Different ways of evolving tool-using brains in teleosts and amniotes.

In mammals and birds, tool-using species are characterized by their relatively large telencephalon containing a higher proportion of total brain neurons compared to other species. Some teleost species in the wrasse family have evolved tool-using abilities. In this study, we compared the brains of tool-using wrasses with various teleost species. We show that in the tool-using wrasses, the telencephalon and the ventral part of the forebrain and midbrain are significantly enlarged compared to other teleost species but do not contain a larger proportion of cells. Instead, this size difference is due to large fiber tracts connecting the dorsal part of the telencephalon (pallium) to the inferior lobe, a ventral mesencephalic structure absent in amniotes. The high degree of connectivity between these structures in tool-using wrasses suggests that the inferior lobe could contribute to higher-order cognitive functions. We conclude that the evolution of non-telencephalic structures might have been key in the emergence of these cognitive functions in teleosts.

ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 days post-fertilization zebrafish following simultaneous visualization with identical orientations.

In recent years, the zebrafish has become a well-established laboratory model. We describe here the ZeBraInspector (ZBI) platform for high-content 3D imaging (HCI) of 5 days post-fertilization zebrafish eleuthero-embryos (EEs). This platform includes a mounting method based on 3D-printed stamps to create a grid of wells in an agarose cast, facilitating batch acquisitions with a fast-confocal laser scanning microscope. We describe reference labeling in cleared fish with a fluorescent lipophilic dye. Based on this labeling, the ZBI software registers. EE 3D images, making it possible to visualize numerous identically oriented EEs on a single screen, and to compare their morphologies and any fluorescent patterns at a glance. High-resolution 2D snapshots can be extracted. ZBI software is therefore useful for diverse high-content analyses (HCAs). Following automated segmentation of the lipophilic dye signal, the ZBI software performs volumetric analyses on whole EEs and their nervous system white matter. Through two examples, we illustrate the power of these analyses for obtaining statistically significant results from a small number of samples: the characterization of a phenotype associated with a neurodevelopmental mutation, and of the defects caused by treatments with a toxic anti-cancer compound.

Fibrillarin is essential for S-phase progression and neuronal differentiation in zebrafish dorsal midbrain and retina.

Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Effect of Nutrient Availability on Progenitor Cells in Zebrafish (Danio Rerio).

In zebrafish brains, populations of continuously proliferating cells are present during an entire life span. Under normal conditions, stem cells give rise to rapidly proliferating progenitors that quickly exit the cell cycle and differentiate. Hence fish are favorable models to study what regulates postembryonic neurogenesis. The aim of this study was to determine if optic tectum (OT) cell proliferation is halted during nutritional deprivation (ND) and whether or not it can be restored with refeeding. We examined the effect of ND on the proliferation of Neuroepithelial/Ependymal Progenitor cell (NeEPC) and transitory-amplifying progenitors (TAPs). Following ND, no PCNA immunostaining was found in OT of starved fish, while positive cell populations of PCNA positive progenitors are found at its periphery in control fish. This indicated that active proliferation stopped. To label retaining progenitor cells, BrdU was applied and a chase-period was accompanied by ND. Positive NeEPCs were detected in the external tectum marginal zone of starved fish suggesting that these progenitors are relatively immune to ND. Moreover in the internal tectum marginal zone labeled retaining cells were observed leaving the possibility that some arrested TAPs were present to readily restart proliferation when nutrition was returned. Our results suggest that neurogenesis was maintained during ND and that a normal proliferative situation was recovered after refeeding. We point to the mTOR pathway as a necessary pathway in progenitors to regulate their mitosis activity. Thus, this study highlights mechanisms involved in neural stem and progenitor cell homeostatic maintenance in an adverse situation. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 26-38, 2017.

zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain.

In studies of brain function, it is essential to understand the underlying neuro-architecture. Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire of the animals used. We describe here a modified version of the passive clearing technique (PACT) ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ; Treweek et al., 2015) , which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish (Danio rerio) ( Frétaud et al., 2017 ; Mayrhofer et al., 2017 ; Xavier et al., 2017 ), but has also been successfully tested on other fish, such as medaka (Oryzias latipes) ( Dambroise et al., 2017 ), Mexican cave fish (Astyanax mexicaus) and African zebra mbuna (Metriaclima zebra), and on other aquatic species, such as Xenopus spp. (Xenopus laevis, Xenopus tropicalis) ( Fini et al., 2017 ) . This protocol, based on the CLARITY method developed and modified by Deisseroth's laboratory and others ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ), was adapted for use in aquatic species, including zebrafish in particular (zPACT). This protocol is designed to render zebrafish specimens optically transparent while preserving the overall architecture of the tissue, through crosslinking in a polyacrylamide/formaldehyde mesh. Most of the lipids present in the specimen are then removed by SDS treatment, to homogenize the refractive index of the specimen by eliminating light scattering at the water/lipid interface, which causes opacity. The final clearing step, consists of the incubation of the specimen in a fructose-based mounting medium (derived from SeeDB) ( Ke et al., 2013 ) , with a refractive index matching that of the objective lens of the microscope. The combination of this technique with the use of genetically modified zebrafish in which green fluorescent protein (GFP) is expressed in specific cell populations provides opportunities to describe anatomical details not visible with other techniques.