An Asymmetric Bilateral Body Plan at the Eight-Cell Stage
We find that after the ABa/ABp spindle skew that breaks the morphological LR symmetry of the embryo (Wood, 1991
), cells are further rearranged to assemble a stereotypical configuration at the 8-cell stage (). Specifically, 5 of the embryo’s 8 cells (ABar, MS, E, C, and P3
) are positioned on a plane that tilts from the embryo’s AP axis to the right side by 22±2° degrees (n=10) (; Movie S1
). The formation of the plane is highly reproducible as on average cells deviate from the plane by less than a quarter of nuclear diameter (, Figure S1B
). Two other cells, namely ABpl and ABpr, are positioned symmetrically on two sides of the tilted plane (, red nuclei). The eighth cell, ABal, is on the left side of the plane.
Formation of a Midline through Blastomere Rearrangements
This stereotypical configuration provides the blueprint for the bilateral body plan of C. elegans. Specifically, the tilted plane serves as the center of bilateral symmetry. According to the invariant cell lineage and fates, the five cells on the plane each generates a bilaterally symmetric structure and essentially contributes equally to the left and right sides of the organism (, see also below), with the minor exception of ABar, where one of its granddaughters (ABarpp) generates a bilateral structure. ABpl and ABpr, the two cells positioned symmetrically on the two sides of the plane, are equivalent fate-wise and give rise to symmetric structures on the left and right side, respectively. Thus, the tilted plane, bisecting the bilaterally symmetric founder cells and hence future bilateral structures, qualifies as the midplane, or the midline as it is commonly referred to. Notably, with the rightward tilt, the midline is uncoupled from the AP axis (i.e. the long axis of the ellipsoidal egg), which has not been seen in other bilaterian organisms.
Furthermore, the eighth cell, ABal, which does not have a bilateral counterpart in the cell lineage, is segregated to the left side of the tilted midline with no cell mirroring it on the right. Thus, the eight cells are organized into an inherently asymmetric bilateral body plan, with more cells on the left side of the midline. This asymmetric bilateral body plan provides the anatomical basis for how the invariant Notch inductions can generate LR fate asymmetry, which was not fully understood before (see Discussion; Hutter and Schnabel, 1995
; Hermann et al., 2000
The tilted midline configuration is maintained until the 88-cell stage (the AB64
stage; , middle panel), as the bilaterally symmetric founder cells divide symmetrically with respect to the tilted midline (). Cell movements then start to gradually adjust the midline over the next two cell cycles, so that by the 350-cell stage (AB256
; , right panel), it aligns with the AP axis (Movie S2
and see Sulston et al., 1983
; Schnabel et al., 1997
; Zhao et al., 2008
). The re-alignment of the midline with the AP axis restores the spatial symmetry of the bilateral structures of the worm. Thus, the dynamic positioning of the midline relative to the AP axis organizes both the LR asymmetric structures and the integration of these asymmetries into a superficially symmetric body plan ().
The Tilted Midline Organizes Body Plan LR Asymmetry and LR Lineage Symmetry
Additionally, the rearrangement of cells during the assembly of the body plan further increases the morphological LR asymmetry caused by the ABa/ABp spindle skew, on top of creating the architectural framework for the bilateral body plan. Quantitatively, the skew increases from 19±5° at the end of the ABa/ABp division to 48±6° after the rearrangement ().
Chiral Morphogenesis Assembles the Asymmetric Bilateral Body Plan
Analyzing time lapse recordings of embryos expressing a plasma membrane marker (PH-domain of PLC1δ1 fused to mCherry; Audhya et al., 2005
), we find that the rearrangement of cells during the assembly of the bilateral body plan involves reproducible LR asymmetric cell behaviors and movements with a specific handedness ( left panel). We term this process chiral morphogenesis. As the ABa/ABp spindle skew finishes and before the contractile ring fully closes, the LR equivalents, ABpl and ABpr, start to exhibit a set of asymmetric behaviors. ABpl undergoes dramatic shape changes: It forms a dorsal lamellipodium, a ventral protrusion as well as anterior filopodial extensions (, upper right panel, marked 1, 2 and 3, respectively), and migrates anteroventrally. Besides these protrusions, ABpl also forms an apparently adhesive contact with C, as the two cells form an hourglass shape (, upper left panel, middle frame and Movie S3
). In contrast, ABpr shows only a rudimentary ventral protrusion and ruffling on its anterior front instead of filopodial extensions (, lower right panel, marked 2’ and 3’, respectively and Movie S3
) and does not move significantly. Another cell with significant shape changes is MS, which forms an elaborate lamellipodial protrusion at its anterior front (, lower left panel).
