Protein phosphatases regulate the growth of developing neurites.

The mechanisms underlying morphogenesis of axons and dendrites are critical for understanding both the structure and function of the nervous system. Since a number of kinases have a well-known effect on neurite outgrowth, we tested the hypothesis that specific phosphatases can also play a role in neurite extension and branching. Both protein phosphatase 1 (PP1) and 2A (PP2A) are present in growing processes and can regulate neuronal outgrowth. Loss-, gain- and recovery-of-function analyses in cultured hippocampal neurons tested the role of PP1 and PP2A in neurite growth. siRNA partially knocked down specific phosphatase isoforms and showed that reducing PP2A increased neurite length. Broad spectrum pharmacologic inhibition of PP1 caused the opposite effect from RNAi of specific phosphatases, indicating that two phosphatase pathways likely affect neurite morphogenesis. Over-expression of PP2A resulted in shorter neurites and decreased dendritic branching. Rescue analysis showed that PP2A homologs could restore the longer neurites caused by RNAi, to their normal size, indicating that both reagents target the same pathway. Thus, the well-known effects of specific kinases can be countered by the activity of phosphatases at different times and locations in the growing neurite. By working together, kinases and phosphatases can play a dynamic role in regulating neurite extension during development.

Activated notch disrupts the initial patterning of dopaminergic spinal cord neurons.

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 (XotchDeltaE) 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.

Establishment of a ventral cell fate in the spinal cord.

The neural plate is induced during gastrulation when the organizer affects the ectoderm around it. Recent experiments show that axial mesoderm can stimulate formation of specific ventral cell types in the spinal cord, including floor plate, motor neurons, and several types of interneurons. We have eliminated or disrupted axial mesoderm by using a variety of methods to show that ventral columns of intermittent dopaminergic neurons in the frog Xenopus also appear to be induced by axial mesoderm. Inversion of the dorsal-ventral neural axis by splitting the presumptive neural plate in vivo, produced two spinal cords with ectopic dopaminergic neurons. The location and number of neurons suggest that even a brief association with axial mesoderm can specify the identity of the first or primary dopaminergic neurons and that notochord retains the ability to induce cells to become secondary dopaminergic neurons.