Positional information: fields, boundaries, and gradients
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Transcript of Positional information: fields, boundaries, and gradients
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Positional information: fields, boundaries, and gradients
Positional information is specified by (1) subdivision of larger fields of cells into smaller fields, and (2) specifying the "address" of each cell within the field.
This is a recursive process that requires translation of gradients of gene expression into sharp boundaries, and initiation of new gradients by these boundaries
What mechanisms are responsible for these transitions?
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The French flag model
Single gradient
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Threshold responses to the Dpp morphogen gradient
(Lost in dpp / - )
hnt ush
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Problems with the morphogen gradient model?
1. Precision: If there is a direct correspondence between morphogen concentration and the activation of downstream genes, then the organism has to precisely control at least three parameters:
Rate of morphogen productionRate of morphogen diffusionRate of morphogen degradation
2. Scale: = D/ regardless of the absolute size of the morphogenetic field.
How can this mechanism deal with variations in size, temperature, random fluctuations, etc.?
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The roles of maternal gradients in Drosophila
Gradients form from maternally deposited transcripts by diffusion or transport in a cell-free environment
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Opposing maternal gradients in the Drosophila egg
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Production of the Bicoid protein gradient in syncytial blastoderm
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Bicoid and the French Flag model
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Quantification of hunchback gradient
Cycle 13 Cycle 14
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Quantification of Bicoid gradient
Average intensity of sliding window the size of 1 nucleus
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Variability of Bicoid gradient
0.23 of max intensity
30% EL
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Variability of Bicoid gradient
Threshold Slope of exponential decay
Half the embryos deviate from the "norm" by more than 5 nuclei
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So what happens to the downstream target of this gradient?
(Bcd) = 0.07 EL(Hb) = 0.01 EL
2/3 of embryos deviate from the "norm" by less than 1 nucleus
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What about scale-independence?
Bcd gradient is not scaled Hb gradient is scaled
Thr
esho
ld
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Can cooperative activation explain the increased precision?
0.1 EL
~30%decrease
100%decrease
Cooperativity of binding is described by Hill coefficient (steepness of saturation curve, plotted against concentration, at 50% saturation)
log( /1-) = log[L] - logKd
HC = 1 means no cooperativity (each molecule binds independently)
HC > 1 means positive cooperativity (binding of one molecule increases affinity for additional molecules)
Bcd / Hb interaction would require HC of at least 10…
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Bcd
Hb
= 0.016
= 0.013
= 0.010
= 0.011
Temperature does not compromise precision (as predicted by cooperativity?)
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What does it take to lose precision?
*
*
*
Regulation by Nanos gradient?Mutual repression by other gap genes?
Autoregulation by Hunchback?
Scaling also remains unaffected (r = 0.7 - 0.76)
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stauHL
stauD3
staur9
staufen affects precision of Bicoid / Hunchback translation
Staufen is an RNA and mtb binding protein localized to both poles of the egg, and required for localizing other mRNAs
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Bicoid binding sites are sufficient for sharpening the expression boundary of an artificial transgene
lacZ expression is driven by a synthetic enhancer that contains 3 artificial high-affinity binding sites for Bcd (and nothing else)
The sharpness is about the same as for native hb and otd genes (5.4 vs 5.8 vs 4.6)
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Deletions of Bcd activation domains compromise sharpening
Mutant Bcd proteins expressed under the control of wild-type regulatory sequences of bicoid
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If does not even have to be Bicoid
Synthetic transcription factors fused to bicoid 3' UTR
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hb: 50.2% 1.5% ELotd: 72.1% 1.4% ELBcd3-lacZ: 71.4% 1.6% EL
Gal4-3CGN4 / UAS-lacZ: 1.7% ELGal4-2Q / UAS-lacZ: 1.9% EL
Precision is maintained by enhancers and transcription factors that have no homology to Bicoid and hunchback
So it must be a general feature of the morphogen gradient…
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Precision is compromised in staufen maternal mutants
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Boundary refinement is a general feature of TF gradient???
Possible explanations: A gradient of general transcriptional repressor?Cooperative interaction between TFs and transcriptional machinery?Changes in chromatin state? Cellularization?Or is this all an artifact of detection?
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Refining the response to a morphogen gradient
Dpp signaling represses the expression of its own antagonist, Brinker
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Brinker, repressed by Dpp, represses Dpp target genes
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brinker silencer causes repression of heterologous enhancers by Dpp signaling
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brinker silencer represses gene expression in response to Dpp signaling in a dosage-sensitive manner
Co-transfection of S2 cells with (1) lacZ reporter, (2) plasmid expressing Su(H) and constitutively active Notch, (3) variable amounts of plasmid expressing constitutively active Thickveins, and (4) luciferase plasmid for normalization of -Gal levels
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Spatial pattern of gene expression is determined by the balance of activator and silencer activities
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Components of Dpp signal transduction required for brk silencing
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TF binding mediates response to Dpp gradient
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This was as far as I got…
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Smo signals to activate Hh targets Ptc, when not bound to Hh ligand,
blocks Smo signalingBinding to Hh inactivates Pct,
releasing Smo to signal
Loss of Ptc leads to ectopic activation of Hh targets
Cells determine their position by measuring the concentration of active (unbound) Ptc, or the ratio of unbound to bound Ptc
Role of receptors and signal transduction in measuring morphogen gradients
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Constitutively active Ptcloop2 cannot bind Hh but can inactivate Smo
Question:Does the minimum amount of Ptcloop2 needed to shut down the Hh pathway depend on the presence of ligand-bound Ptc?
Testing the receptor ratio model
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Different transgenes produce different levels of expression within the normal physiological range
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Unbound Ptc represses Hh targets in a concentration-dependent manner
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Unliganded Ptc blocks Hh transduction in cells lacking endogenous Patched
In L > Ptc2, Hh transduction requires both Hh and endogenous Ptc(since Hh targets are OFF in ptc- clones, or in ptc+ cells away from compartment border)
So ligand-bound Ptc is not equivalent to the absence of Ptc - instead, it titrates out the inhibitory activity of unbound Ptc
y w hsp70–FLP UAS–GFPnls; FRT42D ptc IIW /dpp–lacZ FRT42D Tub1–Gal80; rp49 > CD2, y+ > ptc – hsp70 3'UTR / Tub1–Gal4
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Unliganded Ptc blocks Hh transduction in posterior compartment cells
nub-Gal4 / UAS- CiZnf.GFP
Posterior compartment cells are exposed to uniformly high levels of Hh
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y w hsp70–FLP UAS–GFPnls; rp49 > CD2,y+ > ptcloop2–hsp70 3'UTR FRT42D ptcIIW /dpp–lacZ FRT42D Tub1–Gal80; Tub1 > CD2,y+ > hh–ptc–Tub1 3' UTR
Activation of Hh targets depends on the ratio of ligand-bound and unbound Ptc receptor
Bound and unbound Ptc expressed in different ratios in the absence of endogenous Ptc
A two-fold change in this ratio is sufficient to change Hh targets from ON to OFF state
This range is within the normal range of Ptc concentrations (700% => 100% over a few cells