MINI REVIEW article

Front. Cell Dev. Biol., 24 September 2025

Sec. Morphogenesis and Patterning

Volume 13 - 2025 | https://doi.org/10.3389/fcell.2025.1672263

Left-right symmetry breaking: learning from the chicken

  • Department of Anatomy and Cell Biology, University Medical Center Göttingen, Göttingen, Germany

Abstract

Morphological left-right asymmetry of visceral organs in most cases reveals a typical arrangement. This implies directed symmetry breaking which is suggested to be based on the existence of structural chirality. At early developmental stages many vertebrate model organisms display so-called leftward flow of extracellular fluid which is based on the unidirectional rotation of chiral cilia. Cytoskeletal chirality has been shown to contribute to the left-right asymmetry of invertebrates including Caenorhabditis elegans and Drosophila melanogaster. The mechanisms of left-right symmetry breaking in vertebrates without ciliary flow remain mysterious. Here, we present our perspective on left-right patterning and symmetry breaking in the chick within a broader context.

Meeting point nodal

Left-right asymmetry of viscera is a fundamental anatomical feature of vertebrates and many invertebrates (). Morphological asymmetry is manifested both in bilateral asymmetry or handedness of organs and their asymmetric position and was shown to be preceded by asymmetric gene expression and asymmetrical molecular signaling (referred to here as left-right patterning). Expression of TGF-beta member Nodal in the left lateral plate mesoderm is a common denominator of early left-right molecular patterning in studied vertebrates (; ). Well-studied effector of asymmetric Nodal activity is the transcription factor Pitx2, which has been shown to be directly involved in asymmetric morphogenesis of the heart and the gut (; ). Interestingly, the lateral plate mesoderm is involved in the morphogenesis of asymmetrical organs contributing to heart and connective tissues of the gut. Nodal-Pitx2 module was also reported to be involved in asymmetrical development of amphioxus, snails and sea urchins. Asymmetrical gene expression, however, is preceded by a symmetrical state, displaying either bilateral absence or bilateral presence of gene expression or molecular activity. Assuming original bilateral symmetry of a developing embryo, asymmetric gene expression is preceded by a process described as initial symmetry breaking which is a transition to a state with higher symmetry.

The (chiral) form is the cause

The original idea proposed by Pierre Curie suggested that the symmetry elements in the cause must be found in their effects and asymmetries in the effect are derived from asymmetries in the causes (). Further development of the symmetry breaking concept led to distinction between explicit symmetry breaking, which follows the above described definition and spontaneous symmetry breaking () where the outcomes are distributed equally (outcomes are randomized). An important feature of animal left-right symmetry breaking, however, is its directionality: the resulted sidedness after the symmetry breaking is not random. Hence, the initial animal left-right symmetry breaking cannot be described as spontaneous. This suggests underlying structural molecular asymmetry which may be based on chirality as known in chemistry. Wolpert and Brown () proposed the existence of a chiral molecular determinant denominated as F-molecule which generates initial asymmetry subsequently translated at cellular and organismal level. Afzelius reported a correlation between disturbed laterality and abnormal cilia (): ultrastructural examination of immotile sperm in 4 patients with impaired mucociliary transport and associated recurrent bronchitis revealed the absence of dynein arms in the cilia with three out of four patients also showing situs inversus totalis. The association of bronchiectasis accompanied by sinusitis with situs inversus was already described by Manes Kartagener (). Afzelius proposed that the cilia motility in embryonic tissues critically contributes to the right-left asymmetries of adult organisms (). As cilia reveal structural chirality they fulfill requirements of the hypothetic F-molecule. Indeed, shortly after the proposed role of the hypothetical F-molecule, motile cilia were shown to be present in early mouse embryos during gastrula and early somitogenesis: the ventral surface of epithelialized midline cells assigned to the notochord and node possess long motile monocilia (). Based on results of Afzelius and observation that the comparable developmental stage in rat embryos is critical for the establishment of right-left asymmetry (), Sulik and co-workers suggested a causal contribution of motile monocilia to left-right symmetry breaking (). Screening of mouse strains with complete inverted viscera revealed mutations in cilia-associated axonemal heavy-chain dynein (). Similarly, it was shown that mice depleted for kinesin KIF3B do not form monocilia and reveal randomized left-right asymmetry (). Furthermore, in WT embryos corresponding cilia undergo rotation accompanied by leftward fluid flow as shown by video microscopy of ventral surface of embryos cultured in a medium with fluorescent beads. The experimental reversal of flow direction led to the formation of situs inversus while an artificially generated flow in embryos with defective cilia “rescued” the phenotype ().

