Fixation of <i>Drosophila</i> Embryos

Rothwell, Wendy F.; Sullivan, William

doi:10.1101/pdb.prot4827

Cited by 31 publications

(26 citation statements)

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“…Embryo collection, permeabilization and fixation were based on the protocol described by Rothwell and Sullivan [ 78 ]. The Drosophila melanogaster flies were put on apple juice agar with yeast for egg lay at 25°C overnight.…”

Section: Methodsmentioning

confidence: 99%

ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo

et al. 2015

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Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.

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Section: Methodsmentioning

confidence: 99%

ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo

et al. 2015

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“…Embryos and eggs from the indicated mating were collected from grape juice/agar plates. Embryos were dechorionated in 50% commercial bleach, fixed in methanol/heptane (46), and stored at 4°C until use. Fixed embryos were washed with phosphate buffered saline with 0.1% Tween-20 (PBST) 3 times for 5 min each and blocked with PBST containing 5% vol of normal goat serum (PBST-NGS).…”

Section: Methodsmentioning

confidence: 99%

TheDrosophilaTrpm channel mediates calcium influx during egg activation

Wolfner

2019

Proc. Natl. Acad. Sci. U.S.A.

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Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line–specific RNAi knockdown, and germ-line–specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.

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“…Microtubules were stained using a monoclonal primary antibody (mouse) against β-tubulin (E7, Developmental Studies Hybridoma Bank) at a dilution 1:100, and a secondary antibody conjugated to Alexa 488 dye (Invitrogen, Carlsbad, CA) at a dilution of 1:1000. For phalloidin staining, embryos were fixed for 1 hr using PEMS and a final concentration of 8% formaldehyde, hand-devitellinized as described for D. melanogaster embryos ( Fernández et al, 2007 ; Kaltschmidt et al, 2002 ; Rothwell and Sullivan, 2000 ), and incubated with phallodin-Alexa488 or phalloidin-Alexa563 (Invitrogen, Carlsbad, CA) at a dilution of 1:200 for 1 hr. When double-staining against phalloidin and microtubules, embryos were fixed, hand-devitellinized, and stained for phalloidin first, followed by incubation with the β-tubulin primary and secondary antibodies as above.…”

Section: Methodsmentioning

confidence: 99%

Two consecutive microtubule-based epithelial seaming events mediate dorsal closure in the scuttle fly Megaselia abdita

Fraire-Zamora

Jaeger

Solon

2018

eLife

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Evolution of morphogenesis is generally associated with changes in genetic regulation. Here, we report evidence indicating that dorsal closure, a conserved morphogenetic process in dipterans, evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation. In Drosophila melanogaster, dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis. We find that dorsal closure in Megaselia abdita, a three-tissue system comprising serosa, amnion and epidermis, differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling. In addition to an actomyosin cable, M. abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis. Our study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression.

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Fixation of Drosophila Embryos

Cited by 31 publications

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ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo

ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo

TheDrosophilaTrpm channel mediates calcium influx during egg activation

Two consecutive microtubule-based epithelial seaming events mediate dorsal closure in the scuttle fly Megaselia abdita

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