Geminin is required for left-right patterning through regulating Kupffer's vesicle formation and ciliogenesis in zebrafish

Geminin plays an important role in coordinating the cell cycle with anterior–posterior patterning during embryonic development. However, whether it is involved in the regulation of left–right (LR) patterning remains unknown. Here, we reported that geminin is required for setting up heart and visceral laterality during zebrafish development. Defective heart and visceral laterality was observed in geminin morphants. Further study demonstrated that the left-sided nodal/spaw in the lateral plate mesoderm (LPM) as well as the sideness of its downstream targets lefty2 and lefty1 was perturbed in geminin morphants. Upstream of the left-sided Nodal signal along the regulatory cascade of LR asymmetry, knock down of geminin resulted in defective Kupffer’s vesicle (KV) formation and ciliogenesis rather than middle line defects. Predominant distribution of an antisense morpholino against geminin in dorsal forerunner cells (DFCs) led to defective KV morphogenesis and perturbed LR asymmetry, similar to those of geminin morphants, indicating a cell-autonomous role of geminin in regulating KV formation and ciliogenesis. Our results demonstrate that geminin is required for proper KV formation and ciliogenesis, thus playing an important part in setting up LR asymmetry.     

A variant of fibroblast growth factor receptor 2 (Fgfr2) regulates left-right asymmetry in zebrafish

Many organs in vertebrates are left-right asymmetrical located. For example, liver is at the right side and stomach is at the left side in human. Fibroblast growth factor (Fgf) signaling is important for left-right asymmetry. To investigate the roles of Fgfr2 signaling in zebrafish left-right asymmetry, we used splicing blocking morpholinos to specifically block the splicing of fgfr2b and fgfr2c variants, respectively. We found that the relative position of the liver and the pancreas were disrupted in fgfr2c morphants. Furthermore, the left-right asymmetry of the heart became random. Expression pattern of the laterality controlling genes, spaw and pitx2c, also became random in the morphants. Furthermore, lefty1 was not expressed in the posterior notochord, indicating that the molecular midline barrier had been disrupted. It was also not expressed in the brain diencephalon. Kupffer's vesicle (KV) size became smaller in fgfr2c morphants. Furthermore, KV cilia were shorter in fgfr2c morphants. We conclude that the fgfr2c isoform plays an important role in the left-right asymmetry during zebrafish development.     

Orphan G-Protein Coupled Receptor 22 (Gpr22) Regulates Cilia Length and Structure in the Zebrafish Kupffer’s Vesicle

GPR22 is an orphan G protein-coupled receptor (GPCR). Since the ligand of the receptor is currently unknown, its biological function has not been investigated in depth. Many GPCRs and their intracellular effectors are targeted to cilia. Cilia are highly conserved eukaryotic microtubule-based organelles that protrude from the membrane of most mammalian cells. They are involved in a large variety of physiological processes and diseases. However, the details of the downstream pathways and mechanisms that maintain cilia length and structure are poorly understood. We show that morpholino knock down or overexpression of gpr22 led to defective left-right (LR) axis formation in the zebrafish embryo. Specifically, defective LR patterning included randomization of the left-specific lateral plate mesodermal genes (LPM) (lefty1, lefty2, southpaw and pitx2a), resulting in randomized cardiac looping. Furthermore, gpr22 inactivation in the Kupffer’s vesicle (KV) alone was still able to generate the phenotype, indicating that Gpr22 mainly regulates LR asymmetry through the KV. Analysis of the KV cilia by immunofluorescence and transmission electron microscopy (TEM), revealed that gpr22 knock down or overexpression resulted in changes of cilia length and structure. Further, we found that Gpr22 does not act upstream of the two cilia master regulators, Foxj1a and Rfx2. To conclude, our study characterized a novel player in the field of ciliogenesis.     

Seson, a novel zinc finger protein, controls cilia integrity for the LR patterning during zebrafish embryogenesis

In zebrafish embryos, bilateral symmetry is broken by asymmetric nodal flow generated in Kupffer’s vesicle (KV), the transient cilia-rich organ, analogous to the mouse node. Asymmetric nodal flow induces the asymmetric expression of several genes, which are critical for the determination of correct LR body patterning. seson encoding three consecutive C₂H₂ zinc finger protein is predominantly expressed in the cilia-rich organs including KV. Inhibition of its function by the injection of a seson-specific MO inhibited the left-side biased expression of spaw, and resulted in randomization of the heart, gut looping and brain laterality. Disruption of the LR patterning in seson morphants appeared to be due to severe cilia defects in KV. Seson function was also required for ciliogenesis in other tissues such as the pronephros and olfactory organs. Collectively, our data suggest that Seson has critical roles in ciliogenesis and LR body axis patterning.     

