Meteorins regulate the formation of the left-right organizer and the establishment of vertebrate body asymmetry
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eLife Assessment
This study presents important insights into the regulation of left-right organ formation. By combining genetic perturbation of all three Meteorin genes in zebrafish and timelapse imaging, the authors identify an essential role for this protein family in the establishment of left-right patterning. They provide convincing evidence that Meteorins are required for the morphogenesis of dorsal forerunner cells, the precursors of the left-right organizer (also named Kupffer's vesicle) in zebrafish. In line with this, Meteorins were shown to genetically interact with integrins ItgaV and Itgb1b to regulate dorsal forerunner cell clustering.
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Abstract
While the exterior of vertebrate bodies appears bilaterally symmetrical, internal organ positioning and morphology frequently exhibit left-right (L-R) asymmetries. In several vertebrates, including human, mouse, frog and zebrafish, left-right symmetry-breaking during embryonic development is initiated by a ciliated organ called the Node or left-right organizer. Within the Node, a leftward flow of extraembryonic fluid named the Nodal flow mediates the asymmetric expressions of Nodal factors. Although downstream Nodal pathway components leading to the establishment of the embryonic left-right axis are well known, less is known about the development and formation of the embryonic Node itself.
Here we reveal a novel role for the Meteorin protein family in the establishment of the left-right axis and in the formation of the Kupffer’s vesicle, the Node equivalent structure in zebrafish. We show that the genetic inactivation of each or all three members of the zebrafish Meteorin family ( metrn , metrn-like a and metrn-like b ) leads to defects in properties of the Kupffer’s vesicle, caused by impaired assembly and migration of the Kupffer’s vesicle forming dorsal forerunner cells. In addition, we demonstrate that Meteorins genetically interact with integrins ItgaV and Itgβ1b regulating the dorsal forerunner cell clustering and that meteorins loss-of-function results in disturbed Nodal factor expression and consequently in randomized or symmetric heart looping and jogging.
These results identify a new role for the Meteorin protein family in the left-right asymmetry patterning during embryonic vertebrate development.
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eLife Assessment
This study presents important insights into the regulation of left-right organ formation. By combining genetic perturbation of all three Meteorin genes in zebrafish and timelapse imaging, the authors identify an essential role for this protein family in the establishment of left-right patterning. They provide convincing evidence that Meteorins are required for the morphogenesis of dorsal forerunner cells, the precursors of the left-right organizer (also named Kupffer's vesicle) in zebrafish. In line with this, Meteorins were shown to genetically interact with integrins ItgaV and Itgb1b to regulate dorsal forerunner cell clustering.
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Reviewer #1 (Public review):
Summary:
Meteorin proteins were initially described as secreted neurotrophic factors. In this manuscript, Eggeler et al. demonstrate a novel role for Meteorins in establish left-right axis formation in the zebrafish embryo. The authors generated null mutations in each of the three zebrafish meteorin genes - metrn, metrnla, and metrnlab. Triple mutant embryos displayed phenotypes strongly associated with left-right defects such as heart looping and visceral organ placement, and disrupted expression of Nodal-responsive genes, as did single mutants for metrn and metrnla. The authors then go on to demonstrate that these defects in left-right asymmetry are likely to due to defects in Kupffer's Vesicle and the progenitor dorseal forerunner cells including impaired lumen formation and reduced fluid flow, reduced …
Reviewer #1 (Public review):
Summary:
Meteorin proteins were initially described as secreted neurotrophic factors. In this manuscript, Eggeler et al. demonstrate a novel role for Meteorins in establish left-right axis formation in the zebrafish embryo. The authors generated null mutations in each of the three zebrafish meteorin genes - metrn, metrnla, and metrnlab. Triple mutant embryos displayed phenotypes strongly associated with left-right defects such as heart looping and visceral organ placement, and disrupted expression of Nodal-responsive genes, as did single mutants for metrn and metrnla. The authors then go on to demonstrate that these defects in left-right asymmetry are likely to due to defects in Kupffer's Vesicle and the progenitor dorseal forerunner cells including impaired lumen formation and reduced fluid flow, reduced clustering among DFCs, impaired DFC migration, mislocalization of apical proteins ZO-1 and aPKC, and detachment of DFCs from the EVL. Notably, the authors found that expression of marker genes sox32 and sox17 were not affected, suggesting Meteorins are required for DFC/KV morphogenesis but not necessarily fate specification. Finally, the authors show genetic interaction between Meteorins and integrin receptors, which were previously implicated in left-right patterning. In a supplemental figure, the manuscript also presents data showing expression of meteorin genes around the chick Hensen's node, suggesting that the left-right patterning functions may be conserved among vertebrates.
Strengths:
Strengths of this study include the generation of a triple mutant line that targets all known zebrafish meteorin family members. The experiments presented in this study were rigorous, especially with respect to quantification and statistical analysis.
Weaknesses:
Although the authors convincingly demonstrate a role for Meteorins in zebrafish left-right patterning, data supporting a conserved role in other vertebrates is compelling but limited to one supplemental figure.
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Reviewer #2 (Public review):
Summary:
In this manuscript the authors describe their study on the role of meteorins in establishing the left-right organizer. The left-right organizer is a transient organ in vertebrate embryos in which rotating cilia cause a fluid flow that breaks the left-right symmetry and coordinates lateralization of internal organs such as gut and heart. In zebrafish, the left-right organizer (also named Kupffer's vesicle) is formed by dorsal forerunner cells, but very little is known about how dorsal forerunner cells coalles and form this ciliated vesicle in the embryo. The authors mutated the three meteorin-coding genes in zebrafish and observed that mutations in each one of these causes laterality defects with the strongest defects observed in the triple mutant. Loss of meteorins affects nodal gene expression, …
Reviewer #2 (Public review):
Summary:
In this manuscript the authors describe their study on the role of meteorins in establishing the left-right organizer. The left-right organizer is a transient organ in vertebrate embryos in which rotating cilia cause a fluid flow that breaks the left-right symmetry and coordinates lateralization of internal organs such as gut and heart. In zebrafish, the left-right organizer (also named Kupffer's vesicle) is formed by dorsal forerunner cells, but very little is known about how dorsal forerunner cells coalles and form this ciliated vesicle in the embryo. The authors mutated the three meteorin-coding genes in zebrafish and observed that mutations in each one of these causes laterality defects with the strongest defects observed in the triple mutant. Loss of meteorins affects nodal gene expression, which play essential roles in establishing organ laterality. Meteorins are widely expressed in developing embryos and expression in lateral plate mesoderm and dorsal forerunner cells was observed. The meteorin triple mutant embryos display defects in the migration and clustering of the dorsal forerunner cells impairing kupffer's vesicle formation and cilia rotation. Finally, the authors show that meteorins genetically interact with integrins.
Strengths:
- These authors went through the lengthy process of generating triple mutants affecting all three meteorin genes. This provides robust genetic evidence on the role of meteorins in establishing organ laterality and circumvented that interpretation of the results would be hard due to redundant functions of meteorins.
- The use of life imaging on triple mutants is appreciated
- High-quality imaging of dorsal forerunner to quantify cell migrations and its relation to Kupffer's vesicle formation.Weaknesses:
- Lack of a model how meteorins regulate dorsal forerunner cell migration.
- Only genetic data to suggest a link between meteorins and integrins
- Besides its role in DFC migration, meteorins may also play a more direct role in regulating Nodal signaling, which is not addressed here. -
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