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.
In mice and rabbits monocilia responsible found on cells of the posterior notochordal plate have been shown to play a crucial role in breaking L/R symmetry (1,2). These cilia are able to create a leftward flow of fluid, in a pit-like teardrop shaped space that is not covered by subjacent endoderm (3)., The flow of fluid across this pit stimulates signal transduction that ultimately leads to induction of asymmetric gene expression (1,2).
In the chick embryo, in contrast, the endoderm underlying Hensen’s node (a structure at the rostral end of the primitive streak in the gastrulating embryo) exists as a continuous sheet ventral to the notochordal plate mesoderm (4) and there is no morphological pit on the ventral surface in which a flow of fluid could be established. Prior work has noted cilia at Hensen’s node (5) but these short cilia are on endodermal cells and are unrelated to the motile cilia on the mesodermal cells of the ventral node in the mouse and rabbit. The mesodermal cells at Hensen’s node in the chick are devoid of cilia. In addition, the Talpid chick mutant lacks primary cilia (6) but does not exhibit L/R asymmetry defects. Unlike the mouse and rabbit, the chick node itself becomes morphologically asymmetric, with a marked tilt towards the left around the time the primitive streak reaches full extension, at Stage 4. (7,8) (Fig. 1A-C). Shortly thereafter, a number of small L/R asymmetric expression domains are observed to the right and left of the node (9). However, all of the genes expressed in such a manner are initially expressed in a symmetric fashion, for example, Fgf8 bilaterally along the primitive streak and Shh bilaterally across the top of the node until stage 4, (10) (Fig. 1D_G). Subsequently, concomitant with the development of morphological asymmetries in the node, these gene expression patterns also become gradually asymmetric by stage 5 (Fig. 1H,I).
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.
To investigate cellular rearrangements that could be responsible for establishing the morphological asymmetry of the node, we performed a time lapse analysis of cell movements at Hensen’s node; randomly labeling cells by electroporation of a green fluorescent protein (GFP) reporter. At stage 4, as primitive streak elongation terminates (Fig. 1K, O) but regression has not yet begun (Fig. 1M, Q), the cells that have formed the node exhibit a definitive, albeit brief (3–4 hours) leftward movement (n=10/10, Movies S1,S2,S3 and Fig. 1L, P).
We next attempted to block the leftward movement of cells at the node by the use of drugs that inhibit the function of Rho Kinase (Rok) (Y-27632) and its target MyosinII (Blebbistatin). These two proteins have been shown to provide the force driving various oriented cell rearrangements in several species. After adding these drugs, gastrulation movements continued (Fig. 1R, Fig. S1B,F,J) and most reached at least stage 6, when the notochord has formed, although as previously described (11,12) primitive-streak regression was affected (Fig. 1T, Fig. S1D, H, L). In these drug-treated embryos, he leftward movements of cells at the node was no longer observed (Movie S4, S5 and Fig. 1S, Fig. S1C,G,K). 3D reconstruction of Hensen’s node from stage 5 embryos that were cultured in Blebbistatin or Y-27632 revealed that the node displays a more symmetrical and anteriorly extended shape as compared to control embryos (dotted lines, Fig. 2A-B, E-F, Fig. S2A-B), suggesting that the cell movements we identified are responsible for the morphological asymmetry of the node. We moreover noted that these embryos also displayed symmetrical expression of Shh and FGF8 (Y-27632 n= 16/23 and Blebbistatin n=17/21, Fig. 2G-H, Fig. S2C-D).
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.
We additionally attempted to block the leftward cell movements anterior to the node by two physical methods: manually inserting individual human hairs through the primitive streak extending anterior to the node and surgically bisecting the embryos along the midline. Both manipulations also led to bilateral gene expression (Fig. S3). Thus the leftward cellular movements at the node are necessary to initiate L/R asymmetric expression domains in the chick.
