Cloning and characterization of voltage‐gated calcium channel alpha1 subunits in <i>Xenopus laevis</i> during development

Lewis, Brittany B.; Wester, Matthew R.; Miller, Lauren E.; Nagarkar, Maitreyi D.; Johnson, M. Brittany; Saha, Margaret S.

doi:10.1002/dvdy.22102

Cited by 15 publications

(26 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have previously shown that depletion of Ca 2+ during C. intestinalis neurulation causes profound defects to anterior neural tube development, including an open neural plate phenotype in 40% of embryos, which is consistent with our findings here for CAV3 (Hackley et al 2013). Although T-type calcium channels are known to have early embryonic expression in the developing vertebrate anterior neural tube (Perez-Reyes 2003; Lewis et al 2009), to our knowledge this is the first implication of their involvement in neurulation and potentially neural tube closure. One of the advantages of Ciona compared to their vertebrate cousins is their lower genetic redundancy.…”

Section: Discussionsupporting

confidence: 91%

Exploiting the Extraordinary Genetic Polymorphism ofCionafor Developmental Genetics with Whole Genome Sequencing

et al. 2014

View full text Add to dashboard Cite

Studies in tunicates such as Ciona have revealed new insights into the evolutionary origins of chordate development. Ciona populations are characterized by high levels of natural genetic variation, between 1 and 5%. This variation has provided abundant material for forward genetic studies. In the current study, we make use of deep sequencing and homozygosity mapping to map spontaneous mutations in outbred populations. With this method we have mapped two spontaneous developmental mutants. In Ciona intestinalis we mapped a short-tail mutation with strong phenotypic similarity to a previously identified mutant in the related species Ciona savignyi. Our bioinformatic approach mapped the mutation to a narrow interval containing a single mutated gene, a-laminin3,4,5, which is the gene previously implicated in C. savignyi. In addition, we mapped a novel genetic mutation disrupting neural tube closure in C. savignyi to a T-type Ca 2+ channel gene. The high efficiency and unprecedented mapping resolution of our study is a powerful advantage for developmental genetics in Ciona, and may find application in other outbred species.A valuable attribute of many model organisms is the ability to conduct forward and reverse genetics. The availability of sequenced genomes and transcriptomes have streamlined reverse genetic approaches, but forward genetic approaches remain time consuming and cumbersome. Even for organisms with well-developed mutation mapping strategies and resources, classical linkage analysis can be slow and subject to chance. Genome-wide association studies now provide an alternative approach, but are severely limited by the need for high-frequency alleles and very large samples (Marchini et al. 2007;Cheng et al. 2010). A need remains for additional phenotype-to-genotype strategies in, for example, the investigation of quantitative traits, natural variation, and disease loci (Hillier et al. 2008;Jelier et al. 2011;Liti and Louis 2012;Lehner 2013). In recent years, new and inexpensive deep sequencing technologies have created opportunities for forward genetic approaches (Hobert 2010). By taking a snapshot of variation across the genome of an outbred population, a researcher can now quickly identify a region of homozygosity unique to mutant individuals. Variations of this method then use a fine-mapping parameter to define a high-confidence mapping interval and to retrieve a list of variable sites in the interval as a list of possible causal mutations. Modeling shows that the mapping power using whole-genome sequencing (WGS) is a function of how many genomes are sampled from mutant individuals, the recombination rate, and genome coverage (Leshchiner et al. 2012;Obholzer et al. 2012). This approach has worked efficiently and accurately for the well-assembled, annotated, and inbred genomes of model organisms such as Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus (Blumenstiel et al. 2009;Andersen et al. 2012;Leshchiner et al. 2012).Tunicates, such as the ascidian Ciona intestinalis, are clas...

show abstract

Section: Discussionsupporting

confidence: 91%

Exploiting the Extraordinary Genetic Polymorphism ofCionafor Developmental Genetics with Whole Genome Sequencing

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Calcium ions have been involved in left-right patterning of embryos in zebrafish, Xenopus laevis and mouse (McGrath et al, 2003;Kreiling et al, 2008;Lewis et al, 2009), in left-right determination by means of Notch signaling in chick embryos (Raya et al, 2004), and in the mediation of morphogenetic movements during neural tube formation in axolotl (Moran, 1990). Although no exact data exist on whether Ca 2þ ion kinetics have a specific function in tissue regeneration in vivo, various in vitro studies have suggested that Ca 2þ ions/channels are necessary for this process (Unlu et al, 2002;Nicou et al, 2007).…”

Section: Calciummentioning

confidence: 99%

Ion imaging during axolotl tail regeneration in vivo

Özkucur

Epperlein

Funk

2010

Developmental Dynamics

View full text Add to dashboard Cite

Several studies have reported that endogenous ion currents are involved in a wide range of biological processes from single cell and tissue behavior to regeneration. Various methods are used to assess intracellular and local ion dynamics in biological systems, e.g., patch clamping and vibrating probes. Here, we introduce an approach to detect ion kinetics in vivo using a noninvasive method that can electrophysiologically characterize an entire experimental tissue region or organism. Ion-specific vital dyes have been successfully used for live imaging of intracellular ion dynamics in vitro. Here, we demonstrate that cellular pH, cell membrane potential, calcium, sodium and potassium can be monitored in vivo during tail regeneration in the axolotl (Ambystoma mexicanum) using ion-specific vital dyes. Thus, we suggest that ion-specific vital dyes can be a powerful tool to obtain electrophysiological data during crucial biological events in vivo.

show abstract

“…Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] . Xenopus laevis, a classic model system for the study of early neural development 19,27,29,[31][32][40][41][42] , serves as a particularly suitable system for retinal primary cell culture 10,38,[43][44][45] .…”

Section: Introductionmentioning

confidence: 99%

“…In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,[44][45] .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Dissection, Culture, and Analysis of <em>Xenopus laevis</em> Embryonic Retinal Tissue

McDonough

Allen

Ng-Sui-Hing

et al. 2012

JoVE

Self Cite

View full text Add to dashboard Cite

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,[14][15][16][17][18] .While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,[19][20][21][22][23] . For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,[19][20][21][22][24][25][26][27][28][29][30][31][32][33] . Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] . Xenopus laevis, a classic model system for the study of early neural development 19,27,29,[31][32][40][41][42] , serves as a particularly suitable system for retinal primary cell culture 10,38,[43][44][45] .Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43 . In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,[44][45] .However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1). Video LinkThe video component of this article can be found at

show abstract

Cloning and characterization of voltage‐gated calcium channel alpha1 subunits in Xenopus laevis during development

Cited by 15 publications

References 52 publications

Exploiting the Extraordinary Genetic Polymorphism ofCionafor Developmental Genetics with Whole Genome Sequencing

Exploiting the Extraordinary Genetic Polymorphism ofCionafor Developmental Genetics with Whole Genome Sequencing

Ion imaging during axolotl tail regeneration in vivo

Dissection, Culture, and Analysis of <em>Xenopus laevis</em> Embryonic Retinal Tissue

Contact Info

Product

Resources

About