An effective assay for high cellular resolution time-lapse imaging of sensory placode formation and morphogenesis
BMC Neurosci. 2011 May 9;12:37. doi: 10.1186/1471-2202-12-37.
Shiau CE1, Das RM, Storey KG.
Abstract
BACKGROUND:
The vertebrate peripheral nervous system contains sensory neurons that arise from ectodermal placodes. Placodal cells ingress to move inside the head to form sensory neurons of the cranial ganglia. To date, however, the process of placodal cell ingression and underlying cellular behavior are poorly understood as studies have relied upon static analyses on fixed tissues. Visualizing placodal cell behavior requires an ability to distinguish the surface ectoderm from the underlying mesenchyme. This necessitates high resolution imaging along the z-plane which is difficult to accomplish in whole embryos. To address this issue, we have developed an imaging system using cranial slices that allows direct visualization of placode formation.
RESULTS:
We demonstrate an effective imaging assay for capturing placode development at single cell resolution using chick embryonic tissue ex vivo. This provides the first time-lapse imaging of mitoses in the trigeminal placodal ectoderm, ingression, and intercellular contacts of placodal cells. Cell divisions with varied orientations were found in the placodal ectoderm all along the apical-basal axis. Placodal cells initially have short cytoplasmic processes during ingression as young neurons and mature over time to elaborate long axonal processes in the mesenchyme. Interestingly, the time-lapse imaging data reveal that these delaminating placodal neurons begin ingression early on from within the ectoderm, where they start to move and continue on to exit as individual or strings of neurons through common openings on the basal side of the epithelium. Furthermore, dynamic intercellular contacts are abundant among the delaminating placodal neurons, between these and the already delaminated cells, as well as among cells in the forming ganglion.
CONCLUSIONS:
This new imaging assay provides a powerful method to analyze directly development of placode-derived sensory neurons and subsequent ganglia formation for the first time in amniotes. Viewing placode development in a head cross-section provides a vantage point from which it is possible to study comprehensive events in placode formation, from differentiation, cell ingression to ganglion assembly. Understanding how placodal neurons form may reveal a new mechanism of neurogenesis distinct from that in the central nervous system and provide new insight into how cells acquire motility from a stationary epithelial cell type.
PMID 21554727
<pubmed>21554727</pubmed>| BMC Neurosci.
- File:Chicken placode 01.mp4 Time-lapse imaging of placodal neurons labeled by membrane GFP and nuclear H2B-RFP in the ganglionic anlage show intimate contacts by placodal processes. Movie shows highly dynamic interactions at sites of axon-axon contacts which eventually make a connection at time of growth cone collapse. 336 minutes in real time taken at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.
- File:Chicken placode 02.mp4 Time-lapse movie of a dividing cell labeled by H2B-RFP undergoing a parallel cleavage in the basal side of the placodal ectoderm. Metaphase plate rotates prior to division (arrow). 72 minutes in real time taken at 3-minute intervals; 24 um z-stack; 20×; 6.5 fps.
- File:Chicken placode 03.mp4 Time-lapse movie of a dividing cell labeled by H2B-RFP undergoing a perpendicular cleavage in the apical side of the placodal ectoderm. 60 minutes in real time taken at 3-minute intervals; 24 um z-stack; 20×; 6.5 fps.
- File:Chicken placode 04.mp4 Time-lapse imaging of cell divisions (dotted lines around cells) in the trigeminal placodal ectoderm as labeled by nuclear H2B-RFP and membrane GFP. Dotted white line demarcate basal edge of ectoderm. Yellow arrow points to a placodal cell that divides prior to ingression and a different placodal cell (blue arrow) undergoes division after it is already in the mesenchyme. Video covers 336 minutes of development in real time captured at 3-minute intervals; 45 um z-stack; 20×; 7.5 fps.
- File:Chicken placode 05.mp4 Time-lapse imaging of cell divisions (dotted lines around cells) in the trigeminal placodal ectoderm as labeled by nuclear H2B-RFP and membrane GFP. Dotted white line demarcate basal edge of ectoderm. Yellow arrow points to a placodal cell that divides prior to ingression and a different placodal cell (blue arrow) undergoes division after it is already in the mesenchyme. Video covers 336 minutes of development in real time captured at 3-minute intervals; 45 um z-stack; 20×; 7.5 fps.
- File:Chicken placode 06.mp4 Time-lapse movie of a representative cranial slice in bright-field. The image sequence is taken from the middle slice of a z-stack over the duration of the time-lapse imaging (8.4 hours). For each z-stack, a bright-field image was taken at the middle slice as control to monitor integrity of tissue in parallel with the fluorescence imaging. Images were taken at 3-minute intervals at 20×, and shown at 6.5 fps. ecto, ectoderm; mes, mesenchyme; nt, neural tube
- File:Chicken placode 07.mp4 Time-lapse imaging of actively ingressing cells labeled by membrane GFP and nuclear H2B-RFP from the placodal ectoderm. Movie shows highly interacting placodal cells as they stream out of a discrete exit point on the basal side of the epithelium, forming contacts among themselves and with already ingressed ganglionic placodal neurons. Imaging shows 336 minutes of development captured at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.
- File:Chicken placode 08.mp4 Same as File:Chicken placode 06.mp4 but showing only the membrane GFP channel for clearer visualization of the cell morphology.
- File:Chicken placode 09.mp4 Time-lapse imaging of placodal neurons labeled by membrane GFP and nuclear H2B-RFP in the ganglionic anlage show intimate contacts by placodal processes. Movie shows highly dynamic interactions at sites of axon-axon contacts which eventually make a connection at time of growth cone collapse. 336 minutes in real time taken at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.