It has been previously demonstrated that asymmetric expression of Shh and FGF8 depends on a transient, H+/K+ ATPase-dependent depolarization of membrane potential on the left side of the primitive streak, just before stage 4 (13). We cultured embryos in plates containing the H+/K+ ATPase inhibitors, SCH28080 or Omeprazole, and found that the node displayed a symmetrical morphology comparable to the phenotypes observed with MyosinII and Rok inhibitors, suggesting that in these conditions the cell rearrangements at the node did not occur properly (Fig. 2I-J, Fig. S2E-F). Moreover, when we performed a time lapse analysis on GFP electroporated embryos cultured on plates containing Omeprazole, we did not observe any leftward movements at the node (n=3/4) (Movie S6 and Fig. 1V, Fig. S1O) as observed in control embryos (movie 1 n=10/10 Fig. 1L, P), although the primitive streak elongated and regressed normally (Fig. 1U-W, Fig. S1N-P). Embryos treated with H+/K+ ATPase inhibitors exhibited symmetrical expression of Shh and FGF8 (as previously reported (13)), by stage 5–6 (SCH28080 n=7/12 and Omeprazole n= 6/7, Fig. 2K,L, Fig. S2G-H). These data show that the leftward movement at the node is downstream of the asymmetric H+/K+ ATPase activity.
The mechanism for setting up L/R asymmetric gene expression patterns at the chick node may have relevance beyond avian species. Despite the morphological conservation of the notochordal plate’s ventral surface as seen in mice, rabbits and, possibly, humans (14), histological analysis reveals that, similar to the chick, the cells of the pig notochordal plate do not contain cilia (Fig. 3A-C, Fig. S4D-J). As in the chick, we do not observe any structure in the pig resembling the ventral node of the mouse and rabbit. Moreover, as in the chick, the cells of the pig notochordal plate are completely covered by endoderm. As a consequence, there is no space in which a flow of fluid could be generated (Fig. 3E, Fig. S4A, B). Additionally, like the chick, pig embryos have a morphologically asymmetric node displaced leftward at Stage 5 (n=4/4) (Fig. 3C), and like the chick and unlike the mouse, there are asymmetric gene expression domains adjacent to the node, such as Foxj1 (n=3/3) (Fig. 3A), which precede by several hours the asymmetric expression of Nodal at Stage 6 (Fig. 3D) (Nodal being the first gene known to be asymmetrically expressed in mice and rabbits).
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.
In summary we show here that, in chick embryos, the node is the site of cellular rearrangements that create a leftward movement of cells around it (an observation made independently by Cui et al (15)). The convergence of cells on the right edge of the node and migration away from the midline on the left thereby deform the shape of the node. This movement establishes asymmetric gene expression patterns, not through transcriptional induction or repression but rather in a passive manner by rearranging the relative orientations of cells expressing critical genes (Fig. S5). Moreover, we have found that cell movements at the node arise downstream of a transient depolarization of membrane potential on the left side mediated by the activity of the H+/K+ ATPase. The symmetry-breaking event that leads to asymmetric H+/K+ ATPase activity in chick remains unknown.
Previous mis-expression experiments have put the genes asymmetrically expressed at the chick node into an epistatic pathway. For example, according to current models, Bmp4 (which is expressed in very similar domains to Fgf8, initially bilaterally along the streak and then asymmetrically on the right side) induces Fgf8 and represses Shh. Shh feeds back to repress Bmp4 and induce Nodal, whereas Fgf8 serves to repress Nodal. Our data do not contradict these previously described epistatic relationships. However, since the asymmetric expression domains of these signaling molecules do not form in the absence of cell movements, we suggest that the previously described cross-regulation, for instance the reciprocal inhibition between Shh and Bmp4, functions secondarily to sharpen borders and provide robustness once cells expressing these factors are brought into juxtaposition, rather than as a primary means of establishing their asymmetric gene expression domains.
