30 years after John White et al's EM reconstruction of the C. elegans nervous system, there is still a lack of understanding as to how the neurites actually fasciculate and extend into their respective subtracts during embryogenesis (particularly in the formation of the nerve ring). Here we use high resolution, dual-color fluorescence imaging in live embryos to map the relative spatiotemporal behavior of neurites between and within subtracts as they form the initial ring structure. Cell lineaging with StarryNite and AceTree software enables the identification of each neuron. We have identified 6 spatially distinguishable groups of neurites with stereotypical positions in the nerve ring by the onset of muscular movement, which cover about 30 neurons. The rough order of these groups from anterior to posterior is: 1. RMED, 2. RIH, RIP and RMDD, 3. three sublateral neurons (SMDD, SIAD, SIBV) and three lateral neurons (SAAV, SMDV, and RIV), 4. AVJ and AVD, 5. unknown neurites labeled by a
cnd-1 marker, and 6. ALA. The timing of the initial outgrowth shows a ventral-to-dorsal progression, with ventral (RIH, RMDD) and ventral-lateral (SMDD, SIAD, SIBV) neurons being the earliest, followed by the lateral neurons (SAAV, SMDV, RIV and RIP), the dorsal-lateral neurons (AVJ, AVD), and finally ALA and RMED at the dorsal midline. We infer pioneer-follower relationships or the lack there-of from the (in)consistency of temporal orders among embryos as well as from laser ablation experiments. Overall, the initial neurite extension into the ring begins at late bean stage, about 15 min after the neurons are born. Based a pan-neuronal PH::GFP marker, the ring closes at the dorsal apex at late comma stage, and the overall width grows to 0.75-0.8 microns by the onset of muscular movement (~1.5 hours after the initial extensions). The sum of the 6 neurite groups described above account for 1/2 to 2/3 of the overall size of the ring. Based on this measurement as well as other imaged neurons that do not enter the ring until later, we estimate that about half of the total 140 embryonic nerve ring neurons enter the ring by the onset of muscle movement, and that the 6 distinguishable groups account for half of these.