Antibody staining: CEH-17 expression was strong at the 3-fold to L1 stage and decreased thereafter to reach barely detectable levels in adults. At the L1 stage, CEH-17 was detected exclusively in the nuclei of five neurons of the ring ganglia. One of the five CEH-17+ neurons was dorsal and just posterior to the nerve ring, while the four others were ventral and close to the posterior bulb of the pharynx.
pceh-17::GFP show that the dorsal neuron could be seen projecting two processes towards the ring, which then turned back and coursed along the lateral cords, all the way to the tail of the animal, identifying this neuron as ALA. The four ventral CEH-17 positive neurons were located just posterior to the SMBs, as was described for the SIAs. The four ventral
ceh-17 expressing neurons are believed to be the SIA neurons (SIADR, SIADL, SIAVR and SIAVL). In addition to ALA and the four SIAs, a few other neurons also expressed the
pceh-17::GFP construct. flceh-17::GFP staining resulted in the same expression pattern.
ceh-17 HD::GFP showed effectively restricted GFP expression to the five neurons seen with the antibody (i.e. ALA and the four SIAs). The fluorescence had a granular appearance and was restricted to the cell body, possibly due to trapping of the chimeric protein in the endoplasmic reticulum.
pceh-17::GFP::3 resulted in the same expression pattern (i.e. ALA and the four SIAs), but with axonal staining. The latter construct (
pceh-17::GFP::3 ) was used to examine embryonic expression of
ceh-17. At the comma stage, expression was detected in two dorsal neurons in the head, presumably corresponding to ALA and its sister RMED, and in six cells on the ventral side of the head, just anterior to the excretory pore. These cells probably correspond to the four SIAs, in which the GFP signal was strongest and revealed sprouting axons, and the two SIBVs, sisters of the SIADs. Two other weakly positive cells on the ventral side were detected, one in the mid-body region and one in the tail, at positions fitting those of motor neurons DB5 and DA8, sisters of the SIAVs. The earliest time at which could detect
ceh-17::GFP expression was at the end of gastrulation in four pairs of cells, which could correspond to the four SIAs and their sisters or to the four SIA mothers and their own sisters. Since this is the time when the mothers of SIAs are undergoing their last division, it is not clear whether
ceh-17 expression precedes or follows the last cell division.