Supplementary Material
Supp. Movie 1 The left panel is a movie from a time lapse experiment (described in Fig. 1J-M) showing a dorsal view of a chick embryo electroporated with a GFP reporter construct between stage 3+ and stage 7. After the primitive streak reached its maximum, cells around the node exhibit a leftward movement, and then the primitive streak and the node regress posteriorly. The central panel shows the same movie in which the position of different cells (marked by different colors) has been tracked. The right panel shows the fate of the region of the node defined by the position of cells tracked in the central panel. By the time the node disappears from the movie at around stage 5, the region symmetrical at stage 3 has become asymmetric. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 2 The left panel is a movie from a time lapse experiment showing a dorsal view of a chick embryo electroporated with a GFP reporter construct. This short movie (3.5 hours) shows specifically the leftward movement of cells within the node occurring during stage 4+and 5−. The central panel shows the same movie in which the position of different cells within the node (marked by different colors) has been tracked. The right panel shows the fate of the region of the node defined by the position of cells tracked in the central panel. By the end of the movie at around stage 5−, the region symmetrical at stage 4+ has shifted toward the left side of the embryo. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 3 The left panel is a movie from a time lapse experiment showing a dorsal view of a chick embryo electroporated with a GFP reporter construct. In this movie, only cells from the right side of the Hensen’s node have been electroporated. As the embryo develops, cells on the right side of the node by stage 4+ end up on the left side by stage 5−. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 4 Movie from a time lapse experiment showing a chick embryo electroporated with a GFP reporter construct and cultured on a plate containing the MyosinII inhibitor Blebbistatin. The movie shows that while the movements bringing epiblast cells toward the primitive streak are relatively unaffected, the movements of cells occurring anterior to the node are disorganized; as a consequence the leftward movement around the node is greatly affected. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie5 Movie from a time lapse experiment showing a chick embryo electroporated with a GFP reporter construct and cultured on a plate containing the Rock inhibitor, Y-27632. This movie shows that while the movements bringing epiblast cells toward the primitive streak are relatively unaffected, cells anterior to the node display slow and poorly organized movements, as a consequence the leftward movement is inhibited and the node fails to regress. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 6 The left panel is a movie from a time lapse experiment showing a chick embryo between stage 3+ and stage 6, electroporated with a GFP reporter construct and cultured on a plate containing Omeprazole. The primitive streak elongates properly but once at its maximum, cells around the node fail to move toward the left side of the embryo, then the primitive streak and the node regress posteriorly. The right panel shows the same movie in which the position of different cells (marked by different colors) has been tracked. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp Fig. 1 Effects of Rok, Myosin and voltage gradient inhibitors on cell movements at the node. (A-P) Cumulative images recapitulating the position of electroporated cells between the different time points (noted in the lower right corners). Complete time lapse experiments in which the time series has been coded are shown in a, e, i, and m. The first time points are colored in blue and the last acquired time points are colored in red. (A-D) Movement of cells at the node (square region in a) in control embryos. (B) Cell movement during the primitive streak elongation. (C) Leftward movement of cells around the node. (D) Regression of the node and primitive streak. (E-L) Movement of cells at the node in Y-27632 (E-H) and Blebbistatin (I-L) treated embryos: first, cell movements are relatively unaffected (F, J), then cells exhibit disorganized movements and there is no leftward movement around the node (G, K). Finally, cells display disorganized movements and the primitive streak fails to regress (H, L). (M-P) Cell movement at the node in omeprazole treated embryos: Cells first converge normally toward the primitive streak (N). Cells from the left and right sides then move symmetrically toward the node but not around it (O). (P) The primitive streak regresses normally. Right panels in B-D, F-H, J-L, N-P highlight the trajectories of cells observed in Left panels. The position of the primitive streak and the node pit are represented with a red-dotted line and a red circle, respectively.
Supp Fig. 2 (A, E) 3D reconstruction of confocal views (Z-stacks) of stage 5 embryos (A) Y27632 and Omeprazole (E) treated embryos showing 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) 3D reconstruction of confocal views (Z-stack) of Phalloidin staining of Y-27632 and Omeprazole treated embryos(B, F, respectively) at stage 5. Images are depth-coded: the Phalloidin staining is pseudo-colored such that the dorsal-most staining is blue, the ventral-most staining is red. The shape of the node is outlined by a white-dotted circle. Expression patterns of Shh (C, G,) and FGF8 (D, H,) are more symmetric in drug treated embryos as shown by in situ hybridization.
Supp Fig. 3 (A-F) Embryos in which a human hair has been inserted through the primitive streak and the node in order to prevent asymmetric cell movements. (A, B, C) and (D, E, F) show symmetric expression domains of Shh and FGF8 respectively. (W) and (Z) are pictures from embryos shown in (U, V) and (X, Y) but the hair has been removed to fully reveal the domains of expression of Shh and FGF8. (G-J) Stage 6–7 embryos in which an incision has been made through the node and the anterior primitive streak at stage 4, in order to prevent the leftward movement of cells.(G, H) and (I, J) show bilateral expression of Shh and FGF8 respectively. The position of the node is marked with an asterisk.
