Meteorins regulate the formation of the left-right organizer and the establishment of vertebrate body asymmetry

From the outside, the vertebrate body plan appears bilaterally symmetric. However, internal organs positioning and morphology often display left-right (L-R) asymmetries. For instance, in vertebrates, the heart generally lies on the left side, the liver and the pancreas are positioned on the right, and the gut presents asymmetric rotations.

One major regulator for the establishment of the L-R axis is Nodal, a ligand belonging to the TGFβ protein family (Blum et al., 2014; Blum and Ott, 2018; Grimes and Burdine, 2017; Kawasumi et al., 2011; Branford and Yost, 2004; Zhou et al., 1993). Its signaling is activated in the left lateral plate mesoderm (LPM), whereas Nodal remains inactive in the right LPM, creating an embryo-scale left-right asymmetry (Grimes and Burdine, 2017). In mice, this asymmetry is achieved by an oriented rotation of the cilia of a structure called the Node, generating a leftward flow of extraembryonic fluid named the Nodal flow. This results in an asymmetric expression of several Nodal factors and Nodal signaling. Participating in this patterning, Leftys, soluble inhibitors belonging to a subclass of TGF factors, antagonize Nodal signaling (Chen and Schier, 2002; Chen and Shen, 2004).

The Node and the Nodal flow have been described in numerous vertebrates. For instance, in Xenopus the gastrocoel roof plate and in zebrafish the Kupffer’s vesicle (KV; Essner et al., 2005; Vick et al., 2009; Nonaka et al., 2002; Schweickert et al., 2007; Okada et al., 2005; Tanaka et al., 2005) are key structures of the L-R symmetry breaking during the embryonic development (Nonaka et al., 2002; Okada et al., 2005; Nonaka et al., 1998). Similarly, in humans and other mammals like rabbits and mice, the systematic establishment of left-right body symmetry begins with asymmetric fluid flow driven by rotating cilia (Smith et al., 2019). This occurs within the transient primitive node in humans or the embryonic node in mice and rabbits (Okada et al., 2005; Nonaka et al., 1998; Schoenwolf et al., 2014). However, in other mammalian classes, although left-right symmetry breaking mechanisms are believed to be cilia-independent, they are still poorly understood at present (Blum and Ott, 2018; Hamada et al., 2002). In chicks, the symmetry-breaking structure known as Hensen’s node serves as the ciliated organizer responsible for the left-right asymmetry establishment. In this species, the process of symmetry breaking is believed to occur independently of the non-motile cilia present within Hensen’s node. Instead, the breaking of symmetry is primarily attributed to the leftward movement of cells around the node (Gros et al., 2009; Essner et al., 2002; Stephen et al., 2014).

Compared to the relatively flat-shaped mouse node, the zebrafish KV is a fluid-filled sphere with a ciliated epithelium, and it is formed by dorsal forerunner cells (DFCs; Essner et al., 2005). DFCs first emerge as cells of the epithelium of the dorsal surface in direct contact with the yolk syncytial layer at 6 hours post-fertilization (hpf; Oteíza et al., 2008). Around 8–9 hpf, following the epibolic movement, 20–30 DFCs form a single cluster and migrate towards the vegetal pole in a non-involuting manner at the leading edge (Oteíza et al., 2008; Cooper and D’Amico, 1996). During this migration process, polarized DFCs organize into multiple focal points by retaining long-lasting apical contacts within the cluster, and remaining delaminated DFCs maintain contact with the cluster by cell-cell contact mechanisms (Oteíza et al., 2008; Pulgar et al., 2021). At the final phase of epiboly and after reaching the vegetal pole, the DFC cluster is separating from the dorsal marginal enveloping layer cells by losing the apical contacts (Oteíza et al., 2008; Pulgar et al., 2021). The focal points of the cluster are then rearranged by integrating the unpolarized DFCs to form a single focal point that will expand into a monolayer rosette structure containing a lumen to form the KV by 12 hpf (Essner et al., 2005; Oteíza et al., 2008; Cooper and D’Amico, 1996; Pulgar et al., 2021). Along this process, non-motile and motile monocilia are generated on the apical membrane facing the lumen that create a counterclockwise flow of fluid by the KV, called Nodal flow (Oteíza et al., 2008; Kramer-Zucker et al., 2005). It was shown that the leftward-directed flow within the KV results in asymmetric expression of zebrafish Nodal genes, like spaw, lefty1, and lefty2, which is fundamental for proper L-R patterning (Hirokawa et al., 2006; Harvey, 1998). However, the molecular mechanisms involved in correct DFC migration and clustering that will form the KV remain largely elusive.

In this context, it has been reported that nodal, lefty, as well as p-smad2 expression is downregulated in Meteorin-null ES cells. As such, Meteorin (Metrn) was hypothesized to be a novel important regulator of Nodal transcription (Kim et al., 2014).

