Elsevier

Brain Research

Volume 1344, 16 July 2010, Pages 13-24
Brain Research

Research Report
Adhesion properties and retinofugal expression of chicken protocadherin-19

https://doi.org/10.1016/j.brainres.2010.04.065Get rights and content

Abstract

Protocadherin-19 has been implicated in some neurological diseases, but even the basic properties of this protocadherin have not yet been characterized well. Hence, various basic properties of chicken protocadherin-19 were examined to elucidate its biological role. The protocadherin-19 expressed in L cells was localized at the intercellular contact sites and showed Ca2+-dependent homophilic cell aggregation activity that was relatively weak but showed stringent specificity. The results of a pull-down assay using fusion proteins of the cytoplasmic domain and glutathione S-transferase yielded specifically bound proteins. In the bound fractions, liquid chromatography-mass spectrometry identified Nck-associated protein 1 and cytoplasmic FMP1 interacting protein 2, which have been reported to bind to glutathione S-transferase fused with the cytoplasmic domain of OL-protocadherin, suggesting that these proteins generally have affinity for δ protocadherins. Protocadherin-19 was mainly expressed in the central nervous system. In the chicken retina, protocadherin-19 was expressed as early as embryonic day 5 and was localized in the ganglion cell layer, inner plexiform layer, and optic nerve layer. Chicken protocadherin-19 was co-localized with syntaxin 1 in inner plexiform layer and was also expressed in the optic nerve and in specific layers of optic tectum. These results suggest that protocadherin-19 plays a role as an adhesion protein in optic nerve fiber bundling, optic nerve targeting, and/or synapse formation.

Introduction

Cell–cell adhesion or interaction plays a crucial role in the formation and maintenance of tissue structure. Among the proteins that mediate these activities, Ca2+-dependent intercellular adhesion proteins have much attracted the interest of many investigators, because of their apparent biological importance. Discovered by Yoshida and Takeichi in 1982, E-cadherin was the first Ca2+-dependent cell–cell adhesion protein to be identified. Since then, a vast number of cadherins characterized by their unique cadherin repeat in the extracellular domain have been identified in various tissues and organisms (Takeichi, 1995, Hirano et al., 2003): they constitute a large cadherin superfamily that has been subdivided into several subfamilies.

It has been thought that cadherins play a central role(s) in the formation and maintenance of tissue structure because of their unique characteristics such as stringent specificity of Ca2+-dependent cell–cell adhesion activity, the large number of cadherin species, and characteristic expression pattern as described above. In fact, Larue et al. (1994) showed that the knockout of E-cadherin resulted in the failure of embryonic development. On the other hand, N-cadherin was initially identified as a candidate cadherin with its main function being neural tissue formation (Hatta et al., 1988): N-cadherin is ubiquitously expressed in nervous tissue from the early developmental stages. Despite the initial supposition, however, the knockout of N-cadherin did not severely inhibit the initial step of neural tissue formation (Radice et al., 1997), suggesting that other cadherins may work as a main player in early neural tissue formation and that N-cadherin is involved in other aspects of neural tissue formation and maintenance. Indeed, N-cadherin has been reported to be involved in the synapse formation and plasticity (Fannon & Colman, 1996, Huntley & Bensonm, 1999).

The central nervous system intrinsically requires various types of intercellular adhesion or interaction, because it is composed of complex networks of neurons that assure its intricate function. Hence, a variety of studies on cadherins in central nervous system have been carried out. The results clearly demonstrated that many species belonging to various cadherin subfamilies are expressed in central nervous system (Hirano et al. 2003). The retina has frequently been used for cadherin research as a model system, since it has a simple structure and is relatively easy to handle. Using cell adhesion-inhibitory antibodies, Takeichi's group showed that N-cadherin and R-cadherin play a role in the formation of the laminar structure of the retina (Hatta et al., 1988, Inuzuka et al., 1991). Indeed, zebra fish mutants of N-cadherin did not form laminae in their neural retina (Erdmann et al., 2003, Malicki et al., 2003, Masai et al., 2003). Selective knockout of β-catenin also disrupted retinal formation (Fu et al., 2006). On the other hand, protocadherins (Pcdhs), which constitute the largest cadherin subfamily, are also involved in the formation and function of the central nervous system including the retina (Hirano et al. 2003). Aberrant Pcdh15 is responsible for a degenerative disease of the retina, known as Usher's syndrome (Ahmed et al., 2003, Reiners et al., 2005), although the mechanism is unsolved. Rattner et al. (2001) reported that Pcdh21 was specifically expressed in rod and cone cells and that its knockout resulted in the disruption of photoreceptor cells. Nakao et al. (2005) showed that OL-Pcdh was expressed in developing optic nerve fibers and in the inner plexiform layer. Piper et al. (2008) found that NF-Pcdh regulated retinal axon initiation and elongation.

