Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development

Abstract

Protocadherins, a subgroup of the cadherin superfamily of calcium-dependent cell adhesion molecules, are considered to play important roles in the developing embryo particularly in the central nervous system. The Protocadherin 8 (Pcdh8) gene comprises three coding exons in both human and mouse, and the exon junctions are precisely conserved between these two species. Alternative splicing of Pcdh8 RNA leads to the formation of two isoforms that differ in the length of the cytoplasmic domains. We have investigated the expression of these short and long variants of Pcdh8 during early mouse development by RT/PCR and in situ hybridization. We found that both isoforms were predominantly expressed in the nervous system, and that their expression patterns appeared to be developmentally regulated. However, the short variant had a broader pattern of expression than the long variant and was found in some non-neuronal tissues, such as paraxial mesoderm, developing somites, and in limb interdigital mesenchyme where massive programmed cell death occurs. The differential expression of two alternative cytoplasmic domain variants suggests that Pcdh8 may regulate cell adhesion in a variety of developmental processes, and that this may involve different intracellular interactions.

Introduction

Alternative RNA splicing is a major contributor to protein diversity; however, the functional significance of different isoforms is not always fully understood [1]. Recently, alternative RNA splicing has been found in the protocadherin family of cell adhesion molecules [2], [3], [4], [5], [6]. Protocadherins are transmembrane calcium-dependent adhesion molecules that are structurally similar to the classical cadherins [7], [8], [9], [10], [11]. Protocadherins differ from classical cadherins in the number of extracellular cadherin (EC) repeats that they contain, and by the absence of a β-catenin-binding site in their cytoplasmic domains. β-Catenin links classical cadherins to the cytoskeleton, and also functions as a transcriptional co-activator [12], [13], [14], [15]. It has been reported that the EC domains of protocadherins mediate weak homophilic binding between molecules, similar to classical cadherins [16], [17]. The cytoplasmic domains of protocadherins differ from each other, and this likely reflects the diversity of functional interactions between protocadherins and intracellular components [9], [18].

Protocadherin 8 is highly conserved between human and rodents, and is composed of six EC repeats and a transmembrane domain encoded by a single exon, and a cytoplasmic tail that varies in length due to alternative RNA splicing [2], [4], [5]. In human, alternative RNA splicing yields two isoforms of 974 and 1071 amino acids, respectively [2]. The shorter cytoplasmic domain variant (designated isoform 2 in human) results from the use of a cryptic splice donor site located 291 bp upstream of the 3′ end of the first coding exon, and is homologous to the reported sequence of arcadlin (GenBank acc. no. AB026154) [2]. The longer isoform of human PCDH8 (designated isoform 1 in human) corresponds to the reported sequence of mouse Pcdh8 (GenBank acc. no. AF231125) [19], and to a novel isoform of arcadlin [2]. The additional sequence in the long cytoplasmic domain-containing variant is enriched in alanine (20.6%), glycine (18.5%), and proline (12.4%) residues, and is predicted to adopt a random coil secondary structure [20]. Apart from potential phosphorylation sites for casein kinases 1 and 2, glycogen synthase kinase 3 and protein kinase C [21], the sequence does not contain any obvious motifs or homology to other proteins. An ∼25-amino-acid sequence within the cytoplasmic domain common to both variants of PCDH8 is highly conserved in Danio and Xenopus paraxial protocadherin (PAPC), indicating an orthologous relationship between PCDH8 and PAPC [2], [22] (Fig. 1). In humans, PCDH8 maps to a region on chromosome 13 where weak linkage to schizophrenia has been reported [23]; in mouse, Pcdh8 maps to the syntenic region on chromosome 14 [18].

Although Pcdh8 knockout mice appear to develop normally [22], a recent study, using a soluble form of the molecule, indicates that Pcdh8 is involved in regulating the transition of paraxial mesenchymal cells into epithelial cells during somitogenesis [19]. Other work with rodents indicates that Pcdh8 may be involved in activity-induced synaptic reorganization in the brain. In rat hippocampal granule neurons, Pcdh8 (arcadlin) is rapidly induced by long-term potentiation (LTP) and antibodies directed against Pcdh8 block LTP [17].

Here we report on the expression pattern of the two cytoplasmic variants of Pcdh8 during mouse development. We observed a dynamic and differential expression of the two isoforms, suggesting that alternative RNA splicing of the Pcdh8 gene is developmentally regulated. We found that the long cytoplasmic variant was expressed predominantly in the brain, whereas the short cytoplasmic variant was expressed in the brain, spinal cord, limb, and in paraxial mesoderm in developing somites. The differential expression of two cytoplasmic domain variants suggests that Pcdh8 may regulate cell adhesion in a variety of developmental processes that may involve interactions with different cellular components.