In vivo analysis of morphogenesis of choroid plexus in transgenic zebrafish
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KeywordsTransgenic Line Choroid Plexus EGFP Gene Transgenic Zebrafish Transgenic Embryo
The choroid plexus is a specialized structure for production of cerebrospinal fluid (CSF). Its development has been studied mainly by traditional histological techniques. In result our understanding of this process lacks a continuity and accuracy of the in vivo observations.
Materials and methods
We generated a range of random transgenics by means of enhancer trapping using the Tol2 transposon that carries the EGFP gene controlled by a partial keratin8 promoter. It inserts randomly and reveals activity of enhancers in tissue-specific manner in stable transgenic lines. (Parinov et al., 2004; Choo et al.2006). One of these lines, ET33 expressed GFP in the roof plate, dorsal interneurons and meninx. The injection of the transposase mRNA into ET33 embryos resulted in transposition of Tol2 in germ cells and many novel transgenic lines including ET33E20 ("Gateways") were generated. Gateways embryos demonstrate the GFP expression pattern mainly restricted to the brain and spinal cord, similar to that of several genes located close to the insertion site that we cloned in an attempt to define which genes expression is recapitulated by GFP expression in Gateways. Interestingly, the early GFP expression is localized to sites, where several cranial blood vessels develop later on. GFP also appears in the roof plate and in groups of cells at the midline in contact with brain ventricles, including the choroid plexus. Using the enhancer trap lines we studied the morphogenesis of the choroid plexus of IV ventricle using a combination of histology, immunohistochemistry and in vivo analysis during normal development.
The time-lapse movies of choroid plexus formation in Gateways for the first time illustrated this process in vivo in detail. Based on this we defined three phases of choroid plexus morphogenesis. Our comparative in vivo analysis of mutants affecting major developmental signaling pathways, including Notch and Hedgehog at the Gateways background revealed a role of these pathways during formation of the choroid plexus.
The transgenic embryos of zebrafish represent living markers that allow studying details of brain morphogenesis in vivo. These transgenics expand possibilities to monitor in vivo effect of genetic, toxicological and other experiments performed on embryos of vertebrates.
This article is published under license to BioMed Central Ltd.