The LIM protein Ajuba is required for ciliogenesis and left-right axis determination in medaka

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer’s vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.     

The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination

Analysis of several mutations in the mouse is providing useful insights into the nature of the genes required for the establishment of the left-right axis during early development. Here we describe a new targeted allele of the mouse Tg737 gene, Tg737(Delta)2-3(beta)Gal), which causes defects in left-right asymmetry and other abnormalities during embryogenesis. The Tg737 gene was originally identified based on its association with the mouse Oak Ridge Polycystic Kidney (orpk) insertional mutation, which causes polycystic kidney disease and other defects. Complementation tests between the original orpk mutation and the new targeted knock-out mutation demonstrate that Tg737(Delta)2-3(beta)Gal) behaves as an allele of Tg737. The differences in the phenotype between the two mutations suggest that the orpk mutation is a hypomorphic allele of the Tg737 gene. Unlike the orpk allele, where all homozygotes survive to birth, embryos homozygous for the Tg737(Delta)2-3(beta)Gal) mutation arrest in development at mid-gestation and exhibit neural tube defects, enlargement of the pericardial sac and, most notably, left-right asymmetry defects. At mid-gestation the direction of heart looping is randomized, and at earlier stages in development lefty-2 and nodal, which are normally expressed asymmetrically, exhibit symmetrical expression in the mutant embryos. Additionally, we determined that the ventral node cells in mutant embryos fail to express the central cilium, which is a characteristic and potentially functional feature of these cells. The expression of both Shh and Hnf3(beta) is downregulated in the midline at E8.0, indicating that there are significant alterations in midline development in the Tg737(Delta)2-3(beta)Gal) homozygous embryos. We propose that the failure of ventral node cells to fully mature alters their ability to undergo differentiation as they migrate out of the node to contribute to the developing midline structures. Analysis of this new knockout allele allows us to define a critical role for the Tg737 gene during early embryogenesis. We have named the product of the Tg737 gene Polaris, which is based on the various polarity related defects associated with the different alleles of the Tg737 gene.     

The N-terminal extracellular domain is required for polycystin-1-dependent channel activity

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca(2+)-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca(2+)-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking approximately 2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.     

IP3 signaling is required for cilia formation and left-right body axis determination in Xenopus embryos

Vertebrate left–right (LR) body axis is manifested as an asymmetrical alignment of the internal organs such as the heart and the gut. It has been proposed that the process of LR determination commonly involves a cilia-driven leftward flow in the mammalian node and its equivalents (Kupffer’s vesicle in zebrafish and the gastrocoel roof plate in Xenopus). Recently, it was reported that Ca²⁺ flux regulates Kupffer’s vesicle development and is required for LR determination. As a basis of Ca²⁺ flux in many cell types, inositol 1,4,5-trisphosphate (IP₃) receptor-mediated calcium release from the endoplasmic reticulum (ER) plays important roles. However, its involvement in LR determination is poorly understood. We investigated the role of IP₃ signaling in LR determination in Xenopus embryos. Microinjection of an IP₃ receptor-function blocking antibody that can inhibit IP₃ calcium channel activity randomized the LR axis in terms of left-sided Pitx2 expression and organ laterality. In addition, an IP₃ sponge that could inhibit IP₃ signaling by binding IP₃ more strongly than the IP₃ receptor impaired LR determination. Examination of the gastrocoel roof plate revealed that the number of cilia was significantly reduced by IP₃ signal blocking. These results provide evidence that IP₃ signaling is involved in LR asymmetry formation in vertebrates.     

Xenopus nodal related-1 is indispensable only for left-right axis determination

In Xenopus, multiple nodal-related genes are expressed in the organizer region. Among them, only Xenopus nodal related-1 (Xnr-1) is expressed unilaterally in the left lateral plate mesoderm (LPM) at late neurula-early tailbud stage. To elucidate the essential role of Xnr-1 for left-right specification, loss of function experiments using antisense morpholino oligonucleotides (MOs) targeting three different regions of Xnr-1 were performed. Left-side injection of Xnr-1 MO suppresses the left-side specific genes such as Xnr-1, Xenopus antivin (lefty) and Xenopus pitx2 and randomizes cardiac and visceral left-right orientation. In contrast, paraxial bilateral expression of Xnr-1 along the posterior notochord is not affected by the Xnr-1 MO. In embryos injected with the Xnr-1 MO, morphology of dorsal axial structures is normal and dorsal expression of sonic hedgehog and TGF-beta5 is not changed. Right-side injection of Nodal protein, or polyethyleneimine-based gene transfer of Xnr-1 mRNA in the right LPM induces Xnr-1 and pitx2 in the same side and fully (more than 90%) reverses situs of the internal organs. Left-side injection of Nodal protein restores normal left-right orientation in the embryos that were injected with Xnr-1 MO into the left blastomere and would cause randomization of the left-right axis without the Nodal injection. Taken together, unilateral expression of Xnr-1 in the left LPM directs the orientation of the left-right axis by driving the left-specific gene cascade. Knockdown of Xnr-1 function by the MOs suggests that Xnr-1 is indispensable only for the left-right orientation and dispensable for other embryonic axes probably owing to the redundancy in the function of multiple Xnrs.     

