Histochem Cell Biol (2012) 138:861–879 stages. Cld-1 and -3 staining was already continuous throughout the intercellular regions of the choroidal epi- thelium from the earliest WD8 stage. This is in accordance with a previous study showing the apical localization of Cld-1 in CP of human embryos (Anstrom et al. 2007). Cld- 2 signal displayed a patchy pattern at that early time before it became continuous around WD16. In human as in other mammals, CPs appear in the order of the fourth, then lat- eral ventricle. Given that they form around the WD 6–7 stage (Shuangshoti and Netsky 1966), our data imply that a restrictive and selective barrier exists in human fetuses between blood and CSF, very rapidly after the CP pri- mordium starts to differentiate. The research focus on Clds has always been on their role in tight junction structure. However, the plethora of Clds revealed by this study, their differential expression in the two types of CP and their age- related regulation suggest that there remain other so far undiscovered functions for some of these Clds. With respect to TJ proteins at the BBB, we showed that, in addition to the previously reported Cld-5 (Morita et al. 1999b), brain parenchymal MVs selectively express Cld- 16, as well as Cld-4 at lower levels. Both Cld-5 and -4 genes, considered as sealing Clds, were expressed in E19 or P2 animals, in line with the observation that the BBB is already impermeable to polar tracers in the developing brain (Ek et al. 2006). We also detected mRNAs for Cld-3, -9, -10, -12 and -22 in MVs prepared from P9 and adult rat brain, in accordance with the previous molecular analysis realized on mouse brain endothelial cells (Nitta et al. 2003; Ohtsuki et al. 2008). Cld-1 was not detected in brain ves- sels at any stage of development, neither in rat nor in human. This conﬁrms the ﬁndings of a previous study performed on human embryos (Anstrom et al. 2007). The signiﬁcance of the pore-forming Cl-10 and Cl-16 expres- sion at the BBB, characterized by a high transendothelial electrical resistance is unclear. Much evidence exists for modulation of Clds in response to inﬂammation, infection or oxidative stress, gathered from in vitro cell cultures or in vivo studies in the adult. Cld-3 was down-regulated in the inﬂamed BBB in a mouse model of multiple sclerosis (Wolburg et al. 2003), and was shown to be speciﬁcally sensitive to H2O2 expo- sure in epithelial gastric cells (Hashimoto et al. 2008). Oxidative stress induced by intracerebral HIV gp120 pro- tein injection in the caudate putamen of rats increased the expression of matrix metalloproteinases-2 and -9 which in turn induced a loss of Cld-5 immunoreactivity (Louboutin et al. 2010). Clds can also play a role in infectious diseases by mediating toxin deleterious effects or triggering virus cellular entry. A receptor site for Clostridium perfringens enterotoxin, present on the EL2 segment of Cld-3 and Cld- 4, initiates the process leading to ileal epithelial cell dys- function and necrosis. Cld-1, and possibly Cld-6 and -9 877 have been identiﬁed as co-receptors interacting with the tetraspanin protein CD81 to mediate the internalization of hepatitis C virus in the liver (Evans et al. 2007; Zheng et al. 2007). This mechanism may hold true for the brain or CSF in which the virus has been found (Wilkinson et al. 2009). Brain damage induced by perinatal injury often involves oxidative stress, inﬂammation or infection episodes (Dammann and Leviton 1999; Hagberg et al. 2012). An alteration of choroidal epithelial tight junctions triggered by such events would lead to an impairment of the BCSFB and thus of the cerebral homeostasis that is necessary to normal brain development. As perinatal injury can have dramatic consequences for brain development, a careful evaluation of Cld deregulation at blood–brain interfaces is therefore called for. In summary, the expression pattern of Clds, occludin and zonula occludens proteins in LVCP and 4VCP has revealed a high degree of complexity in the control of the permeability at the BCSFB. The developmental expression proﬁles indicate an early establishment of this barrier, a ﬁnding that was conﬁrmed in human fetuses. Variations in Cld choroidal expression between CPs and during devel- opment probably reﬂect changes in selective blood to CSF transport functions during development, and may be crucial for brain protection, especially in the context of perinatal injuries or exposure to toxins and drugs. Acknowledgments The research leading to these results has received funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement n HEALTH-F2- 2009-241778. We thank Alan S. L. Yu from the Department of Medicine, Division of Nephrology, University of Southern California Keck School of Medicine, Los Angeles, California, who kindly pro- vided the anti-Cld 19 antibody for these studies. We thank Chantal Watrin for her excellent technical help. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Adinolﬁ M (1985) The development of the human blood–CSF–brain barrier. 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