Both Schwann cell and axonal defects cause motor peripheral neuropathy in Ebf2−/− mice
Research Highlights
► Ebf2 is expressed in myelin forming Schwann cells. ► Ebf2−/− mice present a delay in myelination at P2. ► Ebf2−/− mice present morphological abnormalities in Schwann cells and axons at P30. ► Ebf2−/− mice motor system is more affected than the sensory one. ► Ebf2 is an interesting candidate gene for human hereditary peripheral neuropathies.
Introduction
The hereditary motor and sensory neuropathies also defined as Charcot–Marie–Tooth (CMT) disease are a heterogeneous group of genetic disorders affecting the peripheral nervous system (PNS), that are traditionally subdivided into demyelinating and axonal forms. More than 25 genes responsible for inherited neuropathies have been identified in humans, but most cases still await a molecular definition (Nicholson, 2006, Pareyson et al., 2009, Scherer and Wrabetz, 2008, Suter and Scherer, 2003). Several mutations have been identified in structural components of myelin such as Peripheral myelin protein 22 (Pmp22) and Myelin protein zero (Mpz). Moreover, different classes of proteins have been implicated in CMT such as transcription factors, mitochondrial proteins and molecules regulating RNA processing or endosomal trafficking in Schwann cells (SC) (Inoue et al., 2004, Waxman, 1980). Depending on the affected gene and the type of mutation, functional deficits in either SC or neurons, or both, lead either directly or via disturbed axon–SC interactions to axonal atrophy and degeneration (Berger et al., 2006).
SC ensheath and myelinate large calibre axons in the PNS, greatly increasing conduction velocity of action potentials. During PNS development, bundles of axons innervating their motor or sensory targets become surrounded by immature SC. Soon after birth, immature SC differentiate into two different types. SC that are in contact with large calibre axons differentiate into a promyelinating phenotype (1:1 SC to axon relationship), by segregating the associated axon to the periphery of the bundle in a process known as ‘radial sorting’(Court et al., 2006). These cells are the myelin forming SC (MFSC) and start myelinating the axon. A second group of SC, the non-myelin forming SC (NMFSC), associate with several small calibre axons (< 1 μm), which will remain unmyelinated, forming the so-called Remak bundles (Mirsky et al., 2008). Moreover, during development, SC regulate the formation of axonal functional subdomains such as the nodes of Ranvier by controlling Na+ channel (NAV) clusterization and the internodes by regulating their own elongation along the axons (Feinberg et al., 2010, Court et al., 2009).We have previously shown that mice carrying a null mutation in EBF2 transcription factor present, among other phenotypic features, peripheral nerve abnormalities. EBF2 belongs to a class of transcription factors, including four members, highly conserved in evolution and largely expressed in the nervous system during development (Daburon et al., 2008). They are characterized by a non basic Helix Loop Helix dimerization domain and a Zinc finger DNA binding domain. They are implicated in maturation of various cell types with partially overlapping roles, although specific functions have also been discovered for each member of the family. Several studies performed in various species such as Caenorhabditis elegans (Kim et al., 2005, Prasad et al., 1998, Prasad et al., 2008), Drosophila melanogaster (Crozatier et al., 1999, Dubois and Vincent, 2001), Xenopus laevis (Dubois et al., 1998, Pozzoli et al., 2001) and mouse (Garel et al., 1997, Malgaretti et al., 1997, Wang et al., 1997), suggest that Ebf genes are implicated in different steps and pathways of neuronal differentiation, such as neuronal migration (Corradi et al., 2003, Croci et al., 2006, Wang et al., 2004), fasciculation and pathfinding (Prasad et al., 1998). Importantly, motorneuron (MN) development is altered in C. elegans unc-3 mutant in which the nematode ortholog of vertebrate Ebf genes is mutated (Prasad et al., 2008). Several hints on Ebf2 function emerged from the study of the Ebf2−/− mouse phenotype (Corradi et al., 2003, Croci et al., 2006). Although the Ebf2 gene is not essential for completion of embryogenesis, the mutant mice exhibit a combination of neuroendocrine and cerebellar abnormalities due to defective neuronal migration (Corradi et al., 2003, Croci et al., 2006), accompanied by peripheral nerve defects, caused by the expression of Ebf2 in SC precursors and postnatal NMFSC.
