Evidence for the regulation of left-right asymmetry inCiona intestinalis by ion flux

Shimeld, Sebastian M.; Levin, Michael

doi:10.1002/dvdy.20792

Cited by 71 publications

(83 citation statements)

References 49 publications

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“…1). The specifics (e.g., spatial distribution, timing) differ among model species with divergent early embryonic architectures, these and the other biophysical components elucidated in Xenopus have now also been shown to be required for asymmetry in sea urchin (Duboc et al, 2005;Hibino et al, 2006), Ciona (Shimeld and Levin, 2006), chick (Levin et al, 2002;Raya et al, 2004;Adams et al, 2006), zebrafish (Adams et al, 2006), C. elegans (Chuang et al, 2007), and snails (Shibazaki et al, 2004).…”

Section: Intracellular Mechanisms Origins and Evolution Of Early Cytomentioning

confidence: 99%

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

Vandenberg

Levin

2010

Developmental Dynamics

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Consistent laterality is a crucial aspect of embryonic development, physiology, and behavior. While strides have been made in understanding unilaterally expressed genes and the asymmetries of organogenesis, early mechanisms are still poorly understood. One popular model centers on the structure and function of motile cilia and subsequent chiral extracellular fluid flow during gastrulation. Alternative models focus on intracellular roles of the cytoskeleton in driving asymmetries of physiological signals or asymmetric chromatid segregation, at much earlier stages. All three models trace the origin of asymmetry back to the chirality of cytoskeletal organizing centers, but significant controversy exists about how this intracellular chirality is amplified onto cell fields. Analysis of specific predictions of each model and crucial recent data on new mutants suggest that ciliary function may not be a broadly conserved, initiating event in left-right patterning. Many questions about embryonic left-right asymmetry remain open, offering fascinating avenues for further research in cell, developmental, and evolutionary biology. Developmental Dynamics 239:3131-3146,

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Section: Intracellular Mechanisms Origins and Evolution Of Early Cytomentioning

confidence: 99%

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

Vandenberg

Levin

2010

Developmental Dynamics

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“…Although the genetic cascades of secreted signals that occur on only one side of the embryo are being elucidated (Burdine and Schier, 2000;Whitman and Mercola, 2001), upstream mechanisms remain poorly understood (Levin, 2005;Yost, 2001). Recent findings shed light on the events which function upstream of asymmetric transcription (Bunney et al, 2003;Fukumoto et al, 2005b;Kramer et al, 2002;Levin and Mercola, 1998;Levin and Mercola, 1999;Tanaka et al, 2005), and it is now known that ion flux is involved in determining the laterality of several vertebrates and invertebrates (Duboc et al, 2005;Hibino et al, 2006;Levin et al, 2002;Shimeld and Levin, 2006). To gain molecular insight into endogenous ion flows that control asymmetry, we performed a loss-of-function drug screen (Adams and Levin, 2006), implicating the H + /K + -ATPase (Levin et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Adams

Robinson

Fukumoto³

et al. 2006

Development

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Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H + flux. A pharmacological screen implicated the H + -pump H + -V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H + -V-ATPase subunits during the first three cleavages. H + -flux across plasma membranes is also asymmetric at the four-and eight-cell stages, and this asymmetry requires H + -V-ATPase activity. Abolishing the asymmetry in H + flux, using a dominant-negative subunit of the H + -V-ATPase or an ectopic H + pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H + -V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (V mem ) regulation. Varying either pH or V mem , independently of direct manipulation of H + -V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H + -V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H + -V-ATPase induces heterotaxia in both species; in chick, H + -V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H + -V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH-and V mem -dependent model of the early physiology of LR patterning. Development 133, 1657Development 133, -1671Development 133, (2006 DEVELOPMENT 1658 necessary to characterize the endogenous behavior of the relevant pumps in embryos and to place their function in the context of known LR patterning mechanisms. Here, we explore the properties of H + -V-ATPase function in several vertebrate embryos. Through endogenous localization of the H + -V-ATPase and gain-and loss-offunction experiments in chick, frog and zebrafish, we identify the H + -V-ATPase as a novel, conserved, obligate component of LR patterning upstream of asymmetric gene expression. KEY WORDS: Left-right asymmetry, H + -V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage MATERIALS AND METHODS Animal husbandryXenopus embryos were collected according to standard protocols (Sive et al., 2000) in 0.1ϫ Modified Marc's Ringers (MMR) pH 7.8 + 0.1% Gentamicin. Xenopus embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Chick embryos from Charles River Laboratories, maintained at 38°C, were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1992). Zebrafish embryos (Westerfield, 1995) were maintained at 28.5°C in water containing 1 drop per gallon Methyl Blue. Assaying organ situsXenopus e...

