, 2010; Eto et al., 2010). We have shown that the C-terminal aa 856–881 domain of DLK-1L can bind to the kinase domain of both human MAP3K13 and C. elegans DLK-1. Expression of human MAP3K13 in C. elegans neurons can functionally complement dlk-1. The DLK-1L hexapeptide is completely conserved in MAP3K13 but not in MAP3K12. Short isoforms for MAP3K12 are detected as ESTs; MAP3K12 and MAP3K13 can form homomers or heteromers ( Ikeda et al.,

2001; Nihalani et al., 2000). Thus far, most reported studies have focused on the MAP3K12/DLK, and different types of neurons lacking DLK show a range of phenotypes from neurite regeneration to axon degeneration and to neuronal death ( Ghosh et al., 2011; Itoh et al., 2009, 2011; Miller et al., 2009). At present, much less is known about MAP3K13/LZK, ISRIB clinical trial although it has been implicated in neurite outgrowth and may interact with the regrowth inhibitor Nogo ( Dickson et al., 2010). Our discovery of antagonistic C. elegans DLK-1

isoforms, together with our demonstration of functional conservation of DLK-1L and MAP3K13/LZK, suggest that the activation mechanisms of DLK family MAPKKKs in neurons may be conserved. Specifically, we speculate that MAP3K13 could also be kept in an inhibited state by an endogenous inhibitory isoform. As MAP3K12 and MAP3K13 are almost identical buy Screening Library in their kinase and LZ domains, MAP3K12 isoforms could provide this inhibitory function. Clearly, it will be informative to assess the role of MAP3K13 in synaptic development and axon regrowth and its possible crosstalk with MAP3K12. We maintained C. elegans strains on NGM plates at 20°C–22.5°C as described by Brenner Terminal deoxynucleotidyl transferase (1974). The dlk-1 mutations are listed in Table S1; mutations affecting the kinase domain are shown in Figure S1B. We used juIs1[Punc-25-SNB-1::GFP] for viewing GABA motor neuron synapses ( Hallam and Jin, 1998), and muIs32[Pmec-7-GFP] ( Ch’ng et al., 2003) for viewing touch

neuron morphology and axon regeneration studies. Other transgenes and strains are described in Table S2. We scored fluorescent reporters in live animals using a Zeiss Axioplan 2 microscope equipped with Chroma HQ filters. For quantification of touch neuron morphology using muIs32, 100–150 1-day-old adults were analyzed. For quantification of GABAergic motor neuron synapse morphology using juIs1, confocal images of dorsal cords in the midbody were collected on 1-day-old adults immobilized in 1% 1-phenoxy-2-propanol (TCI America) in M9 buffer. For GFP-DLK-1L, GFP-DLK-1S, and CFP-DLK-1L/YFP-DLK-1S, images were collected from 1-day-old adults using a Zeiss LSM510 confocal microscope. For synapse morphology and DLK-1L/S localization, z stack images (1 μm/section) were shown in the figures. We cut PLM axons in anesthetized L4 larvae using a near-infrared Ti-Sapphire laser (KMLabs) as described (Wu et al., 2007).