Initial studies this website using shRNA knockdown of NF186 in neurons in vitro suggested that NF186 coordinates nodal formation and Nav channel accumulation in PNS nodes independently of paranodes, but in an extrinsic manner, implying that external cues are required for node formation (Dzhashiashvili et al., 2007). Other studies performed in transgenic mice expressing NfascNF186 in an Nfasc−/− mutant background revealed that NF186 can facilitate nodal organization independently of paranodes, in both the CNS and PNS ( Zonta et al., 2008). Interestingly, when NfascNF155 was transgenically re-expressed in myelinating glia in

the Nfasc−/− mutant background (mimicking NF186 loss), clustering of Nav channels was observed at the CNS, but not the PNS, nodes ( Zonta et al., 2008). This data suggested

that paranodal PLX4032 research buy domains may suffice in organizing nodes in the absence of NF186 in the CNS. In contrast, in vitro studies utilizing Schwann cells (SCs) and neurons isolated from wild-type and Nfasc−/− mice, respectively, suggested that the paranodal domains were responsible for Nav channel enrichment at mature nodes in the PNS, regardless of NF186 expression ( Feinberg et al., 2010). These conflicting observations have further complicated our understanding of the precise role of NF186 in nodal development and the mechanisms that regulate node formation. Here, using an in vivo genetic ablation approach, we demonstrate that NF186 diglyceride is required for proper nodal organization and function independent of paranodes, and that paranodal domains are not sufficient for nodal coordination in the CNS or the PNS in vivo. Furthermore, in the absence of intact nodes of Ranvier, flanking paranodal domains invade the nodal space, indicating that NF186 plays a vital role in the organization and demarcation of nodes of Ranvier in myelinated axons. To specifically

ablate NfascNF186 from neurons, NfascFlox mice ( Pillai et al., 2009) were crossed to mice expressing Cre recombinase (Cre) under the neuron-specific promoter Neurofilament light chain (Nefl-Cre) ( Leconte et al., 1994); Schweizer et al., 2002). When Cre is expressed, the loxP sites flanking exon 2 of Nfasc are excised, thereby causing a frameshift that results in a premature stop codon in exon 4 (red asterisks in Figure 1A; Figure S1A, available online). PCR amplification of genomic tail DNA was used to identify Nfasc wild-type (+/+), heterozygous (+/Flox), and homozygous floxed (Flox) alleles, as well as Cre ( Figure 1B). To test the efficacy of Nefl-Cre excision of Nfasc during myelination, genomic DNA was isolated from P0, P3, P6, P11, P16, and P19 wild-type (Nefl-Cre;Nfasc+/+) and Nefl-Cre;NfascFlox spinal cords ( Figure 1C). PCR analysis using primers specifically recognizing the Nfasc ablation product (Null) showed recombination of the NfascFlox gene at P0 (birth), indicating early expression of Cre by the Nefl promoter.