the regenerated tissue by a dynamic caudal movement of the epidermis over time, and in turn the loss of epidermal cells is replenished by a global self-replication of basal and suprabasal cells in fin. De-differentiation of non-basal epidermal cells into the basal epidermal cells did not occur during regeneration. Overall, our study reveals the heterogeneous fates of RE cells and a dynamic rearrangement of the epidermis during and after regeneration.

Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin.

Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies ( Shibata et al., 2016 ; Yoshinari et al., 2012 ) is a useful approach for tracking and manipulating the blastema cells during fish fin regeneration.

Colored medaka and zebrafish: transgenics with ubiquitous and strong transgene expression driven by the medaka ß-actin promoter.

Conditional cell labeling, cell tracing, and genetic manipulation approaches are becoming increasingly important in developmental and regenerative biology. Such approaches in zebrafish research are hampered by the lack of an ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the medaka fish (Oryzias latipes) ß-actin (Olactb) promoter, which drives constitutive transgene expression during all developmental stages, and the analysis of adult organs except blood cell types. Taking advantage of the compact medaka promoter, we succeeded in generating a zebrafish transgenic (Tg) line with unprecedentedly strong and widespread transgene expression from embryonic to adult stages. Moreover, the Tg carries a pair of loxP sites, which enables the reporter fluorophore to switch from DsRed2 to enhanced green fluorescent protein (EGFP). We induced Cre/loxP recombination with Tg(hsp70l: mCherry-t2a-Cre(ERt2) ) in the double Tg embryo and generated a Tg line that constitutively expresses EGFP. We further demonstrate the powerful application of Olactb-driven Tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of regenerating fin. Thus, the use of promoter elements from medaka is an alternative approach to generate Tgs with stronger and even novel expression patterns in zebrafish. The Olactb promoter and the Tg lines presented here represent an important advancement for the broader use of Cre/loxP-based Tg applications in zebrafish.

Self-sensing piezoresistive cantilever and its magnetic force microscopy applications.

A newly developed Si self-sensing piezoresistive cantilever is presented. Si piezoresistive cantilevers for scanning microscopy are fabricated by Si micro-machining technique. The sensitivity of the piezoresistive cantilever is comparable to the current laser detecting system. Topographic images are successfully obtained with the piezoresistive cantilever and some comparisons are made with the laser detecting system. Furthermore, the magnetic film (Co-Cr-Pt) is coated on the tip of the piezoresistive cantilever for magnetic force microscopy (MFM) application. The magnetic images are successfully obtained with the self-sensing MFM piezoresistive cantilever. The self-sensing piezoresistive cantilevers have been successfully applied in scanning probe microscopy and MFM.