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.     

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.     

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.     

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.     

Low frequency vibrations disrupt left-right patterning in the Xenopus embryo

The development of consistent left-right (LR) asymmetry across phyla is a fascinating question in biology. While many pharmacological and molecular approaches have been used to explore molecular mechanisms, it has proven difficult to exert precise temporal control over functional perturbations. Here, we took advantage of acoustical vibration to disrupt LR patterning in Xenopus embryos during tightly-circumscribed periods of development. Exposure to several low frequencies induced specific randomization of three internal organs (heterotaxia). Investigating one frequency (7 Hz), we found two discrete periods of sensitivity to vibration; during the first period, vibration affected the same LR pathway as nocodazole, while during the second period, vibration affected the integrity of the epithelial barrier; both are required for normal LR patterning. Our results indicate that low frequency vibrations disrupt two steps in the early LR pathway: the orientation of the LR axis with the other two axes, and the amplification/restriction of downstream LR signals to asymmetric organs.     

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.     

Primary cilia regulate mTORC1 activity and cell size through Lkb1

The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling.     

Laterality defects are influenced by timing of treatments and animal model

The timing of when the embryonic left−right (LR) axis is first established and the mechanisms driving this process are subjects of strong debate. While groups have focused on the role of cilia in establishing the LR axis during gastrula and neurula stages, many animals appear to orient the LR axis prior to the appearance of, or without the benefit of, motile cilia. Because of the large amount of data available in the published literature and the similarities in the type of data collected across laboratories, I have examined relationships between the studies that do and do not implicate cilia, the choice of animal model, the kinds of LR patterning defects observed, and the penetrance of LR phenotypes. I found that treatments affecting cilia structure and motility had a higher penetrance for both altered gene expression and improper organ placement compared to treatments that affect processes in early cleavage stage embryos. I also found differences in penetrance that could be attributed to the animal models used; the mouse is highly prone to LR randomization. Additionally, the data were examined to address whether gene expression can be used to predict randomized organ placement. Using regression analysis, gene expression was found to be predictive of organ placement in frogs, but much less so in the other animals examined. Together, these results challenge previous ideas about the conservation of LR mechanisms, with the mouse model being significantly different from fish, frogs, and chick in almost every aspect examined. Additionally, this analysis indicates that there may be missing pieces in the molecular pathways that dictate how genetic information becomes organ positional information in vertebrates; these gaps will be important for future studies to identify, as LR asymmetry is not only a fundamentally fascinating aspect of development but also of considerable biomedical importance.     

Reduced cell number in the hindgut epithelium disrupts hindgut left–right asymmetry in a mutant of pebble,encoding a RhoGEF,in Drosophila embryos

Animals often show left–right (LR) asymmetry in their body structures. In some vertebrates, the mechanisms underlying LR symmetry breaking and the subsequent signals responsible for LR asymmetric development are well understood. However, in invertebrates, the molecular bases of these processes are largely unknown. Therefore, we have been studying the genetic pathway of LR asymmetric development in Drosophila. The embryonic gut is the first organ that shows directional LR asymmetry during Drosophila development. We performed a genetic screen to identify mutations affecting LR asymmetric development of the embryonic gut. From this screen, we isolated pebble (pbl), which encodes a homolog of a mammalian RhoGEF, Ect2. The laterality of the hindgut was randomized in embryos homozygous for a null mutant of pbl. Pbl is a multi-functional protein required for cytokinesis and the epithelial-to-mesenchymal transition in Drosophila. Consistent with Pbl’s role in cytokinesis, we found reduced numbers of cells in the hindgut epithelium in pbl homozygous embryos. The specific expression of pbl in the hindgut epithelium, but not in other tissues, rescued the LR defects and reduced cell number in embryonic pbl homozygotes. Embryos homozygous for string (stg), a mutant that reduces cell number through a different mechanism, also showed LR defects of the hindgut. However, the reduction in cell number in the pbl mutants was not accompanied by defects in the specification of hindgut epithelial tissues or their integrity. Based on these results, we speculate that the reduction in cell number may be one reason for the LR asymmetry defect of the pbl hindgut, although we cannot exclude contributions from other functions of Pbl, including regulation of the actin cytoskeleton through its RhoGEF activity.     