Supp Fig. 4 (A, B) Median sagittal 1μm plastic sections of the pig node (n), posterior notochord (no) and floorplate (fp) at the late presomite stage (stage 6, A) and at the 5–6 somites stage (B) showing that the node and posterior notochord are completely covered by endoderm (e) throughout this period of development. Transverse section of a pig embryo showing left sided expression of nodal. (D-G) Confocal images of anti-acetylated tubulin staining showing cilia (white arrowheads) and mid-bodies (outlined arrowhead) at different levels (L1, L2, floor plate; L3, notochordal cells; L4, endoderm) of a pig embryo as noted in (C). (H-J) Ventral scanning electron microscopy views of a 5–6 somites rabbit embryo with higher magnifications (I-G) of the notochordal plate and adjacent paraxial mesoderm at the level of the caudal-most somite pair. As opposed to the pig, in the rabbit the endoderm covers the paraxial mesoderm only and is connected medially to the lateral edges of the notochordal plate; consequently, the notochord is free of endoderm and its typical long monocilia (6μm) can be seen on almost every cell. Numbers in C and F indicate the level of somite pairs; pnc: posterior notochord, n: node. Magnification bar: 20μm in A, B, and 5μm in I.
Supp. Fig 5 Alignment of Foxj1 sequences from various species, as shown, indicating conservation of the domain used as a probe in these studies. The grey box indicates overlap of the 832bp probe with the forkhead domain. For more information see the Methods section.
Supp. Figure 6. By stage 3 the node is morphologically symmetric, then a differential transient membrane depolarization drive the leftward movement of cells at the node, leading consequently to asymmetric expression of Shh and FGF8.
Acknowledgments
We thank M. Levin for helpful suggestions for addressing the role of ion channels, R. O’Rahilly for providing information regarding human embryos, and C. Cui, C. Little and B. Rongish for discussing data prior to publication. This work was supported by a Human Frontier Science Program fellowship to JG, a PhD fellowship from the Boehringer Ingelheim Fonds to KF, a grant from the DFG to MB, a grant from the DFG to CV, and R01-HD045499from the NIH to CJT.
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Associated Data
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Supplementary Materials
Supp. Movie 1 The left panel is a movie from a time lapse experiment (described in Fig. 1J-M) showing a dorsal view of a chick embryo electroporated with a GFP reporter construct between stage 3+ and stage 7. After the primitive streak reached its maximum, cells around the node exhibit a leftward movement, and then the primitive streak and the node regress posteriorly. The central panel shows the same movie in which the position of different cells (marked by different colors) has been tracked. The right panel shows the fate of the region of the node defined by the position of cells tracked in the central panel. By the time the node disappears from the movie at around stage 5, the region symmetrical at stage 3 has become asymmetric. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 2 The left panel is a movie from a time lapse experiment showing a dorsal view of a chick embryo electroporated with a GFP reporter construct. This short movie (3.5 hours) shows specifically the leftward movement of cells within the node occurring during stage 4+and 5−. The central panel shows the same movie in which the position of different cells within the node (marked by different colors) has been tracked. The right panel shows the fate of the region of the node defined by the position of cells tracked in the central panel. By the end of the movie at around stage 5−, the region symmetrical at stage 4+ has shifted toward the left side of the embryo. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 3 The left panel is a movie from a time lapse experiment showing a dorsal view of a chick embryo electroporated with a GFP reporter construct. In this movie, only cells from the right side of the Hensen’s node have been electroporated. As the embryo develops, cells on the right side of the node by stage 4+ end up on the left side by stage 5−. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 4 Movie from a time lapse experiment showing a chick embryo electroporated with a GFP reporter construct and cultured on a plate containing the MyosinII inhibitor Blebbistatin. The movie shows that while the movements bringing epiblast cells toward the primitive streak are relatively unaffected, the movements of cells occurring anterior to the node are disorganized; as a consequence the leftward movement around the node is greatly affected. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie5 Movie from a time lapse experiment showing a chick embryo electroporated with a GFP reporter construct and cultured on a plate containing the Rock inhibitor, Y-27632. This movie shows that while the movements bringing epiblast cells toward the primitive streak are relatively unaffected, cells anterior to the node display slow and poorly organized movements, as a consequence the leftward movement is inhibited and the node fails to regress. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp. Movie 6 The left panel is a movie from a time lapse experiment showing a chick embryo between stage 3+ and stage 6, electroporated with a GFP reporter construct and cultured on a plate containing Omeprazole. The primitive streak elongates properly but once at its maximum, cells around the node fail to move toward the left side of the embryo, then the primitive streak and the node regress posteriorly. The right panel shows the same movie in which the position of different cells (marked by different colors) has been tracked. The primitive streak is underlined by a red stippled line and the node by a red circle.
