Dynamic imaging of mammalian neural tube closure

Abstract

Neurulation, the process of neural tube formation, is a complex morphogenetic event. In the mammalian embryo, an understanding of the dynamic nature of neurulation has been hampered due to its in utero development. Here we use laser point scanning confocal microscopy of a membrane expressed fluorescent protein to visualize the dynamic cell behaviors comprising neural tube closure in the cultured mouse embryo. In particular, we have focused on the final step wherein the neural folds approach one another and seal to form the closed neural tube. Our unexpected findings reveal a mechanism of closure in the midbrain different from the zipper-like process thought to occur more generally. Individual non-neural ectoderm cells on opposing sides of the neural folds undergo a dramatic change in shape to protrude from the epithelial layer and then form intermediate closure points to "button-up" the folds. Cells from the juxtaposed neural folds extend long and short flexible extensions and form bridges across the physical gap of the closing folds. Thus, the combination of live embryo culture with dynamic imaging provides intriguing insight into the cell biological processes that mold embryonic tissues in mammals.

Figure 1. Zipper-like closure within the mouse hindbrain

A. Still images from movie 1 captured from time lapse imaging of the hindbrain with the time points in seconds indicated in each panel. The folds come together in a zipper-like manner in a region of the hindbrain rostral to closure point 1. Colored dots track individual cells throughout the time period of imaging. Scale bar 20μm. B. The schematic on the right represents embryo orientation relative to the objective lens and the red dot represents closure point 1. C. Closure in the hindbrain proceeds at a rate of ∼1um/min when the folds are at least 120 um apart through closure. Error bars denote the standard deviation for the distance crossed for each time point in three different embryos.

Figure 2. Secondary closure points in the midbrain

(A) Transition between zippering in the hindbrain to buttoning in the midbrain. White arrowheads indicate thin cell extensions, yellow arrows indicate protruding cells (three neighboring cells protrude from the fold on lower left as observed over time and in a different z plane; not shown). Scale bar 20μm. (B, C) and movies 2 and 3. Imaging of the ∼E9.25 midbrain region schematically shown between closure points 1 and 2 (D, red and purple dots, respectively). Time points in minutes are indicated in each panel. Note a single or a few cells protrude from the non-neural ectoderm layer (demarcated from neural ectoderm by dotted line) on both sides of the neural folds (yellow arrows) and interact with protruding cells on the opposing neural fold. Boxes highlight magnified views showing thin cell extensions in the gap between neural folds (white arrowheads) and which appear to connect the opposing folds (panels B′ and B′) at the position that the secondary closure will form. Scale bar in all panels is 20 μm.

Figure 3. Non-neural ectoderm wraps around neural ectoderm

(A, B) Frontal sections in the midbrain region of an E9.0 embryo fixed immediately after dissection. The image in A shows the two neural folds just preceding neural fold fusion whereas the image in B shows a location where the neural folds have met in the midline. Cryosections in the midbrain region were processed with an anti-E-cadherin antibody (red) to highlight the non-neural ectoderm, phalloidin (green) which marks filamentous actin, and Hoescht (blue) that labels the nuclei. The plane marked by the dotted line denotes the approximate optical plane for the image shown in (C). (C) An embryo that had been cultured and an optical section taken when the neural folds were closely apposed but not yet closed. This optical section was taken on a Zeiss LSM510 META microscope through the single cell layer of non-neural ectoderm (large flattened cells) that wraps around the neural ectoderm (hexagon shaped cells) at the edge of the closing neural folds (the dotted line separates the non-neural from the neural ectoderm). The cell surface facing the physical gap between the folds has a ruffled appearance. Scale bar 20μm.

Figure 4. Flexible, motile extensions and cellular bridges from the non-neural ectoderm

Images of the neural folds in the midbrain region. (A) Numerous long (over 50um) thin cell extensions from the non-neural ectoderm of one fold that extend across the gap towards the converging fold (not visible in this view). Scale bar 20μm. (B) Still image from Movie 4 of the non-neural ectoderm on one fold in the midbrain region that highlights a few of the long flexible cell extensions and short bulbous flexible extensions. Scale bar 20μm. (C) Cellular bridges connect the two juxtaposed folds when they are 20μm apart. In the boxed area, two cellular bridges connect opposing non-neural ectoderm cells. Dotted lines separate the non-neural from neural ectoderm. Scale bar 50μm. (D) Magnification of one of the cell bridges from Figure 4C. Three structures ∼0.5 μm in diameter that are highlighted with the myristoylated Venus fluorescence reporter are present within the cellular bridge (white arrowheads). Scale bar 10μm.