Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development
Introduction
The cerebellum is a prominent part of the vertebrate hindbrain that coordinates posture, locomotion, and a wide range of routine and skilled motor activities. The cells of the cerebellum originate from two different progenitor zones (Altman and Bayer, 1997, Goldowitz and Hamre, 1998). The ventricular zone gives rise to Purkinje cells, Bergmann glia, interneurons, and the neurons of the deep nuclei. In contrast, the upper rhombic lip gives rise to granule cell precursors (GCPs) that migrate rostrally over the cerebellar anlage to form the external granule layer (EGL), which is a heavily proliferating progenitor zone that generates the entire population of cerebellar granule cells. Newborn postmitotic GCPs migrate for a short distance tangentially in the lower EGL, before turning radially inward to settle in the internal granule cell layer (IGL) (Komuro and Yacubova, 2003, Millen and Gleeson, 2008, Sillitoe and Joyner, 2007).
Semaphorins were originally identified as molecules that mediate axon repulsion in insects and vertebrates (Kolodkin et al., 1992, Luo et al., 1993). It was later discovered that Semaphorins are also important regulators of several other biological processes, such as dendrite formation, neural crest migration, vascular development, and activation of the immune response (for reviews, see (Suzuki et al., 2008, Tran et al., 2007, Yazdani and Terman, 2006)). Several Semaphorins have also been described to be involved in tumor formation, acting as regulators of angiogenesis, metastasis, or cell survival (Neufeld and Kessler, 2008).
The mammalian genome contains 20 Semaphorins, which are grouped into classes 3 through 7 (Semaphorin-Nomenclature-Committee, 1999). The main signaling receptors for Semaphorins are Plexins, which are grouped into classes A through D (Tamagnone et al., 1999). The protein structures of Semaphorins and Plexins are characterized by a common large extracellular domain, the “Sema” domain, which mediates binding between ligands and receptors (Gherardi et al., 2004, Janssen et al., 2010). Class 4 Semaphorins (Sema4s) are thought to be ligands for Plexin-B receptors, but apart from the binding of Sema4D to Plexin-B1 (Tamagnone et al., 1999) and of Sema4C to Plexin-B2 (Deng et al., 2007), specific ligand-receptor pairings between the six mammalian Sema4s (Sema4A-D, F, G) and the three Plexin-Bs (B1–B3) are only poorly understood.
The physiological function of Sema4 family members has been well studied for their role in the regulation of the immune response (Suzuki et al., 2008). In neural development, it has been shown that Sema4D plays a key role as a migration promoting factor for a class of hormone secreting neurons of the hypothalamus (Giacobini et al., 2008). A large body of data about Sema4 function has been gathered from cell culture based studies. For example, in cultures of cortical neurons, Sema4D activates Plexin-B1 to regulate the growth and branching of axons and dendrites (Oinuma et al., 2004, Swiercz et al., 2002, Uesugi et al., 2009, Vodrazka et al., 2009). In recent years, several additional molecules that are involved in Plexin-B signaling have been identified, but their in vivo relevance remains to be tested. For example, Plexin-Bs can form complexes with the receptor tyrosine kinases Met or ErbB2, which can determine the outcome of Plexin-B activation (Giordano et al., 2002, Swiercz et al., 2008). The downstream signaling of Plexin-Bs involves small GTPases of the Ras and Rho families that modulate cytoskeletal architecture and integrin adhesion (Zhou et al., 2008). Plexin-B1 and -B2 are widely expressed in the developing nervous system, while Plexin-B3 is restricted to postnatal oligodendrocytes (Perälä et al., 2005, Worzfeld et al., 2004). Targeted deletion of the Plexin-B1 and Plexin-B3 genes in mice resulted in non-detectable or only subtle phenotypes (Deng et al., 2007, Fazzari et al., 2007, Giacobini et al., 2008, Worzfeld et al., 2009). The knockout of Plexin-B2 leads to exencephaly and neonatal lethality on an inbred C57BL/6 genetic background (Deng et al., 2007, Friedel et al., 2007). On an outbred background, surviving Plexin-B2 mutants displayed a severe disruption of the cerebellar cortex that was caused by a disturbed migration of cerebellar granule cell precursors, while overall proliferation and apoptosis rates were largely unchanged (Friedel et al., 2007).
