Ana Catarina Certal

Divergent Protein Kinase Domain 2B (DIPK2B) is a conserved kinase-like protein of the FAM69 family that has been associated with autism spectrum disorder and X-linked intellectual disability. However, its biological function has not yet been described. We previously identified dipk2b as a marker of hemangioblasts and hemogenic endothelial populations in the chick embryo, suggesting a potential role in hematopoietic development. Here, we characterized the expression pattern of dipk2b and the effects of its loss of function during zebrafish development. In line with our observations in the chick embryo, zebrafish dipk2b is expressed in the hematopoietic regions of the developing embryo, including the lateral plate mesoderm, dorsal aorta, and caudal hematopoietic tissue. Using two independent CRISPR/Cas9 loss-of-function alleles, we show that dipk2b homozygous mutant embryos have significantly fewer primitive macrophages than wild-type controls. In addition, dipk2b mutants exhibit a persistent decrease in microglial cell numbers at larval stages, consistent with the reduced macrophage population that gives rise to microglia. Together, these findings uncover a role for Dipk2b in promoting the development of primitive macrophages and microglia from early hematopoietic progenitors. Given the emerging role of microglial dysfunction in neurodevelopmental disorders, our results suggest that DIPK2B deficiency may contribute to autism spectrum disorder and X-linked intellectual disability by impairing microglia development.

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.

The zebrafish is a widely used animal model in biomedical research. Despite this, pathogens continue to be common in laboratory zebrafish. It is important to determine and describe the pathophysiology of cryptic infections on zebrafish to elucidate the impacts on experimental endpoints. Body condition is a basic measurement used experimentally and in health monitoring of animals. We exposed three wild-type zebrafish strains: AB, WIK, and 5D to Pseudoloma neurophilia . After 8 weeks postexposure, we individually imaged and processed fish for histology. Morphometric analysis was performed on images and an index of body condition was calculated based on the ratio of length/width from the dorsal aspect. Histological sections from each fish were examined to establish sex, severity of infection, and tissue distribution. We observed a significant decrease in body condition in female fish that was not observed in males. In addition, we observed a negative correlation between the total gonadal area of P. neurophilia exposed females and unexposed controls. These results illustrate the sex-specific impacts of a common chronic pathogen on zebrafish health and a commonly used experimental endpoint, further supporting the establishment of rigorous health monitoring programs in laboratory zebrafish colonies that include screening for chronic infectious diseases.

Zebrafish (Danio rerio) is one of the top model organisms used in biomedical research. Therefore, it is fundamental that zebrafish facilities continuously improve husbandry methods to provide fish with the best physiological and welfare conditions that suit each experimental purpose. Nutrition is a husbandry aspect that needs further optimization, as it greatly affects growth, reproduction, health and behaviour. Here, we have compared the impact of different feeding regimens on zebrafish survival, growth and reproductive performance. Mutant and wild-type zebrafish were raised using several combinations of two cold-extruded processed feeds—Skretting^®GemmaMicro and Sparos^®Zebrafeed—and one live feed (rotifers). Zebrafeed^® outperformed GemmaMicro^® in terms of survival rate, and embryo viability was also higher when the spawners were fed with Zebrafeed^® either from larval stage or upon sexual maturation. In contrast, GemmaMicro^® favoured growth, both in size and weight. The use of rotifers until 60 days post-fertilization improved survival of fish co-fed with GemmaMicro^®, while delaying their growth. Zebrafeed^® performance was not affected by co-feeding rotifers. Overall, we showed that different nutritional formulas affect physiological parameters, allowing for the establishment of feeding protocols adapted to the objectives of each facility. At the same time, we validated Skretting^®GemmaMicro and Sparos^®Zebrafeed as two commercially available feeds that are well suited for zebrafish nutrition in a laboratory environment.

A husbandry workshop on July 3, 2017, at the 10th European Zebrafish Meeting in Budapest, Hungary (July 3–July 7, 2017), focused on the standardization, optimization, and streamlining of fish facility procedures. Standardization can be achieved for example by developing novel software and hardware tools, such as a fish facility database for husbandry and environmental facility management (Zebrabase, Oltova), or a hand-held, air-pressurized fish feeder for consistent food distribution (Blowfish, Argenton). Streamlining is achieved when work hours are reduced, as with the standardized fish feeder, or by limiting the number and types of fish diets and observing the effect on animal welfare and performance (Barton). Testing the characteristics of new fish diets and observing whether they produce better experimental outcomes (Certal) optimizes diets and improves fish productivity. Collectively, the workshop presentations emphasized how consistency and harmonization of husbandry procedures within and across aquatic facilities yield reproducible scientific outcomes.

Zebrafish are able to regenerate the spinal cord and recover motor and sensory functions upon severe injury, through the activation of cells located at the ependymal canal. Here, we show that cells surrounding the ependymal canal in the adult zebrafish spinal cord express Foxj1a. We demonstrate that ependymal cells express Foxj1a from their birth in the embryonic neural tube and that Foxj1a activity is required for the final positioning of the ependymal canal. We also show that in response to spinal cord injury, Foxj1a ependymal cells actively proliferate and contribute to the restoration of the spinal cord structure. Finally, this study reveals that Foxj1a expression in the injured spinal cord is regulated by regulatory elements activated during regeneration. These data establish Foxj1a as a pan-ependymal marker in development, homeostasis and regeneration and may help identify the signals that enable this progenitor population to replace lost cells after spinal cord injury.

Zebrafish is already one of the most used model organisms in biomedical sciences and other research fields. It is therefore becoming increasingly important to assure that zebrafish maintained in laboratory aquaculture conditions are raised and housed under rigorous standards that promote health and welfare to guarantee the required quality and reproducibility of research data. Specifying the programs each facility is adopting would be the first step to achieve this by allowing other facilities to compare, improve, and discuss their protocols and fish performance. We provide in this article a detailed description of an integrated facility health management program, with protocols and readouts, fully designed and aimed at maximizing fish health, welfare, and performance for research.

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H⁺ ef`flux. The onset and intensity of both V-ATPase expression and H⁺ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H⁺ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.