These studies clearly indicate that a variety of cadherins, especially Pcdhs, are expressed in different parts of the neural retina. Given the complex structure and function of central nervous tissues, it seems quite reasonable to assume that many other Pcdhs are expressed and play various roles in the retina, from the formation of retinal structure to axon guidance to the functional plasticity of retinal function. Indeed, degenerate PCR revealed the expression of various cadherins in the chick retina, including Pcdh19 (Tanabe et al., 2004, Gaitan & Bouchard, 2006); and Lefebvre et al. (2008) reported the localization patterns of various Pcdhs in the retina.

Pcdh19 belongs to the δ2 group of the Pcdh family (Vanhalst et al., 2005, Redies et al., 2005). It was first identified in 2001, at which time its mRNA expression in different tissues was determined (Wolverton and Lalande, 2001). Tanabe et al., 2004, Gaitan & Bouchard, 2006 reported expression of Pcdh19 in chick or mouse retina. Recently, Dibbens et al. (2008) and Hynes et al. (2010) reported that mutation of Pcdh19 resulted in female-limited neurological diseases. Moreover, Emond et al., 2009, Liu et al., in press reported that Pcdh19 was essential for early brain morphogenesis. These results indicate the importance of Pcdh19 in the formation of the central nervous system and thereby the function of it. However, the mechanism of the function and even the basic properties of Pcdh19 are poorly known. Hence, we focused on chicken Pcdh19 (cPcdh19) in this study and characterized its basic properties. Herein we describe our results and discuss its possible function.

Section snippets

Isolation of chicken Pcdh19 cDNA

In order to elucidate the possible role of Pcdhs in the formation and maintenance of retinal structure and function, we first examined what cadherins were expressed in the chicken retina by performing degenerate PCR. The results indicated that a variety of cadherins were expressed in it, as already reported (Tanabe et al., 2004). Among the cadherins expressed, we then subsequently focused on cPcdh19, since this Pcdh was already detected in the human retina, but had not been characterized well (

Discussion

The present study demonstrated that at least 4 splicing variants of cPcdh19 were expressed in the chicken retina. Wolverton and Lalande (2001) already reported that human Pcdh19 was expressed in several forms spliced alternatively in the cytoplasmic domain. Interestingly; however, none of the present clones had the sequence corresponding to a juxtamembrane region in the cytoplasmic domain of the hPcdh19 full-length sequence (denoted by # in Fig. 1A). We examined chicken genomic sequences in the

Chemicals

Antibody against E-cadherin was the product of Takara Bio. Co. (Otsu, Japan). Antibodies against β-catenin and syntaxin 1 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against HA-tag were purchased from Upstate (Charlettesville, CA, USA) and Medical Biological Laboratories (Nagoya, Japan). Anti-mouse and anti-rabbit antibodies conjugated with Alexa Fluor-488 or Alexa Fluor-568 and DiI were obtained from Molecular Probe (Eugene, OR, USA). Alkaline phosphatase-conjugated

Acknowledgments

This study was supported in part by grants-in-aid from the Ministry of Education, Culture, and Technology (the advanced program of high profile research to Department of Science and KAKENHI 18659055 to S.O.), the Ministry of Health, Labour, and Welfare (H21-G-209 to S. T. S.), and from Kwansei Gakuin University (to S.T. S.). We thank Dr. S. Nakagawa (Riken) for providing us with a cDNA library of chicken retina. We are also very grateful to Drs. K. Owaribe (Nagoya University) and S. Hirano

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