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“…More specific compounds targeting different members of various K + and H + channel and pump families ultimately implicated the V-ATPase and H + / K + -ATPase pumps, and the KvLQT-1 and K atp channels; all of these targets were then successfully validated using molecular dominant negative constructs. The identification of this small number of proteins led to the successful characterization of their native expression, uncovering new subcellular localization events in early embryogenesis (Adams et al, 2006;Chen and Levin, 2004;Levin, 2004;Levin et al, 2002); a number of laboratories subsequently extended this work (Duboc et al, 2005;Hibino et al, 2006;Kawakami et al, 2005;Raya et al, 2004;Shimeld and Levin, 2006). This same strategy was then applied to ion transporters in a number of other embryonic patterning events, and to uncovering novel prenervous roles for the neurotransmitter serotonin (Fukumoto et al, 2005a,b;Levin et al, 2006).…”

Section: Sample Previous Applicationsmentioning

confidence: 99%

“…Others have extended them to Ciona and sea urchins (Hibino et al, 2006;Shimeld and Levin, 2006). Each of these model organisms offers a different advantage.…”

Section: Specific Considerations Potential Problems and Trouble-shomentioning

confidence: 99%

“…prior to the formation of neurons (Adams et al, 2006;Bunney et al, 2003;Chen and Levin, 2004;Cheng et al, 2002;Fukumoto et al, 2005a,b;Levin et al, 2002;Nogi et al, 2005;Qiu et al, 2005). The strategy is appropriate for vertebrate and invertebrate systems (Bunney et al, 2003;Hibino et al, 2006;Shimeld and Levin, 2006;Tomlinson et al, 2005), and has revealed a number of important aspects of physiological and morphogenetic regulation. To bring this powerful methodology to wider use in the study of developmental systems, we describe here the strategy, give several specific examples of its use, provide sample databases, and discuss its limitations and future potential.…”

Section: Introductionmentioning

confidence: 99%

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Inverse drug screens: a rapid and inexpensive method for implicating molecular targets

Adams¹,

Levin²

2006

Genesis

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SummaryIdentification of gene products that function in some specific process of interest is a common goal in developmental biology. Although use of drug compounds to probe biological systems has a very long history in teratology and toxicology, systematic hierarchical drug screening has not been capitalized upon by the developmental biology community. This "chemical genetics" approach can greatly benefit the study of embryonic and regenerative systems, and we have formalized a strategy for using known pharmacological compounds to implicate specific molecular candidates in any chosen biological phenomenon. Taking advantage of a hierarchical structure that can be imposed on drug reagents in a number of fields such as ion transport, neurotransmitter function, metabolism, and cytoskeleton, any assay can be carried out as a binary search algorithm. This inverse drug screen methodology is much more efficient than exhaustive testing of large numbers of drugs, and reveals the identity of a manageable number of specific molecular candidates that can then be validated and targeted using more expensive and specific molecular reagents. Here, we describe the process of this loss-of-function screen and illustrate its use in uncovering novel bioelectrical and serotonergic mechanisms in embryonic patterning. This technique is an inexpensive and rapid complement to existing molecular screening strategies. Moreover, it is applicable to maternal proteins, and model species in which traditional genetic screens are not feasible, significantly extending the opportunities to identify key endogenous players in biological processes.

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Evidence for the regulation of left-right asymmetry inCiona intestinalis by ion flux

Cited by 71 publications

References 49 publications

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Inverse drug screens: a rapid and inexpensive method for implicating molecular targets

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