Meteorin (Metrn), a secreted neurotrophic factor highly conserved among vertebrates, is expressed during early mouse development. It is already detected at the blastocyst stage in the inner cell mass and then expands through the extraembryonic ectoderm to the central (CNS) and peripheral nervous system (PNS) during later developmental stages (Kim et al., 2014). It was first described to induce glial cell differentiation and to promote axonal extension in dorsal root ganglion (DRG) explants in vitro (Nishino et al., 2004). The disruption of Metrn function in mice resulted in early embryonic lethality (Kim et al., 2014), preventing the investigation of Metrn protein function during early embryonic development in vivo and, in particular, their role in L-R patterning. Also conserved among vertebrates, its paralog Meteorin-like (Metrnl) has been first reported as a downstream target of the Pax2/5/8 signaling pathway during otic vesicle development (Ramialison et al., 2008) and was later shown to have neurotrophic properties comparable to the ones of Metrn (Jørgensen et al., 2012).

Here, using the zebrafish larva as a model system, we generated knockout lines for all three existing zebrafish metrn genes (metrn, metrnla, and metrnlb). While all three single and compound mutants are viable, metrns mutant embryos displayed organ patterning defects, notably with randomized or symmetric heart looping at a significantly higher frequency. Like in mice, metrn genes are expressed during early development already from the two-cell stage, and transcripts are detected in DFCs as well as in the KV structure. We show that metrns loss of function leads to DFC disorganization and Nodal flow formation defects within the KV. Together, our study reveals a critical role for Metrn proteins in the DFC clustering, KV formation, and in the establishment of the L-R axis.

Proteins of the Meteorin family were first described as secreted neurotrophic factors highly conserved among vertebrates (Kim et al., 2014; Nishino et al., 2004; Ramialison et al., 2008; Jørgensen et al., 2012). In mice, Metrnla mutants display phenotypes as impaired angiogenesis after ischemic injury (Reboll et al., 2022) or dysregulated cytokine production (Ushach et al., 2018). The disruption of Metrn resulted in early embryonic lethality (Kim et al., 2014), preventing the investigation of the combined role of Metrn proteins in early embryonic development in vivo and, in particular, its role in L-R patterning. Here, using the zebrafish larva as a model system, we generated knockout lines for all three zebrafish metrn genes, metrn, metrnla, and metrnlb, which are all viable and fertile, possibly attributed to species-specific differences in compensatory mechanisms, allowing for the study of Meteorins functions during early embryonic development.

Our results demonstrate that Metrns are required for symmetry breaking and the establishment of L-R asymmetry, a novel role for Meteorin proteins during vertebrate embryonic development. All three paralogs analyzed, when genetically deleted, had an effect on heart looping formation, with the strongest effect for Metrnla and Metrn, while Metrnlb had a smaller but still significant effect. In our in situ analysis, we failed to detect early metrnlb expression. We do not exclude that at early stages, metrnlb could be expressed at very low levels or in a very small number of cells that we cannot detect. However, in the metrnlb mutant, the loss of this expression could still be sufficient to induce the low-penetrant phenotype. Interestingly, when analyzing the expression of these genes in a single cell transcriptomic atlas at the earliest available time point (10 hpf), in contrast to the robust expression of the two first genes, metrnlb expression is indeed found in very few cells (Kim et al., 2024).

We show that Metrns play a crucial role in ensuring this function, the genetic interaction with the integrins ItgαV1 and Itgβ1b, known to facilitate the proper clustering and migration of DFCs based on the sufficient accumulation of ZO-1 and apical constriction, essential to form the KV (Oteíza et al., 2008; Ablooglu et al., 2010). Furthermore, our results reveal that Metrns are essential for several aspects of the KV formation and function, including the cilia length, cilia number, KV size, and lumen formation. Consequently, Metrns are important for the regulation of the proper expression of Nodal flow genes, and this regulation is critical for the proper asymmetric positioning and morphology of vital organs like the heart (Figure 6B).

Previous work has shown that zebrafish midline mutants display altered lft1 and lft2 expression patterns as well as discordance among heart, gut, and brain gene expression patterns (Bisgrove et al., 2000). From 9 hpf, metrn expression was visible around the developing LPM close to the midline and the leading edge of the shield. Metrnla expression was found throughout the whole enveloping layer and the developing midline, which plays an important role in vertebrate L-R development. Despite the expression of both paralogs close to or at the midline, we hypothesize that the described body asymmetry defects in triplMut embryos do not originate from impaired midline structures but rather impaired DFC clustering and migration. Indeed, triplMut^-/- embryos display intact tbxta-positive notochords (Figure 4—figure supplement 1C), as well as randomized visceral organs positioning (Figure 1D–E), phenotypes that are not characteristic for defects of the anterior midline (Bisgrove et al., 2000).

