5. Following this stage, a number of neuronal defects are evident, which are consistent with the loss of SCPs in this model, namely, defasciculation, the overt loss of all peripheral projections, and

sensory neuron death. The requirement of SCPs for ERK1/2 and the potential for complex neuron-glial interactions in the context of neural crest Erk1/2 deletion, limited our analysis of neuronal roles for ERK1/2 signaling. To better understand neuronal ERK1/2, we employed two additional Cre lines. Nestin:Cre induces recombination in progenitors throughout the CNS and leads to gene deletion Lumacaftor datasheet in both neuronal and glial populations. However, recombination in the DRG occurs beginning at ∼E10.5 resulting in gene deletion in most DRG neurons, but not in Schwann cells ( Kao et al., 2009, Tronche et al., 1999 and Zhong et al., 2007). The Advillin:Cre line induces recombination in virtually all DRG and trigeminal ganglion neurons beginning at ∼E12.5 and is almost exclusive for these populations ( Hasegawa et al., 2007). Mek1/2CKO(Nes) mice die shortly after birth and major reductions in MEK1/2 expression and ERK1/2 activation were noted in the Mek1/2CKO(Nes) DRG by E14 ( Figure S4A).

Whole-mount neurofilament labeling at mid-embryonic stages revealed a normal pattern of early peripheral nerve development in the absence VX-770 in vivo of Mek1/2 ( Figures 4A and 4B). DRG morphology is grossly normal at birth and the expression of nociceptive markers, P2X3 and TrkA, and the proprioceptive marker, Parvalbumin, are relatively unchanged ( Figures 4C and 4D and data not shown). In the target field, the Protein-histidine tele-kinase main nerve trunks of P0 Mek1/2CKO(Nes) peripheral nerves were relatively normal in size; however, we noted a reduction in the innervation of the subepidermal plexus and the number of cutaneous fibers entering the epidermal field ( Figures 4E–4H). These data show that the early loss

of ERK1/2 signaling in DRG neurons does not modify initial stages of axon outgrowth, but inhibits axon innervation of the cutaneous fields by birth. Erk1/2CKO(Advillin) mice are indistinguishable from controls in the days following birth. However, by the end of the first postnatal week, mutant mice are noticeably smaller and the mice do not survive past three weeks of age. Importantly, the number of fibers innervating the epidermis in P3 Erk1/2CKO(Advillin) hindlimbs was significantly decreased relative to controls ( Figures 4I, 4J, and 4O). At this time point, a relatively normal number of DRG neurons were present, which exhibit a typical pattern of TrkA and CGRP expression and complete loss of ERK2 expression ( Figures 4M and 4N and data not shown). CGRP labeled central afferents also appeared intact in the dorsal spinal cord of mutant mice ( Figures 4K and 4L). In P18 mutants, DRG neuron number was 41.5% ± 3.0% (n = 2) of littermate controls.