Mechanical Ablation of Larval Zebrafish Spinal Cord.

Unlike mammals, adult and larval zebrafish exhibit robust regeneration following traumatic spinal cord injury. This remarkable regenerative capacity, combined with exquisite imaging capabilities and an abundance of powerful genetic techniques, has established the zebrafish as an important vertebrate model for the study of neural regeneration. Here, we describe a protocol for the complete mechanical ablation of the larval zebrafish spinal cord. With practice, this protocol can be used to reproducibly injure upward of 100 samples per hour, facilitating the high-throughput screening of factors involved in spinal cord regeneration and repair.

RNA-induced inflammation and migration of precursor neurons initiates neuronal circuit regeneration in zebrafish.

Tissue regeneration and functional restoration after injury are considered as stem- and progenitor-cell-driven processes. In the central nervous system, stem cell-driven repair is slow and problematic because function needs to be restored rapidly for vital tasks. In highly regenerative vertebrates, such as zebrafish, functional recovery is rapid, suggesting a capability for fast cell production and functional integration. Surprisingly, we found that migration of dormant "precursor neurons" to the injury site pioneers functional circuit regeneration after spinal cord injury and controls the subsequent stem-cell-driven repair response. Thus, the precursor neurons make do before the stem cells make new. Furthermore, RNA released from the dying or damaged cells at the site of injury acts as a signal to attract precursor neurons for repair. Taken together, our data demonstrate an unanticipated role of neuronal migration and RNA as drivers of neural repair.

Midbrain tectal stem cells display diverse regenerative capacities in zebrafish.

How diverse adult stem and progenitor populations regenerate tissue following damage to the brain is poorly understood. In highly regenerative vertebrates, such as zebrafish, radial-glia (RG) and neuro-epithelial-like (NE) stem/progenitor cells contribute to neuronal repair after injury. However, not all RG act as neural stem/progenitor cells during homeostasis in the zebrafish brain, questioning the role of quiescent RG (qRG) post-injury. To understand the function of qRG during regeneration, we performed a stab lesion in the adult midbrain tectum to target a population of homeostatic qRG, and investigated their proliferative behaviour, differentiation potential, and Wnt/ß-catenin signalling. EdU-labelling showed a small number of proliferating qRG after injury (pRG) but that progeny are restricted to RG. However, injury promoted proliferation of NE progenitors in the internal tectal marginal zone (TMZi) resulting in amplified regenerative neurogenesis. Increased Wnt/ß-catenin signalling was detected in TMZi after injury whereas homeostatic levels of Wnt/ß-catenin signalling persisted in qRG/pRG. Attenuation of Wnt signalling suggested that the proliferative response post-injury was Wnt/ß-catenin-independent. Our results demonstrate that qRG in the tectum have restricted capability in neuronal repair, highlighting that RG have diverse functions in the zebrafish brain. Furthermore, these findings suggest that endogenous stem cell compartments compensate lost tissue by amplifying homeostatic growth.

A Modular Millifluidic Homeostatic Imaging Plate for Imaging of Larval Zebrafish.

Zebrafish larvae are suitable in vivo models for toxicological and pharmacological screens due to their transparency, small size, ex utero development, and genetic and physiological similarity to humans. Using modern imaging techniques, cells and tissues can be dynamically visualized over several days in multiple zebrafish larvae. However, precise specimen immobilization and maintenance of homeostatic conditions remain a challenge for longitudinal studies. A highly customizable mounting configuration with inbuilt means of controlling temperature and media flow would therefore be a valuable tool to facilitate long-term imaging of a large number of specimens. Using three-dimensional printing, we have developed a millifluidic, modular homeostatic imaging plate (HIP), which consists of a customizable sample insert and a temperature-controlled incubation chamber that is continuously perfused, providing an ideal environment for long-term experiments where homeostatic conditions are desired. The HIP is cheap to produce, has a standard microtiter well plate format, and can be fitted to most microscopes. We used the device to image dynamic regeneration of spinal cord neurons. The flexibility and adaptability of the HIP facilitate long-term in vivo imaging of many samples, and can be easily adapted to suit a broad range of specimens.