Chiral Morphogenesis Mediates the Formation of the Asymmetric Bilateral Body Plan
With the LR asymmetric protrusive activities, cells show a handed movement and rearrange accordingly. The first major aspect of the cell rearrangement involves three cells, namely ABpl, MS, and ABar (Figure S2A
). Their movements can best be described as a collective rotation around the AP axis ( and Movie S4
). Looking from the anterior end of the embryo, the rotation is clockwise by 83±7° (n=10), and brings ABar and MS onto the midplane ( and S2B
). As other cells do not show circumferential movements (Movie S4
; Figure S2B
), this is not a whole-embryo rotation as previously indicated (Sulston et al., 1983
; Schnabel et al., 1997
). The second major aspect of the cell rearrangement is the juxtaposition of ABar and C. As ABar shifts posteriorly during the three-cell rotation, C shifts anteriorly to meet ABar, likely pulled by ABpl given the hourglass shape (see above). The juxtaposition makes the five-cell midplane contiguous () and separates ABpl and ABpr (red in ), which are sisters and born next to each other, to two sides of the midplane. Besides this, the ABar-C contact is also required for ABar to receive a Wnt signal from C to orient its spindle (Walston et al., 2004
The protrusion formation and the collective cell movement both occur reproducibly and are well coordinated temporally ().
Coordination and Timing of Chiral Morphogenesis
In order to understand how the dynamic protrusions described above mediate the cell movements, we analyzed actomyosin dynamics during chiral morphogenesis. To this end we imaged F-actin by using Lifeact::GFP (Riedl et al., 2008
), and non-muscle myosin II heavy chain (NMY-2::GFP; Munro et al., 2004
). F-actin strongly accumulates in the anterior and dorsal protrusions of ABpl (, upper panel; Movie S5
) and the lamellipodium of MS (, lower panel). Furthermore, as actin polymerization driving protrusion formation requires the Rho GTPase Cdc42 (Pollard et al., 2000
), we observed a loss of protrusions when we deplete embryos for CDC-42 by RNAi (Figure S3A
and data not shown). These findings show that actin polymerization mediates the formation of protrusions and the directional spreading of ABpl and MS. Furthermore, NMY-2 accumulates at the anterior front of ABpl and fills the filopodial protrusions as they grow (, asterisks), indicative for a role of NMY-2 in cell-cell contact formation in these protrusions (Conti et al., 2004
Actomyosin Dynamics and the Coordination of Protrusion Formation
We further investigated the ventral protrusion of ABpl. Temporally and spatially, this protrusion leads the ventral movement of ABpl and the translocation of ABar (): it forms in all embryos examined (n=50) and its size is proportional to the degree of the ventral movement (n=10, ). More specifically, the ABpl ventral protrusion starts to form as the EMS cell forms its cytokinetic contractile ring (, arrow). A small protrusion first forms onto the future MS part of the cell. It then turns into rapid and directional growth along the contact with MS, thus does not seem to simply fill the open space of the EMS furrow (Figure S4
). Interestingly, formation of the ventral protrusion is not associated with an enrichment of F-actin (), but with NMY-2 (). It is therefore possible that it constitutes a ‘cryptic’ lamellipodium (Farooqui and Fenteany, 2004) which has been shown to mediate mechanical coupling during collective migration.
To test if ABpl’s ventral protrusion is triggered by EMS cytokinesis, or if the timing of the two events is coincidental, we delayed EMS division by UV irradiation. We observe that belated EMS divisions delay the formation of the ABpl protrusion, and the delayed protrusion forms when the EMS starts to form the cytokinetic furrow (). In the more severe cases where EMS division is delayed until after ABpl divides, ABpl does not form the protrusion (, left panel). These results suggest that the ABpl ventral protrusion is indeed triggered by EMS cytokinesis. Furthermore, delaying ABpl protrusion delays and reduces ventral movement of ABpl and the ABpl-MS-ABar rotation (, middle and right panels). When the protrusion does not form, there is no ventral movement or collective rotation (, right panel). The irradiation does not affect other protrusions of ABpl (, middle panel, see also below), suggesting that the effect is specific to the ventral protrusion. Furthermore, irradiating other neighbors of ABpl, such as ABal, does not affect the ABpl ventral protrusion or movement (data not shown), suggesting that the irradiation effect is EMS specific. Thus, the results suggest that the ABpl ventral protrusion is triggered by EMS cytokinesis and is required to mediate ABpl’s ventral movement and chiral morphogenesis.