The direction (of flow) decides where it becomes the left side

Leftward flow was observed in frog Xenopus laevis, zebrafish and mouse while morphological signs of a ciliated organizer were reported for many further but not for all studied vertebrates (; ). Successful symmetry breaking requires a temporary formation of a specific “organ”: the ciliary leftward flow is located at the ventral surface of specialized structure called left-right organizer (LRO, cf. Figure 1). Crucially, symmetry breaking by leftward flow is followed by subsequent activation of nodal signaling at the left-side (; ). Two mechanisms were proposed to explain how leftward flow caused left-right patterning. Initial proposal suggested leftward transport of morphogens () while later studies suggested existence of sensory immotile cilia (; ). Indeed, recent studies in mouse and zebrafish embryo strongly support the view that deflection of immotile cilia by flow which lead to polycystin-2 (Pkd2) channel mediated activation of intraciliary Calcium transients (; ). How asymmetrical Calcium transients influence left-right patterning is to be investigated in more detail (). Particularly, it has been suggested that Calcium transients activate RNA binding protein Bicc1 which in turn suppresses the translation of nodal antagonist dand5 mRNA (). The LRO is formed by axial and (in Xenopus) paraxial mesoderm progenitors which form the gastrocoel roof plate in amphibian (; ), the posterior notochord in mouse and rabbit (; ) or the Kupffer’s vesicle in zebrafish embryos (). Function of LRO requires the correct domain architecture and differentiation. The effectively directed flow was suggested to be caused by posterior position and structural features of the motile cilia (). Hence, the correct position of cilia is a function of established planar cell polarity along anterior-posterior axis (; ; ; ; ; ). In Xenopus and mouse embryo LRO includes spatially segregated domains characterized by gene expression and types of cilia (; ; ). Particularly, lateral domains which form at both sides immotile cilia bilaterally express nodal, its antagonist dand5 and in Xenopus somitic marker myoD (; ; ). Importantly, to create the appropriate flow and detect it, ciliated cells should be transiently faced towards a cavity of the LRO whereas after the symmetry breaking event, the surface is covered by endodermal cells. Abnormal development of LRO with a LRO covered by endoderm prevents correct symmetry breaking in Xenopus (; ). Recent observations indicate involvement of morphogen transport in left-right symmetry breaking (; ) suggesting a complex and possibly double-secured mechanism. Flow-mediated transport of PKD to the left of the mouse LRO has been suggested to contribute to left-right symmetry breaking in the mouse embryo (). It must be mentioned that further, earlier asymmetries have been observed in Xenopus and mechanisms based on these have been discussed (; ; ), which still have to be integrated into the existing models.

FIGURE 1

There is another way

Leftward flow has been suggested to be required for directed LR symmetry breaking in many vertebrate models (; ; ) and is one of the most fascinating observations in developmental biology. However, structural elements required for leftward flow are absent in several model organisms. Structural units required for ciliary flow were not detected in chick and pig embryos where the ventral surface of axial mesoderm was shown to be covered by endoderm and subchordal mesoderm (; ) while analysed non-avian reptilian do not form motile cilia (; ). Furthermore, in the chick, the first signs of left-right asymmetry occur at late gastrula stage and include the morphological asymmetry of the node and asymmetric leftward cell movements around the node (; ). These observations also match the asymmetric gene expression in the chick node prior to asymmetry of nodal expression in the lateral plate mesoderm: sonic hedgehog morphogen (shh) was shown to be expressed asymmetrically in the node of notochord stage (stage 5) chick embryo (; ) while this asymmetry was suggested to cause left-sided nodal expression at somitogenesis stages. Further analysis of chicken node suggested asymmetrical morphology of the node itself prior to asymmetric gene expression ().

We suggest that the chick primitive node plays the role of the LRO. In the following we explain the corresponding developmental context.

This is the way (of the chicken)

Gastrulation in the chick starts with the formation of the primitive streak in the posterior area of circle-shaped bilayered embryonic disc (stage 1). Cell intercalation in the posterior epiblast has been suggested as a cellular mechanism of primitive streak formation. Intercalation was shown to be PCP-dependent () and/or be based on Myosin-II based contractions of epiblast cell groups (). Cell division in the anterior pole of the embryo accounts for the tissue fluidity that is necessary for primitive streak formation () while the tensile ring at the margin of the area pellucida is required for tissue flow during primitive streak formation (). Primitive streak undergoes elongation which is accompanied by bilateral flow of cells of dorsal (epiblast) layer towards the streak. The node which gives rise to the axial mesoderm emerges at the anterior tip of the streak and starts to form the prechordal mesoderm (). The maximum of elongation of the primitive streak is followed by the beginning of notochord formation and concomitant shortening of the streak also referred to as streak regression. Global cell movements towards the emerging streak reveal left-right asymmetry, even if the functional significance of this asymmetry remains to be tested (). The first morphological asymmetry arises in the node at the beginning of notochord formation: dorsal views reveal that the right edge of the node is thicker than the left edge while sections demonstrate that this difference is due to mesodermal density at the right side below the epithelial epiblast (). Asymmetric position of density is already detectable prior to the emergence of the notochord. Emergence of the asymmetric notochord is preceded by asymmetrical counterclockwise cell movements within the node (cf. Supplementary Figure). It was suggested that the progressive asymmetry of sonic hedgehog expression is due to displacement of shh domain to the left-side while pharmacological evidence indicates that node rotation requires actomyosin contractility and activity of ATP4a proton pump (). Moreover, N-Cadherin is involved in termination of node rotation (). Further analysis however, revealed, that paraxial asymmetrical expression of nodal is initiated at the beginning of notochord formation while shh is still expressed at both sides of the node () hence indicating that nodal asymmetry is not a direct effect of node and shh domain rotation. Asymmetry of the node and asymmetric shh expression increase concomitant with node regression during stages 5 and 6. Surprisingly shh-expression at these stages is confined to the epiblast of the left node shoulder, to the floor plate, to the prechordal mesoderm but not to the posterior notochord () challenging the widely assumed induction of floor plate by the notochord.

