Developmental Regulation of CPG 15 Expression in Xenopus

Mechanisms controlling dendritic arbor formation affect the establishment of neuronal circuits. Candidate plasticity gene 15 (CPG15) is a glycosylphosphatidyl inositol (GPI)-linked activity-induced protein that has been shown to function as an intercellular signaling molecule that can promote the morphological and physiological development of the Xenopus retinotectal system. A thorough understanding of CPG15 function requires knowledge of the spatiotemporal expression of the endogenous protein. We therefore cloned Xenopus cpg15 and used RNA in situ hybridization and immunohistochemistry to determine the pattern of CPG15 expression. cpg15 mRNA and CPG15 protein are first detectable in the developing spinal cord and become widespread as development proceeds. CPG15 is expressed in sensory regions of the brain, including the visual, auditory, and olfactory systems. Within the retina, CPG15 is only expressed in retinal ganglion cells. CPG15 protein is concentrated in axon tracts, including retinal axons. These data support a model in which CPG15 expressed in retinal ganglion cells is trafficked to retinal axons, where it modulates postsynaptic dendritic arbor elaboration, and synaptic maturation. J. Comp. Neurol. 435:464–473, 2001. © 2001 Wiley-Liss, Inc. Indexing terms: neuritin; ligand; retinotectal; GPI; spinal cord; amphibian; plasticity; visual system Central nervous system (CNS) development is characterized by the coordinate development of presynaptic axonal arbors, postsynaptic dendritic arbors, and synaptic connections between them. Activity-dependent mechanisms regulate multiple aspects of nervous system connectivity during development. One way that activity can influence the development of neuronal connections is through the induction of activity-regulated genes, or candidate plasticity genes (CPGs). Numerous activityinduced genes have been identified through the use of highly selective screens (Nedivi et al., 1993; Qian et al., 1993; Yamagata et al., 1993). These screens have identified genes previously implicated in plasticity such as brain-derived neurotrophic factor (BDNF) and the transcription factors zif268, c-fos, and jun (Nedivi et al., 1993; Qian et al., 1993). They have also identified new functions for previously discovered genes such as the class 1 major histocompatibility (MHC1) molecule (Corriveau et al., 1998) and tissue plasminogen activator (tPA) (Qian et al., 1993), as well as many novel genes with unknown functions such as Homer, Arc, Narp, and cpg15 (Nedivi et al., 1993; Lyford et al., 1995; Tsui et al., 1996; Brakeman et al., 1997) Subsequent analysis has demonstrated that these novel activity-induced genes play a variety of roles in neuronal plasticity (Nedivi, 1999). CPG15, also known as neuritin (Naeve et al., 1997), is a small secreted protein, anchored to the extracellular surface of neurons by a glycosylphosphatidyl inositol (GPI) link. cpg15 was first identified from rat (Nedivi et al., 1996) and has subsequently been cloned from cat (Corriveau et al., 1999) and human (Naeve et al., 1997). In these three mammalian species, the primary amino acid sequence of CPG15 consists of 142 amino acids and shows no homology to other proteins (Nedivi et al., 1996, 1998; Naeve et al., 1997). In situ hybridization studies in mammals show that cpg15 is expressed at peak levels during development, specifically at times of afferent axonal and dendritic growth and synaptogenesis (Nedivi et al., 1996; Grant sponsor: NIH; Grant sponsor: the National Down Syndrome Society; Grant sponsor: the Ellison Medical Foundation. Elly Nedivi and Ashkan Javaherian contributed equally to this article. Elly Nedivi’s present address is the Center for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 50 Ames St., E18-670, Cambridge, MA. 02139. *Correspondence to: Hollis Cline, Cold Spring Harbor Laboratory, P.O. Box 100, 1 Bungtown Road, Cold Spring Harbor, NY 11724. E-mail: cline@cshl.org Received 18 August 2000; Revised 7 February 2001; Accepted 6 April 2001 THE JOURNAL OF COMPARATIVE NEUROLOGY 435:464–473 (2001) © 2001 WILEY-LISS, INC. Naeve et al., 1997; Corriveau et al., 1999). Furthermore, cpg15 is upregulated by visual activity in adult rat visual cortex (Nedivi et al., 1996). Viral expression of cpg15 in Xenopus optic tectal neurons promotes the elaboration of tectal cell dendritic arbors and presynaptic retinal axon arbors in a non-cell autonomous fashion (Nedivi et al., 1998; Cantallops et al., 2000). Electrophysiological recordings indicate that cpg15 promotes synaptic maturation by the insertion of AMPAtype glutamate receptors into synapses (Cantallops et al., 2000). The GPI link is required for these effects of CPG15, because expression of a truncated form of CPG15, lacking the GPI consensus sequence, does not promote axonal or dendritic arbor elaboration or synaptic maturation. A thorough understanding of CPG15 function requires knowledge of the spatiotemporal expression pattern of cpg15 mRNA and protein. To address these issues, we cloned Xenopus cpg15 and mapped the spatiotemporal distribution of cpg15 mRNA and protein during nervous system development in Xenopus. cpg15 mRNA is detectable in Xenopus retina, brain, and spinal cord. Expression is detected early during nervous system development, corresponding to the time of initial morphological development and synaptogenesis. Interestingly, the protein is targeted to projection axons, where it might act as an intercellular signal to promote the development of dendritic arbors of postsynaptic neurons. MATERIALS AND METHODS All experiments were carried out in accordance with animal care protocols approved by Cold Spring Harbor