Paraxial left-sided nodal expression and the start of left–right patterning in the early chick embryo

A common element during early left–right patterning of the vertebrate body is left-sided nodal expression in the early-somite stage lateral plate mesoderm. Leftward cell movements near the node of the gastrulating chick embryo recently offered a plausible mechanism for breaking the presomite-stage molecular symmetry in those vertebrates which lack rotating cilia on the notochord or equivalent tissues. However, the temporal and functional relationships between generation of the known morphological node asymmetry, onset of leftward cell movements and establishment of stable molecular asymmetry in the chick remain unresolved. This study uses high-resolution light microscopy and in situ gene expression analysis to show that intranodal cell rearrangement during the phase of counter-clockwise node torsion at stage 4+ is immediately followed by symmetry loss and rearrangement of shh and fgf8 expression in node epiblast between stages 5− and 5+. Surprisingly, left-sided nodal expression starts at stage 5−, too, but lies in the paraxial mesoderm next to the forming notochordal plate, and can be rendered symmetrical by minimal mechanical disturbance of distant tissue integrity at stage 4. The “premature” paraxial nodal expression together with morphological and molecular asymmetries in, and near, midline compartments occurring at defined substages of early gastrulation help to identify a new narrow time window for early steps in left–right patterning in the chick and support the concept of a causal relationship between a—still enigmatic—chiral (motor) protein, cell movements and incipient left–right asymmetry in the amniote embryo.     

Two novel type II receptors mediate BMP signalling and are required to establish left-right asymmetry in zebrafish

Ligands of the transforming growth factor β (TGFβ) superfamily, like Nodal and bone morphogenetic protein (BMP), are pivotal to establish left-right (LR) asymmetry in vertebrates. However, the receptors mediating this process are unknown. Here we identified two new type II receptors for BMPs in zebrafish termed bmpr2a and bmpr2b that induce a classical Smad1/5/8 response to BMP binding. Morpholino-mediated knockdown of bmpr2a and bmpr2b showed that they are required for the establishment of concomitant cardiac and visceral LR asymmetry. Expression of early laterality markers in morphants indicated that bmpr2a and bmpr2b act upstream of pitx2 and the nodal-related southpaw (spaw), which are expressed asymmetrically in the lateral plate mesoderm (LPM), and subsequently regulate lefty2 and bmp4 in the left heart field. We demonstrated that bmpr2a is required for lefty1 expression in the midline at early segmentation while bmpr2a/bmpr2b heteromers mediate left-sided spaw expression in the LPM. We propose a mechanism whereby this differential interpretation of BMP signalling through bmpr2a and bmpr2b is essential for the establishment of LR asymmetry in the zebrafish embryo.     

Bbs8, together with the planar cell polarity protein Vangl2, is required to establish left-right asymmetry in zebrafish

Laterality defects such as situs inversus are not uncommonly encountered in humans, either in isolation or as part of another syndrome, but can have devastating developmental consequences. The events that break symmetry during early embryogenesis are highly conserved amongst vertebrates and involve the establishment of unidirectional flow by cilia within an organising centre such as the node in mammals or Kupffer's vesicle (KV) in teleosts. Disruption of this flow can lead to the failure to successfully establish left-right asymmetry. The correct apical-posterior cellular position of each node/KV cilium is critical for its optimal radial movement which serves to sweep fluid (and morphogens) in the same direction as its neighbours. Planar cell polarity (PCP) is an important conserved process that governs ciliary position and posterior tilt; however the underlying mechanism by which this occurs remains unclear. Here we show that Bbs8, a ciliary/basal body protein important for intraciliary/flagellar transport and the core PCP protein Vangl2 interact and are required for establishment and maintenance of left-right asymmetry during early embryogenesis in zebrafish. We discovered that loss of bbs8 and vangl2 results in laterality defects due to cilia disruption at the KV. We showed that perturbation of cell polarity following abrogation of vangl2 causes nuclear mislocalisation, implying defective centrosome/basal body migration and apical docking. Moreover, upon loss of bbs8 and vangl2, we observed defective actin organisation. These data suggest that bbs8 and vangl2 act synergistically on cell polarization to establish and maintain the appropriate length and number of cilia in the KV and thereby facilitate correct LR asymmetry.     