Morphological analysis of the node and notochord reveals the continuity of the notochord with the right node shoulder. Assuming that notochord progenitor cells are generated within the node this continuity suggests an asymmetrical notochord formation. This proposal is supported by analysis of notochordal marker expression particularly noggin () and brachyury. The origin of the notochord from the right side of the node results in positioning of the shh expressing floor plate to the left of the notochord and immediately above the paraxial nodal expressing area. This spatial proximity suggests a local induction of the paraxial nodal domain by hedgehog signaling activated by shh ligand secreted from the floor plate. Indeed, the inhibition of hedgehog signaling leads to an absent paraxial nodal expression () while ectopic hedgehog activation () causes bilateral nodal expression. This data strongly suggests that secreted hedgehog is both necessary and sufficient for paraxial nodal induction in the chick while the asymmetry of nodal is due to asymmetry of the notochord formation (Figure 2). Interestingly, prior to asymmetric morphogenesis the node revealed matrix-filled spaces which may be involved in regulation of molecular activity ().

FIGURE 2

Cytoskeleton: hidden egg?

What are the mechanisms leading to the asymmetric morphogenesis of the avian node? Leftward rotation of the node is a robust morphogenetic event and indicates intrinsic tissue chirality which in turn has been suggested to have roots in the cytoskeletal organisation particularly in chiral features of the cytoskeletal constituents such as microtubules or actin (; ; ). Indeed, cytoskeletal asymmetries were shown to be involved in left-right symmetry breaking during development of C. elegans which reveals chiral cortical flow related to contractility of actomyosin () and regulated by RhoA and Cyk1/Formin (; ). Chiral development of snails is also controlled by formins dia1 and dia2 () which regulate actin nucleation and polymerisation while further actin nucleator DAAM () together with unconventional myosin ID (Myo1D) are involved in left-right asymmetry in Drosophila melanogaster (; ). Similar to other members of Myosin I family, Myosin ID was shown to link actin with membrane lipids () and it has been suggested that the role of Myosin ID as a chiral determinant is due to its chiral interaction with actin ().

What's next?

It has been proposed that cellular-scale chirality arises from chiral structures by interaction within “chirality modules” particularly between actin filaments and formin dimers, whereas the understanding of transition to tissue chirality remains challenging ().

How does the cellular chirality contribute to left-right symmetry breaking in the chick? At elongated streak stage only the node tissue displays chiral behaviour which manifests itself during a short period of time. This indicates that the supracellular manifestation of chirality is a subject of regulation. The node which is equivalent of Spemann’s organizer () undergoes different stages: it emerges in the area expressing organizer genes, contributes to the prechordal mesoderm, endoderm, medial paraxial and axial mesoderm. Node rotation takes place after migration of the prechordal mesoderm and clustering of axial mesoderm progenitors in the node mesoderm (). These events may stimulate activation of tissue chirality in the node area. Whether this activation is due to specific molecular pathway or structural constraint has to be investigated in the next step.

Statements

Author contributions

TP: Writing – original draft, Writing – review and editing. NT: Conceptualization, Visualization, Writing – original draft, Writing – review and editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. We acknowledge support by the Open Access Publication Funds of the Göttingen University.

Acknowledgments

We would like to thank Prof. Axel Schweickert for helpful discussion.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The author(s) declare that no Generative AI was used in the creation of this manuscript.

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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2025.1672263/full#supplementary-material

SUPPLEMENTARY FIGURE

Asymmetrical node morphogenesis in the chick embryo (A–C) scanning electron microscopy of the node prior to notochord formation (stage 4: A,B) and at the early notochord stage (stage 5, C,D) embryos. (B) schematic view of asymmetrical node (green) displacement (as reported by and ), (C)– emergence of thicker right node shoulder (rs). (D) floor plate (green) and notochord (red) domains indicated at the surface. A modified from Figure 1F () with permission.

References

Summary

Keywords

left-right symmetry breaking, gastrulation, chirality, vertebrate embryo, cytoskeleton

Citation

Pieper TK and Tsikolia N (2025) Left-right symmetry breaking: learning from the chicken. Front. Cell Dev. Biol. 13:1672263. doi: 10.3389/fcell.2025.1672263

Received

24 July 2025

Accepted

03 September 2025

Published

24 September 2025

Volume

13 - 2025

Edited by

Glenn S. Edwards, Duke University, United States

Reviewed by

Shigenori Nonaka, Graduate University for Advanced Studies (Sokendai), Japan

Updates

Copyright

*Correspondence: Nikoloz Tsikolia,

Disclaimer

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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