After differentiation, Notch signaling inhibits neurite extension in cultured vertebrate neurons and in the

neonatal mouse cortex (Berezovska et al., 1999, Franklin et al., 1999, Redmond et al., 2000 and Sestan et al., 1999) and modulates axon guidance in Drosophila ( Crowner et al., 2003). Our results demonstrate that Notch’s function in regulating the growth potential of neurons is not limited to development. Rather, Notch signaling can function long after development is complete and can act after nerve injury to suppress axon regeneration. Animals were maintained on nematode growth medium agar plates with E. coli OP50 as a source of food ( Stiernagle, 2006). Temperature was controlled at 20°C unless otherwise stated. Null mutations in lin-12 result in sterility, 3-Methyladenine cost so we characterized homozygous mutant progeny that segregated from a balanced heterozygous Afatinib order strain. Maternal contributions of wild-type Notch/lin-12 allow these mutants to survive and develop into viable adults. Many of these adults rupture from their vulva; we used only normally sized, healthy animals in these experiments. Strain names, genotypes, and complete data with p values can be found in Tables S1–S3. All experiments were performed in parallel with a matched control. L4-stage hermaphrodites were mounted in a slurry of 0.1 μm diameter polystyrene beads (Polysciences) or in 50 mM of the GABA

SB-3CT agonist, muscimol, (Sigma M1523) to immobilize the animals. No difference in regeneration rates was observed between beads and muscimol: wild-type animals regenerated at a similar rate under both conditions, and Notch signaling mutants had increased regeneration under both conditions (data not shown). Commissures in the tail region of the animal posterior to the vulva were severed (GABA neurons: VD and DD; acetylcholine neurons: AS and DB). Commissures were visualized with a Nikon Eclipse 80i microscope using a 100× Plan ApoVC lens (1.4 NA) and a Hamamatsu Orca camera. Selected axons were cut

using a Micropoint laser from Photonic Instruments (10 pulses, 20 Hz). Axotomized animals were recovered to agar plates and remounted 18–24 hr later for scoring. At least 30 axons were scored for most genotypes (2–3 cut axons per animal); see Tables S1–S3. Only axons with a distal stump as evidence of a complete cut were scored. Axons with a visible growth cone that had progressed past the cut site, and axons that had regenerated to the dorsal nerve cord, were scored as positive. Axons with no growth or with only filopodial extensions and no progression past the cut site were counted as negative. When scoring full regeneration, only axons that showed visual evidence of reconnection to the dorsal cord 24 hr after axotomy were scored as positive. For growth cone initiation at 4 and 6 hr, axons with a growth cone were scored as positive.