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.     

Polarity proteins are required for left-right axis orientation and twin-twin instruction

Two main classes of models address the earliest steps of left-right patterning: those postulating that asymmetry is initiated via cilia-driven fluid flow in a multicellular tissue at gastrulation, and those postulating that asymmetry is amplified from intrinsic chirality of individual cells at very early embryonic stages. A recent study revealed that cultured human cells have consistent left-right (LR) biases that are dependent on apical-basal polarity machinery. The ability of single cells to set up asymmetry suggests that cellular chirality could be converted to embryonic laterality by cilia-independent polarity mechanisms in cell fields. To examine the link between cellular polarity and LR patterning in a vertebrate model organism, we probed the roles of apical-basal and planar polarity proteins in the orientation of the LR axis in Xenopus. Molecular loss-of-function targeting these polarity pathways specifically randomizes organ situs independently of contribution to the ciliated organ. Alterations in cell polarity also disrupt tight junction integrity, localization of the LR signaling molecule serotonin, the normally left-sided expression of Xnr-1, and the LR instruction occurring between native and ectopic organizers. We propose that well-conserved polarity complexes are required for LR asymmetry and that cell polarity signals establish the flow of laterality information across the early blastoderm independently of later ciliary functions. genesis 50:219-234, 2012. ? 2011 Wiley Periodicals, Inc.     

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.     

Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis.

Determination of the left-right position (situs) of visceral organs involves lefty, nodal and Pitx2 genes that are specifically expressed on the left side of the embryo. We demonstrate that the expression of these genes is prevented by the addition of a retinoic acid receptor pan-antagonist to cultured headfold stage mouse embryos, whereas addition of excess retinoic acid leads to their symmetrical expression. Interestingly, both treatments lead to randomization of heart looping and to defects in heart anteroposterior patterning. A time course analysis indicates that only the newly formed mesoderm at the headfold-presomite stage is competent for these retinoid effects. We conclude that retinoic acid, the active derivative of vitamin A, is essential for heart situs determination and morphogenesis.     

terra is a left-right asymmetry gene required for left-right synchronization of the segmentation clock

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.     

The Arabidopsis voltage-dependent anion channel 2 is required for plant growth

The voltage-dependent anion channels (VDACs), known as a major group of outer mitochondrial membrane proteins, are present in all eukaryotic species. In mammalian cells, they have been established as a key player in mitochondrial metabolism and apoptosis regulation. By contrast, little is known about the function of plant VDACs. Recently, we performed functional analysis of all VDAC gene members in Arabidopsis thaliana, and revealed that each AtVDAC member has a specialized function. Especially, in spite of similar subcellular localization and expression profiling of AtVDAC2 and AtVDAC4, both the T-DNA insertion knockout mutants of them, vdac2–2 and vdac4–2, showed severe growth retardation. These results suggest that AtVDAC2 and AtVDAC4 proteins clearly have distinct functions. Here, we introduced the AtVDAC2 gene into the vdac2–2 mutant, and demonstrated that the miniature phenotype of vdac2–2 plant is abolished by AtVDAC2 expression.     

Smad5 is essential for left-right asymmetry in mice

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.     

A flagellar polycystin-2 homolog required for male fertility in Drosophila

A common inherited cause of renal failure, autosomal dominant polycystic kidney disease results from mutations in either of two genes, PKD1 and PKD2, which encode polycystin-1 and polycystin-2, respectively. Polycystin-2 has distant homology to TRP cation channels and associates directly with polycystin-1. The normal functions of polycystins are poorly understood, although recent studies indicate that they are concentrated in the primary cilia of a variety of cell types. In this report we identified a polycystin-2 homolog in Drosophila melanogaster; this homolog localized to the distal tip of the sperm flagella. A targeted mutation in this gene, almost there (amo), caused nearly complete male sterility. The amo males produced and transferred normal amounts of motile sperm to females, but mutant sperm failed to enter the female sperm storage organs, a prerequisite for fertilization. The finding that Amo functions in sperm flagella supports a common and evolutionarily conserved role for polycystin-2 proteins in both motile and nonmotile axonemal-containing structures.     

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.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

The GBS PI-2a pilus is required for virulence in mice neonates

Background

Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone.

Methodology/Principal Findings

In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity.

Conclusions/Significance

These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.     

Calcium dependence of polycystin-2 channel activity is modulated by phosphorylation at Ser812

Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser(812), a putative casein kinase II (CK2) substrate domain. Ser(812) can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca(2+) but there is a shift in this Ca(2+) dependence such that S812A is 10-fold less sensitive to Ca(2+) activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca(2+) transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser(812) modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.