Dissection of the &lt;em&gt;Drosophila&lt;/em&gt; Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

DeAngelis, Miles W.; Johnson, Ruth I.

doi:10.3791/59299

Cited by 9 publications

(8 citation statements)

References 24 publications

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“…Fly strains were maintained at 25°C on nutrient-rich Drosophila media and prepupae selected and maintained in humidified chambers until dissection at 40 h after puparium formation ( DeAngelis and Johnson, 2019 ). Mouse anti-Arm (1:50; Developmental Studies Hybridoma Bank), and anti-mouse-Cy3 (1:200; Jackson ImmunoResearch) were used to detect AJs and retinas imaged with a Leica DM5500 B fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Perez-Vale

Yow

Johnson

et al. 2021

Journal of Cell Biology

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Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction–cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction–cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

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Section: Methodsmentioning

confidence: 99%

Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Perez-Vale

Yow

Johnson

et al. 2021

Journal of Cell Biology

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“…The larval wing discs [ 69 ], pupal eye discs [ 70 ], and adult posterior midguts [ 71 ] were each dissected and fixed as previously described in the respective references. All of the samples were stained as previously described [ 69 ].…”

Section: Methodsmentioning

confidence: 99%

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

et al. 2021

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Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

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“…Pupal eyes were dissected and fixed as previously described ( DeAngelis and Johnson 2019 ). For 1° antibody staining, rat anti-E-cad (1:20, DSHB, # 528120) was used to visualize cell boundaries.…”

Section: Methodsmentioning

confidence: 99%

“…Between 50 and 70 eyes were dissected from 21 and 40 h APF Canton S pupae at the same time each day, and total RNA was extracted from three biological replicates using the ReliaPrep RNA Tissue Miniprep System (Promega Corporation, Cat # M3001) as described ( DeAngelis and Johnson 2019 ). Barcoded cDNA library prep was performed using the TruSeq library preparation kit, libraries were pooled, balanced pooling was confirmed using qPCR, and 51-bp paired-end sequencing was performed by the University of Michigan Sequencing Core Facility as described ( DeAngelis et al 2020 ).…”

Section: Methodsmentioning

confidence: 99%

Comparative transcriptome analyses of theDrosophilapupal eye

DeAngelis

Coolon

Johnson

2020

G3 Genes|Genomes|Genetics

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Tissue function is dependent on correct cellular organization and behavior. As a result, the identification and study of genes that contribute to tissue morphogenesis is of paramount importance to the fields of cell and developmental biology. Many of the genes required for tissue patterning and organization are highly conserved between phyla. This has led to the emergence of several model organisms and developmental systems that are used to study tissue morphogenesis. One such model is the Drosophila melanogaster pupal eye that has a highly stereotyped arrangement of cells. In addition, the pupal eye is postmitotic that allows for the study of tissue morphogenesis independent from any effects of proliferation. While the changes in cell morphology and organization that occur throughout pupal eye development are well documented, less is known about the corresponding transcriptional changes that choreograph these processes. To identify these transcriptional changes, we dissected wild-type Canton S pupal eyes and performed RNA-sequencing. Our analyses identified differential expression of many loci that are documented regulators of pupal eye morphogenesis and contribute to multiple biological processes including signaling, axon projection, adhesion, and cell survival. We also identified differential expression of genes not previously implicated in pupal eye morphogenesis such as components of the Toll pathway, several non-classical cadherins, and components of the muscle sarcomere, which could suggest these loci function as novel patterning factors.

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Dissection of the <em>Drosophila</em> Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Cited by 9 publications

References 24 publications

Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

Comparative transcriptome analyses of theDrosophilapupal eye

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