DFCs are precursors of the zebrafish KV, organ of laterality, essential for the L-R axis establishment. DFCs develop at the beginning of gastrulation and form the KV by the end of gastrulation (Essner et al., 2005; Cooper and D’Amico, 1996). Given the early onset of metrn and metrnla expression and their specific expression within the DFCs, we investigated their role in the clustering and migration capacity of DFCs during these early embryonic developmental steps. In the course of their migration, DFC progenitors intercalate mediolaterally and result in an oval-rosette shape cluster of DFCs by mid-gastrulation that will form the KV (Oteíza et al., 2008). Metrn^-/-, metrnla^-/-, and triplMut zebrafish embryos displayed DFC assembly defects and delayed migration of these cells during gastrulation. Since DFCs in triplMut still express DFC-specific markers (sox17, sox32, tbxta), we concluded that Metrns are not required for the specification of DFCs per se, but rather that Metrns are important for their proper clustering and migration.

It is important to note that the observed DFC clustering and migration abnormalities in Metrn mutants primarily affect the apico-ventral axis rather than the mediolateral axis. This is evident from the disrupted connection between DFCs and the YSL/EVL in these mutants, while the migration directionality remains intact. Since a proper connection between DFCs and the EVL facilitates a subset of DFCs to establish apical contacts with the EVL and become pre-polarized, one could speculate that Metrns are involved in the polarization and establishment of direct apical contacts of DFCs with the EVL (Oteíza et al., 2008; Cooper and D’Amico, 1996; Pulgar et al., 2021; Solnica-Krezel et al., 1996). This disrupted connection in metrns loss-of-function embryos, which usually links DFCs with the vegetal spread of extra-embryonic tissues (Pulgar et al., 2021), likely results in the observed DFCs vegetal motion defects.

It has been demonstrated that these apical attachments function as tissue connectors, linking DFCs with the spreading of extra-embryonic tissues towards the vegetal pole (Pulgar et al., 2021).

Therefore, the loss of apical attachments in the mutant embryos causes all DFCs to converge less and migrate more slowly than in wild-type controls, as shown by our in vivo imaging analysis.

This would imply that a number of DFCs was simply too slow to reach the vegetal pole in time to form a fully functional KV. Given that KV cilia are monocilia, this subsequently results in a diminished count of KV cells (Figure 5—figure supplement 1A), along with the observed structural abnormalities in the KV lumen and impairments in its overall functionality.

Additionally, it was shown that the formation of the developing KV lumen was directly connected to ciliogenesis as polarized DFCs initiate developing cilia at their membrane domain facing the forming lumen (Oteíza et al., 2008). This indicates that proper DFC polarization is essential for KV formation and for ciliogenesis. In metrns mutants, the reduced cilia length and the lack of PKCζ staining in the developing KV further highlight an impairment of DFC polarization in the absence of Metrns, consequently leading to KV formation and function defects. Further work would be needed to explore whether Metrns directly impact the motility of KV cilia or if the disrupted Nodal flow observed is solely caused by the disturbed KV lumen.

It has been demonstrated that appropriate asymmetrical Nodal flow plays a crucial role in the asymmetrical expression of Nodal genes such as dand5, spaw, lefty1 (lft1), and lefty2 (lft2) (Hirokawa et al., 2006; Harvey, 1998; Hashimoto et al., 2004). As such, the disrupted formation and function of the KV in metrns mutants might contribute to the evident perturbation in Nodal factor expression and may also induce randomization of Nodal factor expression in L-R asymmetry (Essner et al., 2005; Nonaka et al., 1998; Gokey et al., 2016; Ablooglu et al., 2010). We do not exclude, however, that Metrns directly impact the expression of spaw, leftys, and dand5 in addition to their role in DFC cell behaviors and KV formation. One of the earliest functions attributed to Meteorin was as a regulator of mesendoderm development through the enhancement of nodal expression (Kim et al., 2014). In this context, several studies have reported that Nodal signaling was shown to regulate DFC specification (Oteiza et al., 2010; Choi et al., 2007).

In line with the impaired KV formation and function that we observed in metrns mutants, it was shown that knockdown of Integrin αV as well as Integrin β1b in zebrafish results in L-R asymmetry defects (Ablooglu et al., 2010). This was explained by DFC organization and migration defects and disturbed KV formation. IntegrinαVβ1 specific functions have not yet been completely resolved in mammals, mostly due to their broad expression and redundant function with other integrins, as well as the lack of antibodies and inhibitors specific for αVβ1. However, several in vitro studies have already connected αVβ1 to developmental processes. For instance, αVβ1 was shown to be implicated in embryonic astrocytes and oligodendrocyte precursors migration in rodents (Milner et al., 1996) and neural cell adhesion molecule L1 cell binding (Felding-Habermann et al., 1997). Additionally, the fibronectin receptor integrin α5β1 has been found to be crucial for regulating extracellular matrix assembly along tissue boundaries, in coordination with Eph/Ephrin signaling (Jülich et al., 2009).

