Development of mouse spinal cord in tissue culture-III. Quantification of neuron development in neural tube...

Mouse neural tube development in vitro was examined following the isolation and culturing of specific regions of the neural tube. Developmental characteristics of neuron formation and differentiation were assessed both quantitatively and qualitatively. A 1 mm length of embryonic day 10 mouse neural tube was cut into 32 microfragments of equal size and cultured on collagen coated cover-slips. Neuronal cells were observed to emerge after 3 days in culture and to migrate away from the fragment upon an immature astroglial precursor cell layer that had begun to form within the first 24 h of culturing. The extent of neuronal migration, the density and total number of neurons per outgrowth zone, and the size distribution of neurons was quantitated after 21 days in culture. Three distinct patterns of neuronal outgrowth (Types II, III and IV) could be observed with a fourth pattern (Type I) best described as being neuron free. Neuron free outgrowth zones (Type 1) were comprized totally of glial cells and represented approximately 10% of the outgrowth zones examined. Characteristics of Type II outgrowth zones were a relatively restricted neuronal migration and a predominance (85%) of small neurons (< 10μm diameter) scattered around the outgrowth zone. In contrast the area of Type III outgrowth zones was twice that of the Type II, with a neuronal density 2.5 times greater, composed of a near equal proportion of small to medium sized (10-18 μm) neurons. The neuronal density and extent of migration of Type IV zones was similar to that of Type II zones; however the neurons of this fragment type were primarily medium and large sized neurons. In a second experiment the brachial region of E10 neural tube was sectioned into dorsal and ventral halves and microfragments from each half were cultured separately. Dorsal fragments produced primarily Type I and Type II outgrowth zones, and never a Type IV. In contrast, ventral fragments produced primarily Type III and Type IV zones with a small proportion of Type II zones. The results indicate that neuronal precursor cells in vitro maintain an adherence to patterns of neurogenesis programmed in vivo during early stages of neural tube development. The present culture system thus provides a means for the study of the expression in vitro of specific patterns of neuronal development and of the possible environmental influences (epigenetic factors) upon these patterns.