In summary, the data presented here underscore the power of the Tg quail like a novel and powerful tool for studying EC migration patterns and demonstrate that EC motions, in particular during formation of the intersomitic vessels and their asymmetric colonization above the anterior and posterior halves of the somite, are more complex than have been previously reported (101, 102). Analysis of our live imaging data helps a model in which ECs both direct and constrain the migratory behavior and path of NCCs during development of the PNS. Movie 1B shows a reconstructed look at of the sagittal half of the mid-embryonic trunk. The embryo was immunostained with main antibodies that identify NCCs and NCC derivatives (HNK1), and ECs and EC-derived vasculature (QH1), cleared, and then cut in half along the sagittal midline (observe methods). DRG, dorsal root ganglia; ISV, intersomitic vessel; PNVP, perineural vascular plexus; SG, sympathetic ganglia. (Level pub: 120 m) Movie 1C. NCCs are directly apposed to ECs as they enter the intersomitic furrow. St. 15/16 quail. Take flight thru Movie 1C shows a dorsal to ventral progression through the dorsal half of the mid-embryonic trunk (somites 16C21). The embryo was immunostained using main antibodies that identify NCCs and NCC derivatives (HNK1), and ECs and EC-derived vasculature (QH1), SB366791 cleared, and then imaged whole mount SB366791 with the dorsal surface laid down on a cover glass. At this stage, the MSA is definitely densely populated with NCCs having a few entering the intersomitic furrow in limited juxtaposition to ECs comprising the intersomitic vessel. The anterior half of the somite also shows some HNK1 immunoreactivity. DRG, dorsal root ganglia; ISV, SB366791 intersomitic vessel; MSA, migration staging area; NCCs, neural crest cells; NT, neural tube. (Scale pub: 130 m) Movies 2A and 2B. NCCs interact extensively with ECs as they migrate ventrally through the intersomitic furrow. Multispectral time-lapse confocal microscopy of a live, whole mount St. 16 Tg (transgenic quail collection Tg(< 0.0001. D) Schematic showing the basic migration pattern of ECs. ECG) Axial level corresponds to the mid-trunk. E) St. 17. In transverse explants through the posterior half somite, ECs are consistently located on the ventral part of NCCs and appear to block their ventral migration. To aid in the visualization of NCCs, a slice was chosen that includes contralaterally migrating NCCs. F, G) Whole mount embryos viewed from your lateral element. F) At St. 17, ECs are densely populated above the posterior half somite, and sparse above the anterior half (asterisks). G) In contrast, by St. 20C22, ECs are more uniformly distributed along the space of the MSA although some gaps persist (asterisks). HCK) St. 22. Individual frames from Movie 3G. H) A NCC exhibits an elongated morphology as it migrates down a continuous coating Mouse monoclonal to Ractopamine of ECs. ICK) The same NCC stretches filopodia into a space between adjacent ECs (arrows in I, J), followed by realignment of the cell such that the NCC soma techniques into the same space (arrow in K). Contra, contralateral; DA, dorsal aorta; ISF, intersomitic furrow; Ipsi, ipsilateral; NCCs, neural crest cells; NT, neural tube S, somite; VEF, von Ebners fissure. (Level pub: A, 40 m; B, 60 m; E, F, G, 30 m; HCK, 12 m) For Movies 1A,B, the trunks of immunostained whole embryos were slice in half sagittally using a feather cutting tool under a dissecting scope. For Movies 1ACC, embryos were rendered optically transparent (cleared) prior to imaging using a methyl salicylate and benzyl benzoate (MSBB) method explained previously (78). Briefly, embryos were dehydrated using a series of ethanol solutions in distilled water (70%, 95%, 100%), and placed in a methyl salicylate, benzyl benzoate (5:3) remedy for 30 minutes. All 3D images were acquired using the FV300 confocal and 20X objective explained above, having a lateral (xCy) resolution of 0.69 m per pixel and optically sectioned at 2 m in Z. Confocal images were converted to 3D tiff-stacks using ImageJ (http://imagej.nih.gov/), and then imported to Osirix imaging software (http://www.osirix-viewer.com/) to generate rotational and take flight thru movies (Movies 1ACC). For time-lapse imaging in Movies 2A,B, and photomicrographs demonstrated in Numbers 2A and 3F,G, whole embryos were mounted on paper rings and transferred to Millicell-CM 0.4 mm tradition plate inserts (Millipore, Bedford, MA) with the legs removed and saturated in neurobasal press (Invitrogen) supplemented with B27 (Invitrogen) within a 35 mm glass bottom dish (MatTek, Ashland, MA). Images of a single focal plane were captured every 5 minutes using the 20X dry objective on our FV300 (above) within an enclosure heated to 37C. For Movies 3ECG, 4, 5, 6, and Numbers 3E, HCK, and.