Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression
Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development
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.
Section snippets
Whole-mount in situ hybridization
All CD1 mouse embryos were fixed in 4% paraformaldehyde at 4 °C. Antisense RNA probes were labeled with digoxigenin during in vitro transcription, and whole-mount in situ hybridization was performed as previously described [24]. The templates for riboprobes were generated by genomic PCR. To detect both Pcdh8 isoforms, a 1053-bp genomic fragment from the first coding exon was amplified (nucleotides 151–1203 from the initiation codon) using the following PCR primers: forward primer, 5′
Results
To investigate whether the short and long isoforms of Pcdh8 were differentially expressed during mouse development, two different probes were generated for in situ hybridization. A 1053-bp fragment, encompassing part of the extracellular domain, and a 175 bp-fragment, corresponding to most of the additional sequence in the long cytoplasmic domain, were amplified from mouse genomic DNA by PCR. Comparing the pattern of in situ hybridization of the 1053-bp probe, which would hybridize to mRNA of
Discussion
The results from the present study and from Yamamoto et al. [22] demonstrate that Pcdh8 exhibits a dynamic pattern of expression during mouse development. We have further distinguished between the pattern of expression of the two splice variants of Pcdh8, Pcdh8lv and Pcdh8sv for long and short variant, respectively. Pcdh8lv is predominantly expressed in the developing brain in a pattern that may suggest a role in the early patterning of the developing brain. In contrast, we observed that Pcdh8sv
Acknowledgements
We would like to thank Dr. Megumi Adachi for the sequence alignment, and Drs. Kathryn Crossin, Bruce Cunningham, and Joseph Gally for helpful comments. This work was funded by the Neurosciences Research Foundation.
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Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain
2007, NeuroscienceCitation Excerpt :PCDH8 (paraxial PCDH; PAPC) was identified together with PCDH1 (AXPC) in a screen for genes involved in anterior/posterior polarity of prenotochord at the neurular stage in Xenopus (Yamamoto et al., 1998, 2000; Kuroda et al., 2002). PCDH8 has two isoforms, containing 974 and 1071 amino acids by alternative splicing (Makarenkova et al., 2005). The longer variant (designated isoform1) was predominantly expressed in the brain (Makarenkova et al., 2005), and its mRNA is transiently induced in hippocampal granule cells by seizures (Yamagata et al., 1999).