Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis

Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.     

fsi zebrafish show concordant reversal of laterality of viscera, neuroanatomy, and a subset of behavioral responses

Asymmetries in CNS neuroanatomy are assumed to underlie the widespread cognitive and behavioral asymmetries in vertebrates. Studies in humans have shown that the laterality of some cognitive asymmetries is independent of the laterality of the viscera; discrete mechanisms may therefore regulate visceral and neural lateralization. However, through analysis of visceral, neuroanatomical, and behavioral asymmetries in the frequent-situs-inversus (fsi) line of zebrafish, we show that the principal left-right body asymmetries are coupled to certain brain asymmetries and lateralized behaviors. fsi fish with asymmetry defects show concordant reversal of heart, gut, and neuroanatomical asymmetries in the diencephalon. Moreover, the neuroanatomical reversals in reversed fsi fish correlate with reversal of some behavioral responses in both fry and adult fsi fish. Surprisingly, two behavioral asymmetries do not reverse, suggesting that at least two separable mechanisms must influence functional lateralization in the CNS. Partial reversal of CNS asymmetries may generate new behavioral phenotypes; supporting this idea, reversed fsi fry differ markedly from their normally lateralized siblings in their behavioral response to a novel visual feature. Revealing a link between visceral and brain asymmetry and lateralized behavior, our studies help to explain the complexity of the relationship between the lateralities of visceral and neural asymmetries.     

Reversed bodies, reversed brains, and (some) reversed behaviors: of zebrafish and men

Although zebrafish with situs inversus show complete reversal of normal visceral and cerebral asymmetries, they show left-right reversal of only some behaviors, with others continuing to show species-typical lateralization. The implication is that, as in humans, there are at least two independent mechanisms for generating 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.     

The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.     

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.     

The actin nucleator Cordon-bleu is required for development of motile cilia in zebrafish

The cordon-bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. Cobl protein is enriched in the apical domain of ciliated cells, in close proximity to the apical actin cap. Reduction of Cobl by antisense morpholinos reveals an essential role in development of motile cilia in organs such as Kupffer's vesicle and the pronephros. In Kupffer's vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Thus, Cobl represents a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support morphogenesis of motile cilia.     

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.     

Specification of left-right asymmetry in the embryonic gut of Drosophila

Most animals exhibit stable left-right asymmetries in their body. Although significant progress has been made in elucidating the mechanisms that set up these asymmetries in vertebrates, nothing is known about them in Drosophila. This is usually attributed to the fact that no reversals of stable left-right asymmetries have been observed in Drosophila, although relevant surveys have been carried out. We have focused on the asymmetry of the proventriculus in the embryonic gut of Drosophila, an aspect of left-right asymmetry that is extremely stable in wild-type flies. We show that this asymmetry can be reversed by mutations in the dicephalic and wunen genes, which also cause reversals in the antero-posterior axis of the embryo relative to its mother. This is the first observation to suggest that left-right asymmetries in Drosophila can be reversed by genetic/developmental manipulations. It also suggests that maternal signals may initiate the specification of some left-right asymmetries in the embryo.     

The evolution of brain lateralization: a game-theoretical analysis of population structure

In recent years, it has become apparent that behavioural and brain lateralization at the population level is the rule rather than the exception among vertebrates. The study of these phenomena has so far been the province of neurology and neuropsychology. Here, we show how such research can be integrated with evolutionary biology to understand lateralization more fully. In particular, we address the fact that, within a species, left- and right-type individuals often occur in proportions different from one-half (e.g. hand use in humans). The traditional explanations offered for lateralization of brain function (that it may avoid unnecessary duplication of neural circuitry and reduce interference between functions) cannot account for this fact, because increased individual efficiency is unrelated to the alignment of lateralization at the population level. A further puzzle is that such an alignment may even be disadvantageous, as it makes individual behaviour more predictable to other organisms. Here, we show that alignment of the direction of behavioural asymmetries in a population can arise as an evolutionarily stable strategy when individual asymmetrical organisms must coordinate their behaviour with that of other asymmetrical organisms. Brain and behavioural lateralization, as we know it in humans and other vertebrates, may have evolved under basically 'social' selection pressures.     

Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain

How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.     

The orphan G protein-coupled receptor 161 is required for left-right patterning

Gpr161 (also known as RE2) is an orphan G protein-coupled receptor (GPCR) that is expressed during embryonic development in zebrafish. Determining its biological function has proven difficult due to lack of knowledge regarding its natural or synthetic ligands. Here, we show that targeted knockdown of gpr161 disrupts asymmetric gene expression in the lateral plate mesoderm, resulting in aberrant looping of the heart tube. This is associated with elevated Ca²⁺ levels in cells lining the Kupffer's vesicle and normalization of Ca²⁺ levels, by over-expression of ncx1 or pmca-RNA, is able to partially rescue the cardiac looping defect in gpr161 knockdown embryos. Taken together, these data support a model in which gpr161 plays an essential role in left-right (L-R) patterning by modulating Ca²⁺ levels in the cells surrounding the Kupffer's vesicle.     

Regional cell shape changes control form and function of Kupffer's vesicle in the zebrafish embryo

Cilia-generated fluid flow in an 'organ of asymmetry' is critical for establishing the left-right body axis in several vertebrate embryos. However, the cell biology underlying how motile cilia produce coordinated flow and asymmetric signals is not well defined. In the zebrafish organ of asymmetry-called Kupffer's vesicle (KV)-ciliated cells are asymmetrically positioned along the anterior-posterior axis such that more cilia are placed in the anterior region. We previously demonstrated that Rho kinase 2b (Rock2b) is required for anteroposterior asymmetry and fluid flow in KV, but it remained unclear how the distribution of ciliated cells becomes asymmetric during KV development. Here, we identify a morphogenetic process we refer to as 'KV remodeling' that transforms initial symmetry in KV architecture into anteroposterior asymmetry. Live imaging of KV cells revealed region-specific cell shape changes that mediate tight packing of ciliated cells into the anterior pole. Mathematical modeling indicated that different interfacial tensions in anterior and posterior KV cells are involved in KV remodeling. Interfering with non-muscle myosin II (referred to as Myosin II) activity, which modulates cellular interfacial tensions and is regulated by Rock proteins, disrupted KV cell shape changes and the anteroposterior distribution of KV cilia. Similar defects were observed in Rock2b depleted embryos. Furthermore, inhibiting Myosin II at specific stages of KV development perturbed asymmetric flow and left-right asymmetry. These results indicate that regional cell shape changes control the development of anteroposterior asymmetry in KV, which is necessary to generate coordinated asymmetric fluid flow and left-right patterning of the embryo.