874 Fig. 7 Developmental analysis of occludin and zonula occludens protein transcript levels in choroid plexus, brain cortex and cerebral microvessel of the rat. RT-PCR was performed on tissues sampled at four developmental stages. Moderate (ZO-1, ZO-2) to high (occludin) expression levels were found in both CPs and MV. Very low levels of ZO-3 were detected in both tissues. ZO-3, like occludin were distinctively enriched in blood– brain interfaces; see ‘‘Results’’ for a detailed description of the data. Data normalization, data expression, and statistical analysis are as described in Fig. 1 Histochem Cell Biol (2012) 138:861–879 membrane into strands. These proteins contain a short intracellular cytoplasmic amino-terminal sequence fol- lowed by a large extracellular loop (EL1), a short intra- cellular loop, a second extracellular loop (EL2) and a longer C-terminal cytoplasmic tail. EL1 inﬂuences charge and ion selectivity (see infra), while EL2 is important for both Cld oligomerization and cell–cell adhesion (Colegio et al. 2002; Coyne et al. 2003; Daugherty et al. 2007). The identity and relative proportion of Clds within a TJ com- plex, and the identity of the different cytoplasmic and accessory proteins with which they interact, determine the degree of tightness of a cellular barrier (Overgaard et al. 2011). It can be low as in the case of the kidney epithelium (Gunzel and Yu 2009) or high as in the microvascular endothelium of the BBB (Huber et al. 2001; Nitta et al. 2003). Clds have been classiﬁed into two subcategories. The pore-forming Clds, which include Cld-2, -10, -16, allow the charge-selective paracellular diffusion of inor- ganic ions, and decrease transcellular electrical resistance. They may also participate in a size-selective diffusion of non-charged molecules. In contrast, the sealing Clds such as Cld-1, -3 -4 -5, -11 restrict the paracellular permeability and are considered as barrier-tightening Clds (Original references listed in Online resource 3. For review, Van 123 Itallie et al. 2006; Krause et al. 2008; Overgaard et al. 2011; Yu et al. 2009). In CP, the relative proportion of the different Clds forming TJs differs between developing and adult animals, suggesting that the functions supported by these junctions are developmentally regulated. We Fig. 8 Immunohistochemical detection of claudins in choroid plex- c uses of developing human brain. Immunoreactivity of Cld-1, -2, and -3 is presented on the left, central, and right panels, respectively, with hematoxylin counterstaining. a Staining of a WD8 fetal brain. Cld-1 labeling, observed in both LVCP and 4VCP, was continuous and localized apically at intercellular junctions of the epithelium (arrows in inserted high magniﬁcation micrograph). Cld-2 immunoreactivity displayed a similar apical, but patchy pattern in both LVCP and 4VCP. The higher magniﬁcation (insert in LVCP) illustrates the discontinuous labeling (arrows). Cld-3 was also detected in the epithelium of both CP (arrows). Inserts are higher magniﬁcations highlighting the continuous staining pattern. b Staining of a WD22 fetal brain. Cld-1 immunoreactivity was present at all epithelial junctions (shown in 4VCP). A continuous apical labeling of the epithelium was observed for Cld-2 (LVCP is shown). Cld-3 labeling was faint but continuous in LVCP epithelial cells. c Staining of a WD39 fetal brain. A distinct and continuous labeling of the choroidal epithelium was observed for all Cld-1 (shown for LVCP), -2 (shown for 4VCP), and -3. The continuous signal for Cld-3 was only associated with the epithelium (arrows) and not with stromal vessels (arrowheads in LVCP). The insert shows a similar Cld-3 staining for 4VCP. Abbreviations as in Fig. 2. Scale bar 50 lm