Cell movements at Hensen's node establish left/right asymmetric gene expression in the chick

Abstract

In vertebrates, the readily apparent left/right (L/R) anatomical asymmetries of the internal organs can be traced to molecular events initiated at or near the time of gastrulation. However, the earliest steps of this process do not seem to be universally conserved. In particular, how this axis is first defined in chicks has remained problematic. Here we show that asymmetric cell rearrangements take place within chick embryos, creating a leftward movement of cells around the node. It is the relative displacement of cells expressing sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) that is responsible for establishing their asymmetric expression patterns. The creation of asymmetric expression domains as a passive effect of cell movements represents an alternative strategy for breaking L/R symmetry in gene activity.

Fig. 1

Morphological and molecular asymmetries arise in conjunction with a leftward movement around the chick Hensen’s node at stage 4 (A-C) 3D reconstruction of confocal views (Z-stack) of Phalloidin stained embryos at stage 3 (A), 4 (B) and 5 (C). Most dorsal (“D”) staining is depth coded blue; the most ventral (“V”) staining is red. (D-I) In situ hybridizations for Shh (D, F, H) and FGF8 (E, G, I). Red arrows, asymmetric domain of Shh; yellow arrows, asymmetric domain of Fgf8. (J-M) Time series showing movement of electroporated cells. (J) Embryos were electroporated at Stage 3+, and subsequent time points are indicated in the lower right corner. 10–30% of cells are labelled by GFP. Note: cells undergo a leftward movement around the node (L). White arrows (N-Q) show the trajectories of the cells shown in (J-M, respectively). In blebbistatin treated embryos (R-T), early cell movements are relatively unaffected, however there is no leftward movement around the node (S). Later, cells display disorganized movements and the primitive streak fails to regress (T). In Omeprazole treated embryos (U-W) gastrulation movements are normal, however cells from the left and right sides move symmetrically toward the node but not around it. The position of the streak and of the node pit are noted by a red-dotted line and a red circle, respectively.

Fig. 2

Effects of MyosinII and voltage gradient inhibitors on node morphology and asymmetric expression domains. (A, E, I) 3D reconstruction of confocal views (Z-stacks) of stage 5 embryos show the morphology of the node and the primitive streak. Lower panels represent a virtual cross section at the level of the white-dotted line. (B, F, J) Depth-coded 3D reconstruction of confocal views (Z-stack) of Phalloidin staining; the dorsal-most staining is blue, the ventral-most is red. The shape of the node is outlined by a white-dotted shape. In situ hybridization of Shh (C, G, K) and FGF8 (D, H, L). Red arrow, extent of posterior Shh expression; yellow arrow, extent of anterior Fgf8 expression.

Fig. 3

Similarities between the gastrulation node in the pig embryo and Hensen’s node during chick gastrulation. (A-C) Ventral scanning electron microscopy views of a 5–6 somites stage pig embryo with higher magnifications of black box in (B,C) of the notochordal plate and adjacent paraxial mesoderm. The endoderm forms a continuous ventral cover (that carries short stubby cilia of 1.5μm length, red arrowheads) across the entire ventral surface of the embryo. (D) A sagittal section at the level of the presumptive floor plate (fp) and notochord (no), covered by a continuous layer of endoderm (end). Anterior direction indicated by ant., leftward facing arrow. (E) The asymmetric node of a Stage 5 pig embryo, marked by asymmetric expression (arrow heads) of the transcription factor Foxj1, demonstrating a shift of the primitive pit (pp, stippled blackline). R, right; L, left. For more information see Fig. S4.