Quantifying The Gurken Morphogen Gradient in Drosophila Oogenesis

David Schmidt & Mike Robitaille

Lea A. Goentoro, Gregory T. Reeves,Craig P. Kowal, Stanislav Y. Shvartsman, Stanislav Y.

ShvartsmanDepartment of Chemical Engineering and

Lewis-Sigler Institute for Integrative Genomics

Luigi Martinelli,Department of Mechanical and Aerospace

Engineering

Trudi Schϋpbach,Howard Hughes Medical Institute and

Department of Molecular Biology

Introduction: Why is this Important

• Quantifying a morphogen spatial distribution is essential to understand how they induce cell fates during development– Learning specifics about biology’s “control loops,”

how to engineer specific responses• Drosophila oogenesis – one of the most

studied epithelial pattern formation models– Plenty of background for comparison/validation

Introduction: Why is this Important

• Drosophila oogenesis Epidermal Growth Factor Receptor (EGFR) signaling studies show Gurken influences the expression of “tens to hundreds” of genes in follicular epithelium

• Quantifying Gurken’s gradient shape can help determine– Expression thresholds/triggers for specific genes– Estimation/prediction of expression location

Introduction: What to measure

• Quantifying Gurken’s spatial distribution is required to accurately interpret responses by EGFR in the follicular epithelium

Follicular Epithelium

Oocyte

Gurken Secretion

ExtracellularGap

Dorsal

Ventral

Introduction: What to measure

• Previous experiments have studied mutants with varying levels of Gurken secretion deduced a model for EGFR activation

• Have not modeled or visualized the morphogen gradient

Introduction: What to measure

• Staining techniques fail to visualize secreted Gurken– Concentration in ECG negligible compared to

concentration in cell• Attempts at tagging not successful– Originates in oocyte, natural secretion would give

most accurate results

Introduction: What to measure

• EGFR Activation

Oocyte

Introduction: What to measure

• A parameter estimation strategy for quantifying morphogen spatial distribution– Biophysical Modeling– Dimensional Analysis with a single dimensionless

parameter– Quantification of responses in a number of wild-

type genetic backgrounds– Quantification of assays using Gal4/UAS gene

expression system

Results: What can we measure?

• Oocyte physical dimensions & size of Gurken secretion source can be measured

• Binding rates can be experimentally measured• No experimental assays can directly measure

extracellular diffusivity

Results: Gurken Gradient Shape

Results: Gradient CharacterizationL>>λ φ>>1 gv/gd=0

L<<λ φ<<1 gv/gd=1

Bound/R

Dorsal Ventral

gd gv

L>>λ

L<<λ

Oocyte

Follicular Epithelium

0

Follicular Epithelium

Oocyte

So how do we estimate φ?

Need some sort of indicator/marker of the Gurken concentration/level at a specific location.

So how do we estimate φ?

• Use pipe gene as a reporter for monitoring the Gurken gradient why?

• Directly repressed by Gurken-induced Ras/MAPK signaling

• “on” or “off” switch• Sharp and smooth gradient• We can actually measure it

Using Pipe as a reporter

Liga

nd-r

ecep

tor c

ompl

ex

conc

entr

ation

distance

Measuring the Pipe Boundary

• pipeST2 RNA Probe prepared with dig RNA Labeling Kit

• Flied maintained at room temp for 2 days, then placed on yeast at room temp for 2 days

• Ovaries dissected in cold PBS, then fixed in preperation mixture for 20 min before hybridization

• Egg chamberes cross sectioned, imaged at 40x

Using Pipe as a reporter

Introduce dimensionless Gurken concentration at the pipe boundary (denoted by p)

Using Pipe as a reporter

• Φ ~R0.5 depends on EGFR expression levels• γ does not, but it’s location does!• Pipe boundary depends of EGFR expression

if # receptors ↑ less Gurken makes it downstream, CT is reached at a location closer to source

Using Pipe as a reporter

↑ EGFR expression ↑R ↑φ L/λ >>1

Rbound

θ

↓ EGFR expression ↓ R ↓ φ L/λ <<1

Rbound

θ+1

CT

CT

Using Pipe as a reporter

Liga

nd-r

ecep

tor c

ompl

ex

conc

entr

ation

Change EGFR expression level change φ change pipe boundary

Using Pipe as a reporter

The way pipe boundary changes with increase of EGFR expression is different for different initial φ

Using Pipe as a reporter

The way pipe boundary changes with increase of EGFR expression is different for different initial φ

Measure experimentally!

Using Pipe as a reporter

• Use Gal4/UAS system to drive EGFR overexpression• Gal4-E9 weak driver• Gal4-T155 strong driver• How do we measure Change R experimentally???• Measure in egg chambers stage 9-10B– Pipe expression constant during these stages

Using Pipe as a reporter

Estimating φ

• Introduce J(θp, φ): dimensionless gurken concentration at pipe expression domain

• ΔR Δ φ Δθp BUT J does not change

Ro = initialR1 = weak driverR2 = strong driver

Estimating φ• Five measurable inputs

– Widths of pipe domains in 3 genetic backgrounds– Relative strength of two Gal4 Drivers– Egg aspect ratio

• Numerically solved for φ = 2.7, 90% confidence interval (1.5, 5.1)

• Length scale of the signal is ~ 1/3 the size of the patterned field (L~3λ)

• Confirms hypothesis that L ~ λ

Gurken Gradient

• Ventral side ~10% of dorsal• Supports Gurken as a long-

range signal• Also agrees with known

kekkon and sprouty expression

• Gradient can allow both high & low threshold targets of Gurken signalling

CT

Cks~ 3 CT

Discussion

• Qualitative depiction of Gurken gradient, characterized by φ

• Quantitative understanding how Gurken gradient regulates to perturbations

• EGFR transduces & reglates spacial range of Gurken (not new, found in many other systems i.e. Casanova 1993)

• Can be expanded to other Gurken targets, quantify their thresholds

Discussion

• Gurken gradient sensitive to EGFR expression levels downstream processes can buffer significant variations of pipe domain

• Model predicts ↑ EGFR expression ↑ pipe domain, but real life is more complicated

• Spitz-positive feedback exists in dorsal region, there is ligand-independent EGFR activation

Discussion

• Details on receptor activation, trafficking dynamics, feedback loops left out

• Seems L ~ λ in all patterning systems where morphogen gradient needs localized production and uniform degradation

• Future work: determine L/λ for other morphogens