Zebrafish Bmp4 regulates left-right asymmetry at two distinct developmental time points

Left-right (LR) asymmetry is regulated by early asymmetric signals within the embryo. Even though the role of the bone morphogenetic protein (BMP) pathway in this process has been reported extensively in various model organisms, opposing models for the mechanism by which BMP signaling operates still prevail. Here we show that in zebrafish embryos there are two distinct phases during LR patterning in which BMP signaling is required. Using transgenic lines that ectopically express either noggin3 or bmp2b, we show a requirement for BMP signaling during early segmentation to repress southpaw expression in the right lateral plate mesoderm and regulate both visceral and heart laterality. A second phase was identified during late segmentation, when BMP signaling is required in the left lateral plate mesoderm to regulate left-sided gene expression and heart laterality. Using morpholino knock down experiments, we identified Bmp4 as the ligand responsible for both phases of BMP signaling. In addition, we detected bmp4 expression in Kupffer's vesicle and show that restricted knock down of bmp4 in this structure results in LR patterning defects. The identification of these two distinct and opposing activities of BMP signaling provides new insight into how BMP signaling can regulate LR patterning.     

β-Catenin 1 and β-catenin 2 play similar and distinct roles in left-right asymmetric development of zebrafish embryos

β-Catenin-mediated canonical Wnt signaling has been found to be required for left-right (LR) asymmetric development. However, the implication of endogenous β-catenin in LR development has not been demonstrated by loss-of-function studies. In zebrafish embryos, two β-catenin genes, β-catenin 1 (ctnnb1) and β-catenin 2 (ctnnb2) are maternally expressed and their zygotic expression occurs in almost all types of tissues, including Kupffer's vesicle (KV), an essential organ that initiates LR development in teleost fish. We demonstrate here that morpholino-mediated knockdown of ctnnb1, ctnnb2, or both, in the whole embryo or specifically in dorsal forerunner cells (DFCs) interrupts normal asymmetry of the heart, liver and pancreas. Global knockdown of ctnnb2 destroys the midline physical and molecular barrier, while global knockdown of ctnnb1 impairs the formation of the midline molecular barrier. Depletion of either gene or both in DFCs/KV leads to poor KV cell proliferation, abnormal cilia formation and disordered KV fluid flow with downregulation of ntl and tbx16 expression. ctnnb1 and ctnnb2 in DFCs/KV differentially regulate the expression of charon, a Nodal antagonist, and spaw, a key Nodal gene for laterality development in zebrafish. Loss of ctnnb1 in DFCs/KV inhibits the expression of charon around KV and of spaw in the posterior lateral plate mesoderm, while ctnnb2 knockdown results in loss of spaw expression in the anterior lateral plate mesoderm with little alteration of charon expression. Taken together, our findings suggest that ctnnb1 and ctnnb2 regulate multiple processes of laterality development in zebrafish embryos through similar and distinct mechanisms.     

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.     

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.     

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.     

Roles for fgf8 signaling in left-right patterning of the visceral organs and craniofacial skeleton

Laterality is fundamental to the vertebrate body plan. Here, we investigate the roles of fgf8 signaling in LR patterning of the zebrafish embryo. We find that fgf8 is required for proper asymmetric development of the brain, heart and gut. When fgf8 is absent, nodal signaling is randomized in the lateral plate mesoderm, leading to aberrant LR orientation of the brain and visceral organs. We also show that fgf8 is necessary for proper symmetric development of the pharyngeal skeleton. Attenuated fgf8 signaling results in consistently biased LR asymmetric development of the pharyngeal arches and craniofacial skeleton. Approximately 1/3 of zebrafish ace/fgf8 mutants are missing Kupffer's vesicle (KV), a ciliated structure similar to Hensen's node. We correlate fgf8 deficient laterality defects in the brain and viscera with the absence of KV, supporting a role for KV in proper LR patterning of these structures. Strikingly, we also correlate asymmetric craniofacial development in ace/fgf8 mutants with the presence of KV, suggesting roles for KV in lateralization of the pharyngeal skeleton when fgf8 is absent. These data provide new insights into vertebrate laterality and offer the zebrafish ace/fgf8 mutant as a novel molecular tool to investigate tissue-specific molecular laterality mechanisms.     

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left–right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer's vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left–right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads to coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left–right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects.     

Wnt/Axin1/beta-catenin signaling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/beta-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries.     

