Patterns of spon1b:GFP expression during early zebrafish brain development.

OBJECTIVE: F-spondin is part of a group of evolutionarily conserved extracellular matrix proteins in vertebrates. It is highly expressed in the embryonic floor plate, and it can bind to the ECM and promote neuronal outgrowth. A characterization of F-spondin expression patterns in the adult zebrafish brain was previously reported by our group. However, given its importance during development, we aimed to obtain a detailed description of green fluorescent protein (GFP) expression driven by the spon1b promotor, in the developing zebrafish brain of the transgenic Tg(spon1b:GFP) line, using light sheet fluorescence microscopy (LSFM). RESULTS: Images obtained in live embryos from 22 to 96 h post fertilization confirmed our earlier reports on the presence of spon1b:GFP expressing cells in the telencephalon and diencephalon (olfactory bulbs, habenula, optic tectum, nuclei of the medial longitudinal fasciculus), and revealed new spon1b:GFP populations in the pituitary anlage, dorso-rostral cluster, and ventro-rostral cluster. LSFM made it possible to follow the dynamics of cellular migration patterns during development. CONCLUSIONS: spon1b:GFP larval expression patterns starts in early development in specific neuronal structures of the developing brain associated with sensory-motor modulation. LSFM evaluation of the transgenic Tg(spon1b:GFP) line provides an effective approach to characterize GFP expression patterns in vivo.

Xenotransplantation of Human glioblastoma in Zebrafish larvae: in vivo imaging and proliferation assessment.

Glioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However, patient prognosis remains very poor, prompting the search for new models for drug discovery and testing, especially those that allow assessment of in vivo responses to treatment. Zebrafish xenograft models have an enormous potential to study tumor behavior, proliferation and cellular interactions. Here, an in vivo imaging and proliferation assessment method of human GBM xenograft in zebrafish larvae is introduced. Zebrafish larvae microinjected with fluorescently labeled human GBM cells were screened daily using a stereomicroscope and imaged by light sheet fluorescence microscopy (LSFM); volumetric modeling and composite reconstructions were done in single individuals. Larvae containing tumors were enzymatically dissociated, and proliferation of cancer cells was measured using dye dilution by flow cytometry. GBM micro-tumors formed mainly in the zebrafish yolk sac and perivitelline space following injection in the yolk sac, with an engraftment rate of 73%. Daily image analysis suggested cellular division, as micro-tumors progressively grew with differentiated fluorescence intensity signals. Using dye dilution assay by flow cytometry, at least three GBM cells' division cycles were identified. The combination of LSFM and flow cytometry allows assessment of proliferation and tumor growth of human GBM inside zebrafish, making it a useful model to identify effective anti-proliferative agents in a preclinical setting.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique's relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.