Supp Fig. 1 Effects of Rok, Myosin and voltage gradient inhibitors on cell movements at the node. (A-P) Cumulative images recapitulating the position of electroporated cells between the different time points (noted in the lower right corners). Complete time lapse experiments in which the time series has been coded are shown in a, e, i, and m. The first time points are colored in blue and the last acquired time points are colored in red. (A-D) Movement of cells at the node (square region in a) in control embryos. (B) Cell movement during the primitive streak elongation. (C) Leftward movement of cells around the node. (D) Regression of the node and primitive streak. (E-L) Movement of cells at the node in Y-27632 (E-H) and Blebbistatin (I-L) treated embryos: first, cell movements are relatively unaffected (F, J), then cells exhibit disorganized movements and there is no leftward movement around the node (G, K). Finally, cells display disorganized movements and the primitive streak fails to regress (H, L). (M-P) Cell movement at the node in omeprazole treated embryos: Cells first converge normally toward the primitive streak (N). Cells from the left and right sides then move symmetrically toward the node but not around it (O). (P) The primitive streak regresses normally. Right panels in B-D, F-H, J-L, N-P highlight the trajectories of cells observed in Left panels. The position of the primitive streak and the node pit are represented with a red-dotted line and a red circle, respectively.
Supp Fig. 2 (A, E) 3D reconstruction of confocal views (Z-stacks) of stage 5 embryos (A) Y27632 and Omeprazole (E) treated embryos showing 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) 3D reconstruction of confocal views (Z-stack) of Phalloidin staining of Y-27632 and Omeprazole treated embryos(B, F, respectively) at stage 5. Images are depth-coded: the Phalloidin staining is pseudo-colored such that the dorsal-most staining is blue, the ventral-most staining is red. The shape of the node is outlined by a white-dotted circle. Expression patterns of Shh (C, G,) and FGF8 (D, H,) are more symmetric in drug treated embryos as shown by in situ hybridization.
Supp Fig. 3 (A-F) Embryos in which a human hair has been inserted through the primitive streak and the node in order to prevent asymmetric cell movements. (A, B, C) and (D, E, F) show symmetric expression domains of Shh and FGF8 respectively. (W) and (Z) are pictures from embryos shown in (U, V) and (X, Y) but the hair has been removed to fully reveal the domains of expression of Shh and FGF8. (G-J) Stage 6–7 embryos in which an incision has been made through the node and the anterior primitive streak at stage 4, in order to prevent the leftward movement of cells.(G, H) and (I, J) show bilateral expression of Shh and FGF8 respectively. The position of the node is marked with an asterisk.
Supp Fig. 4 (A, B) Median sagittal 1μm plastic sections of the pig node (n), posterior notochord (no) and floorplate (fp) at the late presomite stage (stage 6, A) and at the 5–6 somites stage (B) showing that the node and posterior notochord are completely covered by endoderm (e) throughout this period of development. Transverse section of a pig embryo showing left sided expression of nodal. (D-G) Confocal images of anti-acetylated tubulin staining showing cilia (white arrowheads) and mid-bodies (outlined arrowhead) at different levels (L1, L2, floor plate; L3, notochordal cells; L4, endoderm) of a pig embryo as noted in (C). (H-J) Ventral scanning electron microscopy views of a 5–6 somites rabbit embryo with higher magnifications (I-G) of the notochordal plate and adjacent paraxial mesoderm at the level of the caudal-most somite pair. As opposed to the pig, in the rabbit the endoderm covers the paraxial mesoderm only and is connected medially to the lateral edges of the notochordal plate; consequently, the notochord is free of endoderm and its typical long monocilia (6μm) can be seen on almost every cell. Numbers in C and F indicate the level of somite pairs; pnc: posterior notochord, n: node. Magnification bar: 20μm in A, B, and 5μm in I.
Supp. Fig 5 Alignment of Foxj1 sequences from various species, as shown, indicating conservation of the domain used as a probe in these studies. The grey box indicates overlap of the 832bp probe with the forkhead domain. For more information see the Methods section.
Supp. Figure 6. By stage 3 the node is morphologically symmetric, then a differential transient membrane depolarization drive the leftward movement of cells at the node, leading consequently to asymmetric expression of Shh and FGF8.