Testing Retina Fate Commitment in Xenopus by Blastomere Deletion, Transplantation, and Explant Culture

Moody, Sally A.

doi:10.1007/978-1-61779-848-1_7

Cited by 6 publications

(3 citation statements)

References 17 publications

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“…As detailed protocols are available on the identification and dissection of blastomeres (Moody, 2012 ; Grant et al, 2013 ), only a brief summary of the major steps follows.…”

Section: Methodsmentioning

confidence: 99%

High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo

Lombard‐Banek

Moody

Nemes

2016

Front. Cell Dev. Biol.

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Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of the basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in the 16-cell embryo of the South African clawed frog (Xenopus laevis) that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

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“…As detailed protocols are available on the identification and dissection of blastomeres (Moody, 2012 ; Grant et al, 2013 ), only a brief summary of the major steps follows.…”

Section: Methodsmentioning

confidence: 99%

High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo

Lombard‐Banek

Moody

Nemes

2016

Front. Cell Dev. Biol.

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“…Lineage Tracing and Fate Mapping in Xenopus Embryos [Moody 2018] and Moody [2012a;2012b]). Place the embryos in sterile 1× culture solution (SS, MMR, or MBS) and incubate at 14˚C-20˚C until reaching the desired stage for injection.…”

Section: Selecting and Microinjecting Embryosmentioning

confidence: 99%

Microinjection of mRNAs and Oligonucleotides

Moody

2018

Cold Spring Harb Protoc

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Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage. Xenopus blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsAgarose, 2% in 1× culture medium (if preparing agarose injection dishes in Step 1)Add 2 g of electrophoresis-grade agarose to 100 mL of 1× culture medium in a screw-cap glass bottle. Autoclave to dissolve agarose. This can be stored in the refrigerator, and microwaved to liquefy the agarose when it is next needed.

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“…Elevated Hh signaling often reduces the expression of eye markers and decreases the size of the eye (Amato et al, 2004;Koide et al, 2006;Rorick et al, 2007;Min et al, 2011). We injected RUSC 1233-C (1 ng) at the 8-cell stage into dorsal animal blastomeres of Xenopus embryos, which give rise to the neural tube and retina (Moody, 1987(Moody, , 2012. To assess changes in Hh signaling, we monitored the expression of gli1, a direct target of Hh signaling (Lee et al, 1997).…”

Section: Rusc1 Inhibits Hh Signaling During Xenopus Embryonic Developmentioning

confidence: 99%

Members of the Rusc protein family interact with Sufu and inhibit vertebrate Hedgehog signaling

Jin

Schwend

et al. 2016

Development

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Hedgehog (Hh) signaling is fundamentally important for development and adult tissue homeostasis. It is well established that in vertebrates Sufu directly binds and inhibits Gli proteins, the downstream mediators of Hh signaling. However, it is unclear how the inhibitory function of Sufu towards Gli is regulated. Here we report that the Rusc family of proteins, the biological functions of which are poorly understood, form a heterotrimeric complex with Sufu and Gli. Upon Hh signaling, Rusc is displaced from this complex, followed by dissociation of Gli from Sufu. In mammalian fibroblast cells, knockdown of Rusc2 potentiates Hh signaling by accelerating signaling-induced dissociation of the Sufu-Gli protein complexes. In Xenopus embryos, knockdown of Rusc1 or overexpression of a dominant-negative Rusc enhances Hh signaling during eye development, leading to severe eye defects. Our study thus uncovers a novel regulatory mechanism controlling the response of cells to Hh signaling in vertebrates.

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Testing Retina Fate Commitment in Xenopus by Blastomere Deletion, Transplantation, and Explant Culture

Cited by 6 publications

References 17 publications

High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo

High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo

Microinjection of mRNAs and Oligonucleotides

Members of the Rusc protein family interact with Sufu and inhibit vertebrate Hedgehog signaling

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