Abolishing the ventral protrusion also reveals the nature of the other behaviors of ABpl: The anterior and dorsal protrusions are not affected, suggesting that their formation does not depend on the interaction with the EMS furrow (, middle panel). Furthermore, as ABpl does not move ventrally in this case, the result suggests that the dorsal protrusion is an active process, rather than a passive adhesion patch being stretched by ABpl’s movement. In contrast, the stretching of C by ABpl disappears, suggesting that it is the result of traction force as ABpl’s movement pulls on C.
Handedness of Chiral Morphogenesis
As described above, ABpr, the bilateral equivalent of ABpl fate-wise, has a much more reduced ventral protrusion in terms of size and duration compared to that of ABpl. Given that the ABpl ventral protrusion is required for chiral morphogenesis, this difference may determine the handedness of chiral morphogenesis, that is, the clockwise direction of the cell rearrangement. We therefore investigated how the asymmetry and handedness are brought about.
Given that EMS cytokinesis triggers the ventral protrusion, we first focused on the EMS furrow. EMS undergoes an asymmetric abscission with the contractile elements coalescing faster on the left side and the midbody forming on the right (). Thus, we tested if the asymmetric furrow, with faster contraction on the left, may direct ABpl to grow its ventral protrusion faster and/or more significantly than ABpr. Specifically, we perturbed the asymmetric furrow by depleting embryos of the septins UNC-59/61 with RNAi, which are required for asymmetric abscission (Maddox et al., 2007
). The perturbation led to cases where the EMS midbody appears on the left instead of the right side (Movie S6
). The result suggests that there is a novel mechanism that aligns the intrinsically asymmetric contractile ring with the LR body axis and that septins are required for the alignment. However, the asymmetry between ABpl and ABpr ventral protrusions is not affected and chiral morphogenesis occurs as in the wild type. This result further confirms that the ABpl ventral protrusion is not a passive response to fill the open space of the EMS furrow as the space on the left side is much smaller than the wild type. More importantly, the result suggests that while the EMS furrow triggers the ventral protrusion formation, its LR asymmetry does not regulate the handedness of chiral morphogenesis.
Handedness of Chiral Morphogenesis
We then tested if the handedness of the ABa/ABp spindle skew sets the handedness of the ABpl-ABpr asymmetry and chiral morphogenesis. To do so, we reversed the handedness of the ABa/ABp spindle skew with two different conditions. First, we cultivated worms at low temperature, which induces reversal of the ABa/ABp spindle skew in a fraction of embryos by impairing an unknown process before fertilization of the oocyte (Wood et al., 1996
). In the cases where we observed reversed spindle skew, mirror-imaged chiral morphogenesis occurs: ABpr instead of ABpl undergoes a dramatic shape change, forms protrusions and the cellular rearrangement is a counterclockwise rotation (, lower panel; Movie S7
). As a result, the midplane is tilted to the left and C induces a spindle rotation in ABal instead of ABar. Second, we analyzed a temperature-sensitive mutant for a Gαi gene, gpa-16
(it143). At non-permissive temperature, gpa-16
(it143) randomizes ABa/ABp spindle orientation, thus gives rise to a fraction of embryos where the spindles lie in the reversed direction (Bergmann et al., 2003
). We observed 4 such reversed cases out of a total of 65 embryos. Consistent with the cold-induced reversals, all 4 embryos showed mirror-image chiral morphogenesis ( and Movie S8
). These results suggest either that the ABa/ABp spindle skew is an upstream event that sets the handedness of chiral morphogenesis, or that the two events are in parallel under the assumption that the two experimental conditions both perturb an early symmetry breaking event to which the spindle skew and chiral morphogenesis react identically.
LR Asymmetry in Cortical Contractility
Given that ABpl and ABpr are bilateral equivalents fate-wise, we reason that their asymmetric behaviors might lie in mechanical LR asymmetries. Indeed, ABpl and ABpr show different cortical morphologies and actomyosin dynamics. Spreading of both cells is accompanied by cortical flow and the formation of an apical NMY-2 cap-like structure (Movie S9
). Specifically, cortical NMY-2 flow starts as cells exit cytokinesis, from the periphery towards the center of the apical surface. When cortical flows reach a steady state, NMY-2 forms a torus-like structure in ABpl (, left panel). In ABpr, however, the torus collapses into a central patch (, right panel). As it has been shown that cortical flows originate from dynamic contractions of actomyosin (Munro et al., 2004
), we reasoned that the observed cortical asymmetry might also reflect differential contractility of cells. We therefore quantified the spreading of the two cells by measuring the maximal apical extension of ABpl and ABpr along the AP axis. We find that ABpl occupies more space on the surface of the embryo than ABpr (). Thus, the molecular dynamics and the morphological differences suggest asymmetric contractility between ABpl and ABpr.
