Rotation of organizer tissue contributes to left-right asymmetry

Abstract

Current hypotheses regarding vertebrate left-right asymmetry patterns are based on the presumption that genetic regulatory networks specify sidedness via extracellular morphogens and/or ciliary activity. We show empirical time-lapse evidence for an asymmetric rotation of epiblastic nodal tissue in avian embryos. This rotation spans the interval when initial symmetric expression of Shh and Fgf8 becomes asymmetrical with respect to the midline.

Figure 1. Whole-mounted chicken embryos subjected to time-lapse imaging after H2B-GFP electroporation

A-C. Three brightfield image frames extracted from a 360 min time-lapse sequence (Movie 1) demonstrate the extensive degree of tissue-level motion and expansion around a region of interest (ROI; white box), which is centered on Hensen's node, at HH stages 4-6. The brightfield Movie 1 is a collage composed of six image frames (2×3) blended into a single wide-field view using TiLa-KUMC software. D. A corresponding H2B-GFP epifluorescence image within the ROI. The node (asterisk) and the presumptive anterior-posterior axis (dashed line) are indicated. The epifluorescence image depicts one 10X image field. E and F. Epifluorescence cellular tracking within the same ROI as Panels A-C demonstrate the counter-clockwise vortical motion of some nodal tissue indicated by the white arrow (see corresponding Movie 1). The cellular (nuclear) tracks from 20 consecutive frames are colored yellow and superimposed with the last 5 frames colored green. Simple inspection confirms that the motion pattern within the node is not bilaterally equivalent with respect to the anterior-posterior axis (dashed line in Panel D; see Movies 1 and 2). Magnification bars=250 μm.

Figure 2. In situ mRNA hybridizations of whole-mounted embryos and corresponding cross-sections

A and B. In situ mRNA hybridization for Shh and Fgf8, respectively, at HH stage 5⁺ shows that gene expression occurs during the time and at the place where epiblastic nodal cells are vigorously moving. The insert in the upper left shows a ROI denoted by a white box. Magnification bars=250μm. Asterisks denote the position of Hensen's node. C-H. Serial cross-sections of the HH stage 5⁺ specimens in Panels A and B reveal that Shh and Fgf8 mRNAs are asymmetrically expressed in the epiblastic layer (ep) of the node and in some mesodermal cells (mes), but not in the hypoblastic layer (hy). The white dashes in A and B denote the axial position of the tissue sections in C-H. Black arrowheads denote the midline. Magnification bars = 50μm.

Figure 3. A model of motion in and near Hensen's Node

An interpretative diagram of epiblastic cellular/tissue motion patterns in and near Hensen's node, based on empirical time-lapse data (n=10). Also depicted are two possible scenarios whereby gene products, Fgf8 and Shh, initially expressed in a symmetrical manner, could be moved into asymmetrical positions as a direct result of the empirically observed biophysical/mechanical tissue deformations. Purple color denotes the respective distribution of a gene product. At HH stage 4 the avian node is a quasi-discoidal shaped tissue with a slight asymmetry toward the anatomical left (dorsal view). As embryogenesis progresses the empirical time-lapse data show that the cranial-most nodal/peri-nodal cells move counterclockwise. The arrows correspond to the color scheme of the Movies 1 and 2, with yellow depicting early image frames and green depicting later time points. On the anatomical right we envision a stream of cells approaching the node and then forking, with one stream moving in a cranially or counterclockwise fashion, while the other cellular stream flows caudally or somewhat clockwise — this latter motion pattern can be observed in Movie 2. Let the morphogen Fgf8 be symmetrically expressed (purple) at HH stage 4. After being subjected to the motion patterns indicated by the arrows, for six hours — the morphogen producing cells would be positioned asymmetrically as shown at HH stage 6. Similarly, let Shh expressing cells be distributed symmetrically at HH stage 4 (purple), then be moved to an asymmetrical position at HH stage 6. Note that the molecular distribution patterns proposed in this model correspond to the cognate mRNA expression patterns shown in Figure 2 and by Dathe and colleagues (2002). It must be kept in mind that the tissue patterns we depict in this diagram occur within a continuously deforming biophysical framework in vivo.