Figure 1. A Gallery Representing Discoveries of Developmental Dynamics that Were Possible only because of Intravital Timelapse Imaging Methods.
(Drawn from work in the authors' laboratories.)
(A) Dynamics of growing retinal ganglion cell axon in larval zebrafish optic tectum (Meyer and Smith, 2006).
(A1) Construction of the axon arbor involves extensive and contemporaneous retraction and elimination of newly formed branches, while formation of presynaptic active zones appears to promote branch stabilization. Moreover, newly formed synaptic puncta are preferential sites of new branch formation (arrowheads). Two-photon fluorescence images were acquired at 10 min intervals. Soluble DsRed fluorescence (red) marks axonal cytoplasm; A synaptophysin:GFP fusion protein (yellow puncta) marks cites of putative nascent presynaptic active zones. Scale bar: 10 μm.
(A2) Histogram representing observed lifetimes of newly formed branches. Most nascent axonal arbor branches have short lifetimes of less than 1 hr.
(A3) Histogram representing observed lifetimes of synaptophysin-gfp puncta. Most newly formed puncta are short lived.
(B) Dynamics of growing tectal neuron dendrite in larval zebrafish optic tectum (Niell et al., 2004).
(B1) Dendritic arbor growth occurs contemporaneously with synapse formation. Two-photon fluorescence images of the same tectal neuron were acquired at intervals as indicated in dpf (days post-fertilization). Soluble DsRed fluorescence (red) marks dendritic cytoplasm; A PSD-95:GFP fusion protein (green puncta) marks cites of putative nascent postsynaptic active zones. Scale bar: 10 μm.
(B2) Construction of a dendritic arbor involves extensive and contemporaneous retraction and elimination of newly formed branches, while formation of postsynaptic active zones appears to promote branch stabilization, often with an arrest of branch retraction at the exact site of a synaptic punctum (arrow). Time points indicated in minutes; scale bar: 3 μm.
(B3) Parallel time courses of dendritic arbor growth and synapse formation are consistent with a “synaptotrophic” model of dendrite growth, where the formation of synaptic puncta plays a causal role in the stabilization of newly formed branches. Quantitation from images similar to (B1) (six cells).
(C) Dramatic reversals in nerve terminal area during synapse elimination (Walsh and Lichtman, 2003). The four panels show four timelapse views of the same neuromuscular junction imaged between postnatal days 11 and 14. One axon expressing CFP (blue) loses and then regains postsynaptic territory. Between postnatal day (P)11 and P12 the CFP axon relinquished some of its territory to the YFP (yellow) input (compare arrows in top left and right panels). By P14, the CFP-expressing axon had reclaimed the upper right portion of the junction but continued to retreat from the lower part of the junction (compare arrows in P12 and P14 panels). At P18, the CFP input had reclaimed all of its former territory and had taken over the postsynaptic territory previously occupied by the YFP input. The thinner appearance of the junction after P11 is due to slight muscle fiber rotation. This kind of nonmonotonic behavior can only be appreciated by imaging of the same specimen over time. Scale bars equal 10 μm.
(D) Dynamics of dendritic filopodia on a DiI-labeled motorneuron in embryonic zebrafish spinal cord (Jontes et al., 2000).
(D1) Left column is time series (intervals in minutes as indicated) of images acquired using a laser-scanning confocal microscope. Right column is similar specimen (same intervals) imaged using two-photon microscope. Note the superior quality of the image acquired using two-photo excitation in comparison to the single-photon excitation confocal. In this case, image quality of the confocal was limited by the need to limit excitation energy to avoid photodamage to DiI-stained neurons. Higher excitation rates were possible without photodamage using two-photon excitation.
(D2) Rapid dynamics of dendritic filipodia are consistent with a role in exploration for suitable presynaptic partners. Histogram represents the very short lifetimes typical of motorneuron dendritic filopodia at times of developmental synapse formation. Scale: 20 μm panel width.