Our results suggest that one mechanism by which Metrns act is through their genetic interaction with Integrin αV/β1b to mediate the proper clustering of DFCs. These interactions could influence Integrin’s adhesive functions (or other properties) necessary for DFC clustering. If so, the absence of Meteorins would lead to disturbed adhesion and/or Integrin signaling, resulting in DFC migration defects and disturbed KV development. Interestingly, it was shown that in αV and β1b morphants, DFC-EVL connections remain intact (Ablooglu et al., 2010), unlike in our meteorin mutants. This contrast suggests that Meteorins alone are crucial for DFC polarization and that their interaction with integrins αVβ1b may rather be important for the subsequent steps leading to KV formation. While our genetic interaction studies provide strong evidence of a link between Meteorins and Integrins, further biochemical analyses are required to fully elucidate this interaction.

Despite the recent advancements in uncovering the multiple functions of Meteorins, their receptors during early development are still unknown. It was recently shown that METRNL is promoting heart repair through its binding to the KIT tyrosine kinase receptor, establishing METRNL as a KIT receptor ligand in the context of ischemic tissue repair (Reboll et al., 2022). In another study, it was reported that Metrn is a ligand for the 5-hydroxytryptamine receptor 2b (Htr2b; Dai et al., 2022). Additionally, Metrn was associated with the regulation of reactive oxygen species levels in hematopoietic stem/progenitor cells via directing phospholipase C signaling (Dai et al., 2022). When we analyzed the expression of kita, one of the two zebrafish orthologs to KIT tyrosine kinase receptor, at 9 hpf, we found that kita is solely expressed in the prechordal plate and later (from 11 hpf) in the lateral borders of the anterior neural plate (Figure 6—figure supplement 2B, left panel). Its paralog kitb is expressed from 11 hpf in the anterior ventral mesoderm (Figure 6—figure supplement 2B, right panel), as previously reported (Mellgren and Johnson, 2005). Additionally, the analysis of htr2b expression during zebrafish development revealed that its expression only starts from around 2 dpf in the heart (Figure 6—figure supplement 2C). The absence of kita/b as well as htr2b expression at early embryonic stages in or at proximity of DFCs and in the forming KV suggests that Metrns rather act through other receptor(s) to ensure their L-R patterning functions.

Mechanisms underlying L-R patterning are known to be highly conserved within vertebrates. Hence, we raised the question of whether the Meteorin protein family is conserved across different species. Genes coding for metrn and metrnl exist in several vertebrates classes but could not be found in the genomes of invertebrates (Zheng et al., 2016). Metrnl ortholog genes could instead be found in amphioxus and tunicates. Therefore, we propose that Metrns function in the establishment of the L-R axis that we have uncovered here emerged during Chordate evolution (Figure 6—figure supplement 2D).

All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for Figure 1—figure supplement 1, Figure 2 and Figure 6—figure supplement 2.

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Author details

1. Fanny Eggeler

   Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0001-6803-1984

2. Jonathan Boulanger-Weill

   Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

   Contribution

   Conceptualization, Software, Formal analysis, Validation, Investigation, Visualization, Methodology

   Contributed equally with

   Flavia De Santis and Laura Belleri

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1580-0778

3. Flavia De Santis

   1. Institut Curie, PSL Research University, Paris, France
   2. ZeClinics SL. Sant Feliu de Llobregat, Barcelona, Spain

   Contribution

   Conceptualization, Resources, Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

   Contributed equally with

   Jonathan Boulanger-Weill and Laura Belleri

   Competing interests

   is affiliated with ZeClinics SL. The author has no other competing interests to declare

   "This ORCID iD identifies the author of this article:" 0000-0002-0988-6560

4. Laura Belleri

   Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Jonathan Boulanger-Weill and Flavia De Santis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0001-5475-7821

5. Karine Duroure

   Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

   Contribution

   Data curation, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5959-569X

6. Thomas O Auer

   1. Institut Curie, PSL Research University, Paris, France
   2. Department of Biology, University of Fribourg, Fribourg, Switzerland

   Contribution

   Conceptualization, Resources, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3442-0567

7. Shahad Albadri

   Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

   Contribution

   Conceptualization, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   For correspondence

   shahad.albadri@inserm.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3243-7018

8. Filippo Del Bene

   Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   filippo.del-bene@inserm.fr

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-8551-2846

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