Functional and genetic analysis of choroid plexus development in zebrafish

Abstract

The choroid plexus, an epithelial-based structure localized in the brain ventricle, is the major component of the blood-cerebrospinal fluid barrier. The choroid plexus produces the cerebrospinal fluid and regulates the components of the cerebrospinal fluid. Abnormal choroid plexus function is associated with neurodegenerative diseases, tumor formation in the choroid plexus epithelium, and hydrocephaly. In this study, we used zebrafish (Danio rerio) as a model system to understand the genetic components of choroid plexus development. We generated an enhancer trap line, Et(cp:EGFP)sj2, that expresses enhanced green fluorescent protein (EGFP) in the choroid plexus epithelium. Using immunohistochemistry and fluorescent tracers, we demonstrated that the zebrafish choroid plexus possesses brain barrier properties such as tight junctions and transporter activity. Thus, we have established zebrafish as a functionally relevant model to study choroid plexus development. Using an unbiased approach, we performed a forward genetic dissection of the choroid plexus to identify genes essential for its formation and function. Using Et(cp:EGFP)sj2, we isolated 10 recessive mutant lines with choroid plexus abnormalities, which were grouped into five classes based on GFP intensity, epithelial localization, and overall choroid plexus morphology. We also mapped the mutation for two mutant lines to chromosomes 4 and 21, respectively. The mutants generated in this study can be used to elucidate specific genes and signaling pathways essential for choroid plexus development, function, and/or maintenance and will provide important insights into how these genetic mutations contribute to disease.

CNS angiogenesis and barriergenesis occur simultaneously

“…This expression pattern was maintained into adulthood (data not shown). To examine mCherry expression in vasculature of circumventricular organs that lack BBB properties (Saunders et al, 2008), we crossed glut1b:mCherry to cp:EGFP, an enhancer trap line that expresses GFP in the choroid plexus (Henson et al, 2014), and injected albumin Alexa Fluor 647 conjugate to visualize the vasculature. We found that mCherry was not expressed in the vasculature of the choroid plexus (Figure 1G–J).…”

“…Et(cp:EGFP) was previously generated in our lab (Henson et al, 2014). Embryos and larvae were maintained at 28.5°C in egg water (0.03% Instant Ocean in reverse osmosis water).…”

Development and functions of the choroid plexus–cerebrospinal fluid system

“…Third, the model that systemic signals regulate ChP 129 , together with findings that CSF composition and mixing with the interstitial fluid maintain the health of the brain via convective flux more generally than previously believed 137–139 (BOX 3), raise tantalizing hypotheses for the myriad ways by which CNS health can be regulated. Collectively, pairing actively evolving technologies with available models, including recently developed fluorescent ChP zebrafish lines 140 , should accelerate steps towards further elucidating the biology of the ChP–CSF system as well as advancing future therapies, including the transplantation of engineered ChP epithelial cells.…”

Frizzled 4 regulates ventral blood vessel remodeling in the zebrafish retina

“…Similar to the ocular vasculature in mammalian embryos, zebrafish embryos possess choroidal and hyaloid networks that function similarly to their mammalian counterparts . In order to better understand how the fzd4 mutation affects retinal vascularization, and to determine the time point at which fzd4 vasculature formation deviates from its control counterpart, we followed the developmental progression of the retinal vasculature in control and fzd4 fish for the first 12 weeks of development (n = 10 fish/genotype/week).…”