mTOR-rictor is the Ser473 kinase for AKT1 in mouse one-cell stage embryos

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. The mTORC2 containing mTOR and rictor is thought to be rapamycin insensitive and it is recently shown that both rictor and mTORC2 are essential for the development of both embryonic and extra embryonic tissues. To explore rictor function in the early development of mouse embryos, we disrupted the expression of rictor, a specific component of mTORC2, in mouse fertilized eggs by using rictor shRNA. Our results showed that one-cell stage eggs that were lack of rictor could not enter into the two-cell stage normally. Recent biochemical studies suggests that TORC2 is the elusive PDK2 (3'-phosphoinositide-dependent kinase 2) for AKT/PKB Ser473 phosphorylation, which is deemed necessary for AKT function, so we microinjected AKT-S473A into mouse fertilized eggs to investigate whether AKT-S473A is downstream effector of mTOR.rictor to regulate the mitotic division. Our findings revealed that the rictor induced phosphorylation of AKT in Ser473 is required for TORC2 function in early development of mouse embryos.     

Directional asymmetry reversal of male copulatory organs in chafer beetles (Coleoptera: Scarabaeidae): implications on left–right polarity determination in insect terminalia

During large‐scale examinations of male genitalia for taxonomic studies of Sericini chafers (Coleoptera: Scarabaeidae), we have found that reversals of directed left–right (LR) asymmetry in male copulatory organs are compared to other insects not common. However, such inversions do occur occasionally and they must be considered a significant ‘source of error’ in taxonomic studies. Our data indicate asymmetry reversal of male genitalia affects only the abdominal segments (genitalia) as changes in asymmetric structures of the head and thorax were not observed. Based on these observations, we believe that this partial abdominal LR asymmetry reversal of terminalia could be due to a local change in the polarity determining LR patterning in the growth zone of posterior abdominal segments as development of these segments begins.     

Identification of Rictor as a Novel Substrate of Polo-like kinase 1

Plk1 has been essentially described as a critical regulator of many mitotic events. However, increasing evidence supports the notion that its molecular functions are not restricted to the cell cycle. In particular, recent reports suggest the existence of a molecular and functional link between Plk1 and the mammalian target of rapamycin (mTOR) pathway, which controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Herein, we have identified rapamycin-insensitive companion of mTOR (Rictor), a core component of mTORC2, as a new Plk1 substrate and have shown that Plk1 phosphorylates Rictor at Ser1162 in vitro and in vivo. Surprisingly, cells expressing the unphosphorylatable mutant (S1162A) of Rictor did not show any effect on well characterized canonical PI3K-mTOR pathway. However, we found that cells expressing the unphosphorylatable form of Rictor have an elevated level of mSin1 isoform (mSin1.5). Considering that mSin1.5-containing mTORC2 was reported to associate with stress signaling, we propose that phosphorylation of Rictor at Ser1162 by Plk1 might be involved in a novel signaling pathway by regulating the mSin1.5-defined mTORC2.     

ALK4 functions as a receptor for multiple TGF beta-related ligands to regulate left-right axis determination and mesoderm induction in Xenopus

In Xenopus, several TGF betas, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left-right (LR) defects when ectopically targeted to right mesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGF betas. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGF beta receptor(s) are involved in mediating left-side TGF beta signaling. We report here that similar to effects of ectopic TGF betas, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGF beta(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGF betas, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGF betas is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGF betas to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.     

The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.     

mTOR Signalling in Health and Disease

The TOR (target of rapamycin) proteins are found in all eukaryotes. TOR has a protein kinase domain, as well as other domains through which it interacts with partner proteins to form at least two types of multiprotein complex, TORC1 and TORC2 (TOR complexes 1 and 2). Rapamycin, an antibiotic and immunosuppressant, inhibits functions of TORC1. Use of this drug has revealed roles for TORC1 and its mammalian counterpart, mTORC1, in promoting many anabolic processes. mTORC1 signalling is activated by growth factors and nutrients. It is highly active in many cancers and plays a role in tumorigenesis and in other diseases. Much less is known so far about the functions and regulation of (m)TORC2. The goal of this meeting was to bring together researchers studying the roles of mTORC1/2 in normal cell and animal physiology in diverse systems, as well as scientists exploring the therapeutic value of inhibiting mTOR (mammalian TOR) signalling.