Colonizing while migrating: how do individual enteric neural crest cells behave?
1 Department of Anatomy & Neuroscience, University of Melbourne, Melbourne 3010 VIC, Australia
2 School of Mathematical Sciences, Queensland University of Technology, GPO Box 2434, Brisbane 4001 QLD, Australia
3 RIKEN Center for Developmental Biology, Laboratory for Neuronal Differentiation and Regeneration, Kobe, Japan
4 Division of Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan
BMC Biology 2014, 12:23 doi:10.1186/1741-7007-12-23Published: 26 March 2014
Explant of colon from an E12.5 Ednrb-hKikGR mouse in which a group of ENCCs that were 600 to 700 μm from the wavefront were photoconverted from green to red. Some of the photoconverted ENCCs migrated along longitudinally-oriented strands with high speed (cells tracks indicated by blue and purple lines). Z-series images using a X10 objective lens through the ENCC network were captured every four minutes for 10 hours, and then each z-series projected. Caudal is to the right.
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Same explant as shown in Movie 1. One of the photoconverted ENCCs exhibited a complex, circular pathway as indicated by the white line. This ENCC migrated at an average of 70 μm/h, but it advanced caudally only 140 μm after 16 hours. In the middle of the movie when it is migrating rostrally, it collides with a brighter green ENCC that is migrating caudally, but still proceeds rostrally. Caudal is to the right.
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Explant of colon from an E12.5 Ednrb-hKikGR mouse. Red channel showing only four groups of photoconverted ENCCs, which were 80, 200, 600 and 1,000 μm from the most caudal cell at the commencement of imaging. ENCC do not retain their spatial order, and there is significant intermixing of cells photoconverted at different locations. Images were captured every 10 minutes for five hours using a X10 objective lens. Caudal is to the right.
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Additional file 4: Figure S1:
Morphology of ENCCs and filopodia and lamellopodia extension. Figure S2: Interactions between the filopodia and/or lamellipodia at the front of one chain and cells or filopodia from another chain resulted in three general outcomes: adhesive, “walk-past” and repulsive (contact inhibition of locomotion). Figure S3: There were no significant differences in the number of Hu + neurons per area of Kik + cells in control explants (n = 12) and in explants cultured in the presence of BQ788 (20 μM) (n = 13) for 10 hours. At the end of the culture period, explants were fixed, processed for immunohistochemistry using an antibody to the pan-neuronal marker, Hu. The number of Hu + cells per area of Kik + cells was quantified using Fiji software.
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Higher magnification movie showing a photoconverted ENCC in the middle of the field of view, which is the daughter cell of a cell that was photoconverted when it was undergoing mitosis; the nucleus is red and the cytoplasm is orange because of the presence of newly synthesized (green) protein after cell division. This ENCC, and other ENCCs in the field of view at the fronts of chains, extended filopodia and lamellopodia in a variety of directions. After the red ENCC encountered another chain, a number of processes adhered to the chain and the cell then joined the chain. The movement between images is due to spontaneous contractions of the developing external muscle. Images were captured every 2.5 minutes for five hours using a X40 objective lens for two hours. Caudal is to the right.
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Red channel only after ENCCs 600 μm from the wavefront of the E12.5 colon had been photoconverted. Longitudinally projecting neurites are present on the caudal side. Images were captured every five minutes for 5 hours using a X20 objective lens for 9.5 hours. Caudal is to the right.
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Movie in which all ENCCs, except those approximately 100 μm from the wavefront, were photoconverted from green to red. ENCCs use the red neurite in the middle of the field of view as a substrate to advance caudally. Images were captured using a X40 objective lens every four minutes for 16 hours, but this movie only shows a 3-hour period starting 11 hours after photoconversion and the commencement of imaging. The brightness of the green channel is reduced. Caudal is to the right.
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