Retinoic acid signaling sequentially controls visceral and heart laterality in zebrafish

During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the left-sided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality.     

Characterization of the medaka (Oryzias latipes) primary ciliary dyskinesia mutant, jaodori: Redundant and distinct roles of dynein axonemal intermediate chain 2 (dnai2) in motile cilia

Cilia and flagella are highly conserved organelles that have diverse motility and sensory functions. Motility defects in cilia and flagella result in primary ciliary dyskinesia (PCD). We isolated a novel medaka PCD mutant, jaodori (joi). Positional cloning showed that axonemal dynein intermediate chain 2 (dnai2) is responsible for joi. The joi mutation was caused by genomic insertion of the medaka transposon, Tol1. In the joi mutant, cilia in Kupffer's vesicle (KV), an organ functionally equivalent to the mouse node in terms of left-right (LR) specification, are generated but their motility is disrupted, resulting in a LR defect. Ultrastructural analysis revealed severe reduction in the outer dynein arms in KV cilia of joi mutants. We also found the other dnai2 gene in the medaka genome. These two dnai2 genes function either redundantly or distinctly in tissues possessing motile cilia.     

hemingway is required for sperm flagella assembly and ciliary motility in Drosophila

Cilia play major functions in physiology and development, and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to primary ciliary dyskinesia, with upper-airways recurrent infections, left–right asymmetry perturbations, and fertility defects. In Drosophila, we identified hemingway (hmw) as a novel component required for motile cilia function. hmw encodes a 604–amino acid protein characterized by a highly conserved coiled-coil domain also found in the human orthologue, KIAA1430. We show that HMW is conserved in species with motile cilia and that, in Drosophila, hmw is expressed in ciliated sensory neurons and spermatozoa. We created hmw-knockout flies and found that they are hearing impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, is required for elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, we propose that HMW is not a structural component of the motile axoneme but is required for proper acquisition of motile properties. This identifies HMW as a novel, evolutionarily conserved component necessary for motile cilium function and flagella assembly.     

The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry

We have identified and characterized a new zebrafish gene, southpaw, that is required for visceral and diencephalic left-right asymmetry. southpaw encodes a new member of the nodal-related class of proteins, a subfamily within the transforming growth factor beta superfamily of secreted factors. southpaw is expressed bilaterally in paraxial mesoderm precursors and then within the left lateral plate mesoderm. At late somite stages, left-sided southpaw expression transiently overlaps the left-sided expression domains of other genes that mark the developing heart, such as lefty2. We have injected morpholinos to block the translation of the southpaw mRNA or to block splicing of the southpaw pre-mRNA. These morpholinos cause a severe disruption of early (cardiac jogging) and late (cardiac looping) aspects of cardiac left-right asymmetry. As the left-right asymmetry of the pancreas is also affected, southpaw appears to regulate left-right asymmetry throughout a large part of the embryo. Consistent with the morphological changes, the left-sided expression domains of downstream genes (cyclops, pitx2, lefty1 and lefty2) are severely downregulated or abolished within the lateral plate mesoderm of Southpaw-deficient embryos. Surprisingly, despite the absence of southpaw expression in the brain, we find that early diencephalic left-right asymmetry also requires Southpaw activity. These observations lead to a model of how visceral organ and brain left-right asymmetry are coordinated during embryogenesis.     

Dynein axonemal intermediate chain 2 is required for formation of the left-right body axis and kidney in medaka

Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left-right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility.     

Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.     

Autotaxin in embryonic development

Autotaxin (ATX) is a secreted lysophospholipase D that generates the multifunctional lipid mediator lysophosphatidic acid (LPA). LPA signals through six distinct G protein-coupled receptors, acting alone or in concert to activate multiple effector pathways. The ATX–LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes and plays a vital role in embryonic development. Disruption of the ATX-encoding gene (Enpp2) in mice results in intrauterine death due to vascular defects in the extra-embryonic yolk sac and embryo proper. In addition, Enpp2 (−/−) embryos show impaired neural development. The observed angiogenic defects are attributable, at least in part, to loss of LPA signaling through the Gα_12/13-linked RhoA-ROCK-actin remodeling pathway. Studies in zebrafish also have uncovered a dual role for ATX in both vascular and neural development; furthermore, they point to a key role for ATX–LPA signaling in the regulation of left–right asymmetry. Here we discuss our present understanding of the role of ATX–LPA signaling in vertebrate development. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

A Highlights from MBoC Selection: Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left–right asymmetry

The establishment of left–right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites—cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer’s vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite–associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.