To further elucidate the function of the Plexin-B2 in cerebellar development, we have set out to identify its functional in vivo ligands. We have identified here Sema4C and Sema4G as candidate ligands for Plexin-B2 by expression and binding studies. Sema4C and Sema4G share a high degree of sequence homology with each other, and both genes are broadly expressed in the developing nervous system (Inagaki et al., 1995, Li et al., 1999). Interestingly, Sema4C and Plexin-B2 show also largely complementary expression patterns in the adult vascular and endocrine systems (Zielonka et al., 2010). Sema4C has been previously shown to bind to Plexin-B2 in cell culture and to promote migration of granule cells (Deng et al., 2007). However, little is known otherwise about the biological function of these genes in neural development. We have generated mouse mutants for the Sema4C and Sema4G genes, and we provide evidence, based on phenotypic analysis, genetic interaction studies, and migration assays with cerebellar explant cultures, that Sema4C and Sema4G act as ligands of Plexin-B2 to regulate the development of cerebellar granule cells.
Section snippets
Expression of Sema4 and Plexin-B genes in the developing cerebellum
To identify candidate ligands for Plexin-B2 in cerebellar development, we examined the expression of Sema4 genes in the developing cerebellum at postnatal day P10, during the peak period of cerebellar granule cell neurogenesis (Mares et al., 1970) (Fig. 1A). Expression patterns were correlated to cerebellar cell types by comparison with sections that were co-labeled for specific marker proteins (Suppl. Fig. 1). Of the six murine Sema4 genes, Sema4a, Sema4b, and Sema4d are mainly expressed in
Discussion
Dissecting the Plexin–Semaphorin signaling network is important in understanding how the nervous system is built up. Here we provide evidence that Sema4C and Sema4G are functional in vivo ligands for Plexin-B2 regulating the migratory properties of cerebellar granule cells during development.
Mouse genetics
The Sema4C gene was mutated by the targeted trapping method (Friedel et al., 2005). A targeting vector was constructed by flanking a secretory trap cassette (Leighton et al., 2001) with 5′ and 3′ homology arms of 5 kb and 3 kb size, respectively. Correct homologous recombination in E14Tg2a mouse embryonic stem cells resulted in the insertion of the trap cassette between exons 12 and 13 of the Sema4C gene (exon 1 counted as the exon containing the start codon), which is upstream of the exons
Acknowledgments
This work was supported by the NIH grants RO1MH60612 and U01HL66600 and by the European Union FP6 EUCOMM program. We thank S. Strittmatter, Yale University, for a Sema4C-AP expression plasmid, and L. Tamagnone, University of Turin, for a Plexin-B1 expression plasmid. We thank A. Chedotal, University Paris, M. Hatten, Rockefeller University, and A. Huber, C. Haupt, A. Kurz-Drexler, and R. Koester, Helmholtz Center Munich, for protocols and advice.
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2018, Molecular and Cellular NeuroscienceCitation Excerpt :Second, we determined that the percentage of total DAPI+ cells labeled as inhibitory interneurons, excitatory neurons, and astrocytes are in agreement with published studies (Fig. S1A) (Bekkers, 2011; Chamberland and Topolnik, 2012; Degen et al., 2012; Seifert and Steinhauser, 2017). Third, we confirmed that the signal obtained from the Plxnb1, Plxnb2, Sema4a, and Sema4d probes in cerebellum was in agreement with published studies describing the expression of these genes in that brain region ((Fig. S2) (Maier et al., 2011)). Coronal sections of postnatal day 14 (P14) wildtype mouse brains containing the hippocampus were probed for a gene of interest and a cell type specific marker simultaneously (e.g. Plxnb1 and Aldh1l1; Fig. 1).
- 1
Present address: Institut de Recherches Cliniques de Montréal (IRCM), Cellular Neurobiology Research Unit, Québec, Canada H2W 1R7.
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Present address: Department of Neurosurgery, Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.