In view of the possibility that Ebf2 represents a new molecule implicated in peripheral nerve function and a potential new candidate gene for inherited peripheral nerve disorders, we analyzed the postnatal expression of Ebf2 in peripheral nerves and characterized in detail the peripheral neuropathy affecting the Ebf2−/− mice. Here, we found that the gene is expressed not only in NMFSC as previously known, but also in MFSC and perineurial cells throughout postnatal development. As a consequence, we observed that the process of myelination is delayed after birth. Later in development, myelination appears less affected; however, the deletion of the gene causes several morphological defects such as shortening of the internodal length and lack of larger calibre axons, which leads to a functional impairment of the peripheral motor system. Interestingly, a child with delayed mental and motor development associated with a peripheral neuropathy has been described to be associated with an interstitial deletion on the short arm of chromosome 8, including the EBF2 human locus (Klopocki et al., 2006).
These results provide a detailed characterization of the neuropathological phenotype of Ebf2−/−mutants, and indicate Ebf2 gene as a new potential candidate for CMT disease.
Section snippets
Mouse genetics
The targeting construct, described in Corradi et al. (2003), contains a lacZ cDNA. All experiments were carried out on F1 hybrids obtained by crossing Ebf2+/− pure-bred FVB/N females with Ebf2+/− pure-bred C57BL/6 males. All studies were conducted using coisogenic control littermates. This hybrid strain was chosen to obviate the low fertility and poor maternal behaviour of C57BL/6J heterozygous mothers (Corradi et al., 2003). Mice were housed in groups under constant temperature and humidity
Ebf2 is expressed in postnatal MFSC
To characterize Ebf2 expression during postnatal peripheral nerve development, we performed Bluo-Gal staining on Ebf2+/− nerves, taking advantage of the beta galactosidase gene knocked into exon 1 of the Ebf2 genomic locus (Corradi et al., 2003). At two days after birth (P2), SC are completing the sorting of axons (pro-myelinating SC) and begin to acquire their mature phenotype starting the formation of myelin. We found Ebf2 gene expression (Fig. 1, dark signals) both in pro-myelinating SC (
Discussion
Over the last years several mutations in a variety of genes have been identified that lead to different forms of CMT disease. However, research focused on the identification of new genes responsible of this disease is very active to find genetic causes to the numerous cases that still remain without a molecular definition.
In the previous paper describing different features of Ebf2−/− mice phenotype, we showed that Ebf2 is expressed postnatally in NMFSC and that Ebf2−/− mice present a decreased
List of abbreviations
- CASPR
Contactin associated protein 1
- CMT
Charcot–Marie–Tooth
- IL
internodal length
- MFSC
myeling forming Schwann cells
- MN
motorneurons
- MNCV
motor nerve conduction velocity
- Mpz
Myelin protein zero
- NMFSC
non-myeling forming Schwann cells
- NCV
nerve conduction velocity
- MNCV
motor nerve conduction velocity
- Pmp22
Peripheral myelin protein 22
- PNS
peripheral nervous system
- SC
Schwann cell
- SNCV
sensory nerve conduction velocity
- TEM
transmission electron microscopy
- WT
wild type
The following are the supplementary materials related to
Acknowledgments
The authors wish to thank Dr. Laura Croci (San Raffaele Scientific Institute Milano, Italy) and Michèle Carnaud (Inserm UMR-S 839, Paris, France) for technical help and suggestions; Dr. Lawrence Wrabetz (HSR, Milano Italy) and Dr. Maria Pennuto (IIT, Genova Italy) for critical reading of this manuscript. This work was supported by Compagnia di San Paolo, Torino (to A.C. and F.B.) and by the Italian Ministry of Research (to F.B). The support of Telethon-Italy (GGP09134 to F.B) is also
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These two authors contributed equally to this work.