Abstract
BackgroundDendrites differ from axons in patterns of growth and development, as well as in morphology. Given that microtubules are key structural elements in cells, we assessed patterns of microtubule stability and polymerization during hippocampal neuron development in vitro to determine if these aspects of microtubule organization could distinguish axons from dendrites.ResultsQuantitative ratiometric immunocytochemistry identified significant differences in microtubule stability between axons and dendrites. Most notably, regardless of developmental stage, there were high levels of dynamic microtubules throughout the dendritic arbor, whereas dynamic microtubules were predominantly concentrated in the distal end of axons. Analysis of microtubule polymerization using green fluorescent protein-tagged EB1 showed both developmental and regional differences in microtubule polymerization between axons and dendrites. Early in development (for example, 1 to 2 days in vitro), polymerization events were distributed equally in both the anterograde and retrograde directions throughout the length of both axons and dendrites. As development progressed, however, polymerization became biased, with a greater number of polymerization events in distal than in proximal and middle regions. While polymerization occurred almost exclusively in the anterograde direction for axons, both anterograde and retrograde polymerization was observed in dendrites. This is in agreement with predicted differences in microtubule polarity within these compartments, although fewer retrograde events were observed in dendrites than expected.ConclusionBoth immunocytochemical and live imaging analyses showed that newly formed microtubules predominated at the distal end of axons and dendrites, suggesting a common mechanism that incorporates increased microtubule polymerization at growing process tips. Dendrites had more immature, dynamic microtubules throughout the entire arbor than did axons, however. Identifying these differences in microtubule stability and polymerization is a necessary first step toward understanding how they are developmentally regulated, and may reveal novel mechanisms underlying neuron maturation and dendritic plasticity that extend beyond the initial specification of polarity.
Highlights
Dendrites differ from axons in patterns of growth and development, as well as in morphology
Tyrosinated microtubules are abundant in distal ends of axons and tips of developing dendrites To assess spatial and temporal patterns of microtubule stability in axons and dendrites of developing hippocampal neurons, the distribution of dynamic and stable microtubules was determined using antibodies to tyrosinated (Tyr) and acetylated (Ac) tubulin, respectively
Key stages of dendritic development chosen for quantitative analysis were, first, minor process growth following axon specification (2 days in vitro (DIV); Figure 2A–C), and second, differentiation of the dendritic arbor characterized by the development of taper and formation of branches (4 and 7 DIV; Figure 2D–F and 2G–I, respectively)
Summary
Dendrites differ from axons in patterns of growth and development, as well as in morphology. For hippocampal neurons developing in vitro, polarization occurs as structurally equivalent immature minor processes differentiate into mature axonal and dendritic arbors [1,2]. During this developmental progression, dendrites diverge from axons through a stereotypic sequence of morphogenesis, suggesting that at some level the cytoskeleton is being organized differently [3]. It may be that differences in local regulation of microtubule polymerization or stability underlie the faster rate of growth observed in axons [2,9,10], as well as contribute to the delayed formation and maturation of the dendritic arbor [3,11,12]. Few studies have tested whether dendritic growth depends on tubulin subunit addition to the same extent, but overexpression of cypin, a protein that promotes microtubule polymerization, has been shown to increase the size of the dendritic arbor [22,23]
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