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.

Mesencephalic origin of the inferior lobe in zebrafish.

BACKGROUND: Although the overall brain organization is shared in vertebrates, there are significant differences within subregions among different groups, notably between Sarcopterygii (lobe-finned fish) and Actinopterygii (ray-finned fish). Recent comparative studies focusing on the ventricular morphology have revealed a large diversity of the hypothalamus. Here, we study the development of the inferior lobe (IL), a prominent structure forming a bump on the ventral surface of the teleost brain. Based on its position, IL has been thought to be part of the hypothalamus (therefore forebrain). RESULTS: Taking advantage of genetic lineage-tracing techniques in zebrafish, we reveal that cells originating from her5-expressing progenitors in the midbrain-hindbrain boundary (MHB) participate in the formation of a large part of the IL. 3D visualization demonstrated how IL develops in relation to the ventricular system. We found that IL is constituted by two developmental components: the periventricular zone of hypothalamic origin and the external zone of mesencephalic origin. The mesencephalic external zone grows progressively until adulthood by adding new cells throughout development. CONCLUSION: Our results disprove a homology between the IL and the mammalian lateral hypothalamus. We suggest that the IL is likely to be involved in multimodal sensory integration rather than feeding motivation. The teleost brain is not a simpler version of the mammalian brain, and our study highlights the evolutionary plasticity of the brain which gives rise to novel structures.

X-FaCT: Xenopus-Fast Clearing Technique.

Accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. Confocal imaging and tissue clearing on whole organs allow for 3D imaging of cellular structures after being subjected to in-toto immunohistochemistry. Lately, the passive CLARITY technique (PACT) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. We recently demonstrated tissue clearing on one-week old tadpole brain (Fini et al., Sci Rep 7:43786, 2017). We here describe a further simplified version with clearing of small tissue samples (thickness inferior to 500 µm)) carried out by immersion in a fructose-based high-refractive index solution (fbHRI). By refining steps of the protocol, we were able to reduce the overall procedure time by two thirds. This offers the advantages of reducing the time of experimentation to a week and minimizes procedure-induced tissue deformations. This protocol can be easily adapted to be performed on thick section. We present an example of immunohistochemistry performed on NF45 Xenopus laevis brains with anti-pH 3 (phosphorylated histone H3) antibody used to stain chromatin condensation commonly associated with proliferation.

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.