Redundant functions but temporal and regional regulation of two alternatively spliced isoforms of Semaphorin 3F in the nervous system

Abstract

SEMA3F is a secreted semaphorin that affects axon and cell guidance in the developing nervous system, and is also thought to have anti-tumor activity. Two spliced forms of SEMA3F have been identified that differ by the insertion of 31 amino acids in the sema domain. Here, we investigated the bioactivity of these isoforms and show, using coculture and binding assays, that they share common axonal chemorepulsive properties and binding to neuropilin receptors. SEMA3F isoforms were also found to regulate endothelial cell morphology by remodeling lamellipodial protrusions. Although Sema3F expression globally decreased during mouse development, we noted an enrichment of the longest isoform at postnatal stages in some territories such as the brainstem and spinal cord. These results indicate that although functionally redundant in cell culture assays, Sema3F spliced forms are characterized in vivo by a temporal and regional specific regulation during maturation of the nervous system.

Introduction

Semaphorins are a large family of secreted and transmembrane molecules that play an important role in the formation of neuronal connectivity (He et al., 2002). Secreted semaphorins repel and collapse axonal growth cones by binding to receptors composed of neuropilin (NP) and plexin complexes (Tamagnone and Comoglio, 2000) and activating intracellular pathways that control actin dynamics (Castellani and Rougon, 2002).

Several reports suggest that semaphorins and their receptors also regulate cell morphology and motility, and contribute to organogenesis in a variety of non neuronal tissues Brown et al., 2001, Loes et al., 2001, Bismuth and Boumsell, 2002, Fujii et al., 2002, Ginzburg et al., 2002, Kagoshima et al., 2001, Neufeld et al., 2002, Takashima et al., 2002. In addition, semaphorins have been implicated in tumorigenic processes. The human secreted semaphorins, SEMA3B and SEMA3F, were both isolated within a common 3p21 chromosomal region deleted in lung cancer Roche et al., 1996, Xiang et al., 1996, Sekido et al., 1996. Introduction of SEMA3B or SEMA3F in a lung cancer cell line expressing predominantly the NP1 receptor led to growth inhibition Tse et al., 2002, Xiang et al., 2002, Tomizawa et al., 2001, and SEMA3B was shown to induce apoptosis in this setting (Tomizawa et al., 2001). Although the functional properties of semaphorins responsible for these activities remain to be clarified, SEMA3F expression patterns and localization of the protein in cell protrusions are suggestive of a possible role in the control of adhesion or movement Brambilla et al., 2000, Nasarre et al., 2003. Such functions were also suggested by the observation, based on differential display, that metastatic lung adenocarcinoma cells overexpress H-SemaE, now called SEMA3C (Martin-Satue and Blanco, 1999). The recent finding that SEMA3B gene expression is inducible by p53 raises the possibility for the semaphorin proteins to influence cell growth associated with tumor progression (Ochi et al., 2002).

In mouse, Sema3F is expressed from early developmental stages in various neuronal and nonneuronal tissues Eckhardt and Meyerhans, 1998, Xiang et al., 1996. In the nervous system, Sema3F was shown to inhibit neurite outgrowth, to collapse several populations of axons Chédotal et al., 1998, de Castro et al., 1999, and to exert chemotactic attractive effect on oligodendrocyte precursor cell migration (Spassky et al., 2002).

Intringuingly, two alternative spliced forms of human and mouse SEMA3F mRNA have been identified Roche et al., 1996, Eckhardt and Meyerhans, 1998, one of which contains an extra exon encoding a 31-amino acid addition within the sema domain. No particular motif was found in these additional amino acids and its function, heretofore, has been largely uninvestigated except in a tumor suppression model where both spliced forms behaved similarly (Xiang et al., 2002). Several transcriptional variants exist for the class VI semaphorins, SEMA6C and SEMA6D, although their functional consequences are also unknown Correa et al., 2001, Qu et al., 2002. An interesting feature concerning the SEMA3F isoforms is that their ratio strongly differs in various lung tumors (Roche et al., 1996), suggesting functional or regulatory differences. In the present study, we investigated the biological properties of these SEMA3F isoforms using a hippocampal axon repelling test, and the morphology of endothelial cells. In addition, their expression pattern in the mouse developing central nervous system was studied.