LR Asymmetries in ABpl/pr Cortical Contractility
To further test the hypothesis of mechanical LR asymmetry, we perturbed the cortical actin dynamics by depleting WAVE-Arp2/3 complex components with RNAi. The WAVE-Arp2/3 complex (Figure S3E
) is required for polymerization of branched actin filaments and for strengthening of the cortex, hence reduction of WAVE-Arp2/3 should enhance contractility. Indeed, we find that RNAi of arx-2/-3/-4/-7
(summarized as WAVE-Arp2/3 RNAi) leads to large ruptures of the cortex and blebbing (Severson et al. 2002
; Figure S3F and G
). With RNAi of the WAVE-Arp2/3 complex, the ABpl cortex starts to resemble ABpr: The torus-like NMY-2 structure collapses into a solid patch ( and S3B
), and ABpl’s flattening is reduced (, upper panel).
According to our hypothesis, the reduction of actin polymerization and actomyosin contractility, should also abolish cortical asymmetries. To this end, we used RNAi to deplete CYK-1, which is a formin homolog responsible for linear actin polymerization and the Arp2/3-independent assembly of the actomyosin cortex (Severson et al, 2002
). We find that ABpl and ABpr now resemble each other. In both cells, apical NMY-2 is greatly reduced, the apical surface is round (), and the two cells occupy comparable space on the surface of the embryo because ABpl now flattens less than the wild-type (, lower panel).
These results suggest that ABpl and ABpr regulate their cortical contractility differently and exert mechanical asymmetry during chiral morphogenesis. The observed direction of NMY-2 flow further suggests that cells do not roll freely into their new positions. Specifically, NMY-2 flows towards the apical center without a circumferential component in the flow, which would be expected if cells roll. Consistent with the notion that deformation-based forces are involved in cell movement, perturbation of actomyosin contractility in the above experiments greatly reduce cell movement and consequently the tilt of the midline decreases from 22±2° in wild-type embryos to 6±3° (WAVE-Arp2/3 RNAi) or 3±3° (cyk-1
RNAi) (Figure S3D
More importantly, in 6 out of 21 embryos depleted for WAVE-Arp2/3, ABpr instead of ABpl undergoes significant ventral movement () and the collective cell movement is counterclockwise, the opposite of normal chiral morphogenesis (; Movie S10
). In contrast, the initial spindle skew of ABa/ABp in these cases is normal, suggesting that the chiral forces driving the handed collective cell movement is perturbed, rather than the upstream LR cue. Interestingly, in these cases ABpr forms a large ventral protrusion similar to that of wild-type ABpl. While the RNAi would affect actomyosin in all cells, a possible interpretation is that the enlarged ventral protrusion in ABpr competes with the ventral protrusion of ABpl, leading to a randomization in terms of the direction of the collective cell movement. Such an interpretation is consistent with the various results in the above sections, which all attest to an essential role of the ABpl ventral protrusion in chiral morphogenesis.
Non-Canonical Wnt Activates Cortical Dynamics and Chiral Morphogenesis
Non-Canonical Wnt Signaling is Required for the Formation of the Asymmetric Bilateral Body Plan
We find that reduction of MOM-2 (Wnt) by RNAi greatly reduces the dynamic shape change of ABpl. The ventral protrusion as well as the dorsal protrusion connects ABpl to ABar in the clockwise rotation and the anterior filopodia onto ABal, are reduced or abolished in 30% of the mom-2
RNAi embryos (n=20). Meanwhile, the global translocation of the 4 AB cells is reduced by about 15% (n=5) (). Consequently, the tilt of the midplane is reduced from 22±2° to 6±3°. In 75% of the embryos (n=20), ABar and C fail to contact each other, which in turn leads to a failure of ABar spindle rotation (, upper panel). Other genes in the Wnt signaling pathway, namely gsk-3
, and mig-5
(Disheveled) give similar phenotypes (, middle panel and data not shown). However, RNAi of pop-1
, the sole homolog of the TCF/Lef transcription factor downstream of Wnt in C. elegans
, does not affect the protrusions or chiral morphogenesis (, lower panel), even though the RNAi leads to the known phenotype of the MS to E fate transformation (Lin et al., 1995
). Thus, our results suggest that it is the non-canonical Wnt pathway that regulates chiral morphogenesis without a transcriptional response. In mom-2
RNAi embryos the apical flow of NMY-2 and the torus-like NMY-2 cap is impaired (), although NMY-2::GFP background levels in ABpl are similar to the wild-type (). As the spindle skew in ABa/ABp is not perturbed in these experiments, our results suggest that the non-canonical Wnt pathway acts as a permissive signal to activate the dynamics of the actomyosin cortex and chiral morphogenesis.