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#experimental evidence

2 public questions tagged with this topic.

Morphogen Induction-l

Which experimental finding supports the existence of morphogen gradients?

Existence of morphogen gradients was substantiated by visualizing Bicoid protein distribution in Drosophila early syncytial blastoderm using antibody staining. Immunostaining revealed anterior-high posterior-low exponential gradient correlating with fate map and hunchback expression boundary. Manipulating bicoid dosage by genetics or mRNA injection shifted gap gene expression boundaries anteriorly or posteriorly in predictable threshold manner, and ectopic gradient induced mirror-image anterior structures posteriorly. This quantitative correlation between protein concentration, enhancer occupancy and transcriptional outcome provided direct evidence that graded distribution of single factor could impart positional values and specify distinct segment identities along embryonic axis without additional signals.

Ref: Driever and Nusslein-Volhard, Cell 1988, Bicoid Gradient Defines Anterior Pattern in Drosophila.

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Cytoskeleton

Which experimental evidence would best demonstrate that dynein is required for Golgi positioning?

Perinuclear positioning of Golgi ribbon relies on continuous dynein-dependent transport of Golgi stacks toward microtubule minus ends clustered at centrosome. Direct causality requires selective dynein perturbation while monitoring microtubules. Approach involves inducible expression of p150Glued CC1 fragment that competes for dynein binding to dynactin, siRNA against dynein heavy chain DYNC1H1, or pharmacologic ciliobrevin D that inhibits AAA+ ATPase without depolymerizing tubulin. Outcome assessed by immunofluorescence for cis marker GM130, medial Golgin-84, trans TGN46 shows rapid conversion of compact juxtanuclear ribbon to dispersed ministacks throughout cytoplasm, quantified by distance from nucleus and fragment number. Maintaining intact microtubules visualized by alpha-tubulin staining confirms defect due to motor loss not track loss. Depolymerizing microtubules with nocodazole also disperses Golgi but nonspecifically. Inhibiting kinesin would cause tighter perinuclear clustering opposite prediction, mutating actin tests different cytoskeleton. Thus dynein-specific inhibition leading to dispersion provides strongest mechanistic proof. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

Ref: Burkhardt Cell 1997; dynein-dynactin perturbation fragments Golgi, demonstrates retrograde positioning requirement.

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