Asymmetric nodal signaling in the zebrafish diencephalon positions the pineal organ

The vertebrate brain develops from a bilaterally symmetric neural tube but later displays profound anatomical and functional asymmetries. Despite considerable progress in deciphering mechanisms of visceral organ laterality, the genetic pathways regulating brain asymmetries are unknown. In zebrafish, genes implicated in laterality of the viscera (cyclops/nodal, antivin/lefty and pitx2) are coexpressed on the left side of the embryonic dorsal diencephalon, within a region corresponding to the presumptive epiphysis or pineal organ. Asymmetric gene expression in the brain requires an intact midline and Nodal-related factors. RNA-mediated rescue of mutants defective in Nodal signaling corrects tissue patterning at gastrulation, but fails to restore left-sided gene expression in the diencephalon. Such embryos develop into viable adults with seemingly normal brain morphology. However, the pineal organ, which typically emanates at a left-to-medial site from the dorsal diencephalic roof, becomes displaced in position. Thus, a conserved signaling pathway regulating visceral laterality also underlies an anatomical asymmetry of the zebrafish forebrain.     

Shaping the zebrafish heart: From left-right axis specification to epithelial tissue morphogenesis

Although vertebrates appear bilaterally symmetric on the outside, various internal organs, including the heart, are asymmetric with respect to their position and/or their orientation based on the left/right (L/R) axis. The L/R axis is determined during embryo development. Determination of the L/R axis is fundamentally different from the determination of the anterior-posterior or the dorsal-ventral axis. In all vertebrates a ciliated organ has been described that induces a left-sided gene expression program, which includes Nodal expression in the left lateral plate mesoderm. To have a better understanding of organ laterality it is important to understand how L/R patterning induces cellular responses during organogenesis. In this review, we discuss the current understanding of the mechanisms of L/R patterning during zebrafish development and focus on how this affects cardiac morphogenesis. Several recent studies have provided unprecedented insights into the intimate link between L/R signaling and the cellular responses that drive morphogenesis of this organ.     

The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish

The vertebrate body plan features a consistent left-right (LR) asymmetry of internal organs. In several vertebrate embryos, motile cilia generate an asymmetric fluid flow that is necessary for normal LR development. However, the mechanisms involved in orienting LR asymmetric flow with previously established anteroposterior (AP) and dorsoventral (DV) axes remain poorly understood. In zebrafish, asymmetric flow is generated in Kupffer's vesicle (KV). The cellular architecture of KV is asymmetric along the AP axis, with more ciliated cells densely packed into the anterior region. Here, we identify a Rho kinase gene, rock2b, which is required for normal AP patterning of KV and subsequent LR development in the embryo. Antisense depletion of rock2b in the whole embryo or specifically in the KV cell lineage perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ LR asymmetries. Analyses of KV architecture demonstrated that rock2b knockdown altered the AP placement of ciliated cells without affecting cilia number or length. In control embryos, leftward flow across the anterior pole of KV was stronger than rightward flow at the posterior end, correlating with the normal AP asymmetric distribution of ciliated cells. By contrast, rock2b knockdown embryos with AP patterning defects in KV exhibited randomized flow direction and equal flow velocities in the anterior and posterior regions. Live imaging of Tg(dusp6:memGFP)(pt19) transgenic embryos that express GFP in KV cells revealed that rock2b regulates KV cell morphology. Our results suggest a link between AP patterning of the ciliated Kupffer's vesicle and LR patterning of the zebrafish embryo.     

Laterotopic representation of left-right information onto the dorso-ventral axis of a zebrafish midbrain target nucleus

The habenulae are part of an evolutionarily highly conserved limbic-system conduction pathway that connects telencephalic nuclei to the interpeduncular nucleus (IPN) of the midbrain . In zebrafish, unilateral activation of the Nodal signaling pathway in the left brain specifies the laterality of the asymmetry of habenular size . We show "laterotopy" in the habenulo-interpeduncular projection in zebrafish, i.e., the stereotypic, topographic projection of left-sided habenular axons to the dorsal region of the IPN and of right-sided habenular axons to the ventral IPN. This asymmetric projection is accounted for by a prominent left-right (LR) difference in the size ratio of the medial and lateral habenular sub-nuclei, each of which specifically projects either to ventral or dorsal IPN targets. Asymmetric Nodal signaling directs the orientation of laterotopy but is dispensable for the establishment of laterotopy itself. Our results reveal a mechanism by which information distributed between left and right sides of the brain can be transmitted bilaterally without loss of LR coding, which may play a crucial role in functional lateralization of the vertebrate brain .     

Bmp and nodal independently regulate lefty1 expression to maintain unilateral nodal activity during left-right axis specification in zebrafish

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.     

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.     

D-JNK signaling in visceral muscle cells controls the laterality of the Drosophila gut

Although bilateral animals appear to have left-right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. Here, we showed that Drosophila Jun N-terminal kinase (D-JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a D-JNK phosphatase, showed random laterality of the AMG. Directional LR looping of the AMG required D-JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of D-JNK signaling was required for the LR asymmetric looping. We also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) was regulated by D-JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augmented D-JNK signaling in this process. Our results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates.