Differential expression of individual gamma-protocadherins during mouse brain development
Introduction
Proper processing of information in the vertebrate central nervous system is crucially dependent on the correct formation of neuronal connections at synapses. Different groups of adhesion molecules have been implicated in synaptogenesis and synaptic adhesion (for a review, see Yamagata et al., 2003). Of these, the unique features of cadherins have attracted particular attention. Distinct neuronal populations and functional circuits are defined by the expression of a specific set of cadherins (Redies and Takeichi, 1996, Takeichi, 1988). Cadherins are integral parts of the synaptic junctional complex, and different synaptic subsets are marked by distinct cadherins and their associated molecules, i.e., the catenins (Fannon and Colman, 1996, Uchida et al., 1996; for a review, see Redies, 2000). Moreover, alterations of cadherin adhesion influences synaptic transmission (Bozdagi et al., 2000, Tang et al., 1998, Togashi et al., 2002) and, conversely, changes in synaptic excitability change the molecular properties of the cadherin adhesion complex (Murase et al., 2002, Tanaka et al., 2000). Thus, cadherins are prime candidates for the molecular determination of neuronal connectivity as envisioned in the chemoaffinity hypothesis by Roger Sperry (Sperry, 1963; for a review, see Shapiro and Colman, 1999).
Protocadherins constitute the largest subgroup within the cadherin superfamily (Angst et al., 2001, Nollet et al., 2000). A typical protocadherin includes five to seven cadherin ectodomains, a single transmembrane domain and an individual cytoplasmic part that differs strikingly from the well-conserved cytoplasmic domain of classical cadherins (Obata et al., 1995, Sano et al., 1993, Suzuki, 1996, Wolverton and Lalande, 2001). About 70 protocadherin genes are known in mouse and human, and many of these are expressed in the nervous system (for a review, see Frank and Kemler, 2002). More than 50 genes are contained in three sequential gene clusters, termed protocadherins alpha, beta, and gamma (Pcdh-α, Pcdh-β, Pcdh-γ) (Wu and Maniatis, 1999, Wu et al., 2001). Gene-specific mRNAs each encoding a complete protocadherin with an individual cytoplasmic domain are derived from these arrayed exons by alternative promotor choice. Pcdh-α and Pcdh-γ transcripts are cis-spliced to three small exons located at the 3′ end of these clusters which code for a family-specific “constant” cytoplasmic domain (Tasic et al., 2002, Wang et al., 2002a). In contrast, the Pcdhs-β are single-exon genes, lacking such constant domain exons (Vanhalst et al., 2001, Wu et al., 2001).
Pcdh-γ transcripts are abundantly detected in the human and mouse nervous system using RT-PCR and probes against the constant domain (Kallenbach et al., 2003, Sano et al., 1993, Tasic et al., 2002, Wang et al., 2002a, Wang et al., 2002b), but limited information is available on the expression patterns of individual transcripts (Hirano et al., 1999, Hirano et al., 2002, Wang et al., 2002b). Deletion of all variable exons in the Pcdh-γ cluster leads in homozygous knockout mice to apoptotic degeneration of interneurons and neonatal lethality, while synapse formation appeared grossly unaffected (Wang et al., 2002b). Recently, Pcdhs-γ were shown to be localized to synapses and nearby tubulovesicular structures, suggesting adhesive and modulatory functions at the synaptic scaffold (Phillips et al., 2003). Now, it is important to understand how the molecular diversity of Pcdhs-γ translates into the presumptive functions in neuronal differentiation and synaptogenesis.
Here, we ask whether individual Pcdhs-γ are differentially expressed in the developing mouse brain. Specifically, how does the expression of the three subgroups of Pcdhs-γA, -γB, and -γC compare across various brain regions? Our results suggest that multiple Pcdhs-γ are expressed on the level of individual neurons and that their expression and subcellular localization are possibly regulated cell autonomously. The distribution and properties of Pcdhs-γ support the idea that they locally modify adhesion or signaling complexes involved in neuronal differentiation and synaptogenesis.
Section snippets
Pcdhs-γ promote calcium-dependent cell adhesion
To test some functional properties of Pcdh-γ proteins, the coding sequences of γA3 and γC3 were cloned in frame with an eGFP reporter fused to the C-terminal end and then expressed in cell lines and neurons (Fig. 1, Fig. 2). Adhesive properties of Pcdhs-γ were investigated in HEK 293 cells, which showed a robust expression of the tagged-Pcdhs at the cell surface (Fig. 1). In mouse fibroblast L cells, which are frequently used to analyze classical cadherins, Pcdh-γ fusion proteins were mostly
Discussion
Following the idea that differential and combinatorial expression of classical cadherins is crucially involved in the specification of neurons and their synaptic connections, similar functions have been postulated for the genomically clustered protocadherins (Serafini, 1999, Shapiro and Colman, 1999, Redies, 2000, Yagi and Takeichi, 2000). We show that Pcdhs-γ exhibit adhesive properties and are expressed at synapses, reminiscent of features of classical cadherins. Comparing the expression of
Animals and tissues
Swiss Webster and NMRI mice were used in this study. Tissues were collected from various embryonal and postnatal developmental stages [embryonic day (E) 0.5 = appearance of vaginal plug; postnatal day (P) 0 = birth]. Mice were killed by CO2 inhalation and decapitated. Whole embryos or brains were rapidly dissected and either snap-frozen on dry ice for subsequent RNA or protein isolation, or embedded in O.C.T. compound (Sakura) and frozen in liquid isopentane for in situ hybridization. For
Acknowledgments
We thank the late Lazar Fidler and Melanie Volk for excellent technical support. The contributions of Björn Haker to the characterization of the peptide-antisera are acknowledged. Many thanks to Drs. Ingrid Haas and Randy Cassada for fruitful discussions and corrections. M.F. was supported by a fellowship of the German Academic Exchange Service (DAAD, Hochschulsonderprogramm III; D/99/18054) during his postdoctoral stay in the laboratory of D.R.C. and grant NS20147 from NINDS to D.R.C.; G.R.P.
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Present address: Boehringer Ingelheim, CNS pharmacology, 88397 Biberach, Germany.