In the frog Xenopus laevis, gamma-aminobutyric acid (GABA)-immunoreactive spinal cord neurons (Kolmer-Agduhr cells) formed a dispersed pattern within two columns on either side of the midline. The cellular pattern became established during embryonic and larval development. The GABA-immunoreactive cells are cerebrospinal fluid (CSF)-contacting neurons that began to appear by 1.2 days (st 26) of development. This stage occurred shortly after neural tube closure (0.9 days, st 21) and followed the appearance of ultrastructural characteristics of CSF-contacting neurons. The pattern of GABA-immunoreactive cells emerged during embryogenesis, as their density increased. Each longitudinal column was heterogeneous, containing cells with and without GABA immunoreactivity. Spatial analysis at several embryonic and larval stages showed that the cells in each column formed a nonrandom, dispersed pattern even at early stages of differentiation. This one-dimensional pattern resembled that of dopamine-immunoreactive neurons, which are also located in the ventral spinal cord. The patterning of both cell types followed a different time course, but the ultimate spacing of the neurons remained comparable. These results suggested that the mechanism patterning the two cell types within the same region was similar but not identical and may involve related molecular mechanisms.

Dopaminergic spinal cord neurons differentiate in the ventral spinal cord in a nonrandom dispersed pattern. To test whether Notch signaling was involved in generating the pattern of this neuron population as with others, we overexpressed a constitutively active form of Xenopus Notch (XotchΔE) in developing frog embryos. Overexpression was targeted to half the spinal cord by injecting activated Notch RNA into one blastomere at the two-cell stage. Injected animals showed morphological differences on the injected side including reduced numbers of dopaminergic spinal cord neurons. This is consistent with a role for Notch signaling in establishing the fate of this population in the developing spinal cord. At a later stage of development, dopaminergic neurons continued to differentiate on both sides of the spinal cord, but the difference between experimental and control columns remained constant. This is consistent with transient activation of Notch disrupting the fate of the earliest (primary) but not later (secondary) dopaminergic neurons. The precursors to secondary neurons appear to be refractory to Notch signaling at earlier stages of development.