Lecture 5

• Anterior Posterior axis formation: Cell Biology

• Bicoid is a morphogen

Lab 5 schedule

• On Monday and Tuesday come in at the regular time.

• On Tuesday and Wednesday come to B&G 3015 to mount your embryos. Elyse will discuss the time of day to come.

Exams

• Midterm Tuesday Feb. 28, 2012

UCC 63, 7-10PM

• Tentative Final exam date Thursday April 19, 2012 at 9:00AM

Structure of the egg

Dorsal appendage

Chorion

VitellineMicropyle

D

V

A P

Cellular Blastoderm

Hartenstein 1993

Blastoderm fate map

Hartenstein 1993

Simplified Fate Map

Nusslein-Volhard et al., Science 238, 1675-1681

Remove posterior pole plasm

Classical Embryology

Remove 5-10% of the anterior cytoplasm

Nusslein-Volhard et al., Science 238, 1675-1681

Transplant anterior cytoplasm to posterior

Nusslein-Volhard et al., Science 238, 1675-1681

Transplant posterior cytoplasm to anterior

Nusslein-Volhard et al., Science 238, 1675-1681

Transplant posterior cytoplasm to anterior

Nusslein-Volhard et al., Science 238, 1675-1681

Transplant posterior cytoplasm to an anterior that lacks the anterior signal

Nusslein-Volhard et al., Science 238, 1675-1681

Properties of the polar center signals

• Both exert long range effects

• The anterior and posterior activities inhibit each other

• Both activities have inductive properties

Properties of the polar center signals

• Both exert long range effects

Remove posterior pole plasm

Classical Embryology

Remove 5-10% of the anterior cytoplasm

Nusslein-Volhard et al., Science 238, 1675-1681

Properties of the polar center signals

• The anterior and posterior activities inhibit each other

Transplant posterior cytoplasm to anterior

Nusslein-Volhard et al., Science 238, 1675-1681

Transplant anterior cytoplasm to posterior

Nusslein-Volhard et al., Science 238, 1675-1681

Properties of the polar center signals

• Both activities have inductive properties

Transplant anterior cytoplasm to posterior

Nusslein-Volhard et al., Science 238, 1675-1681

Transplant posterior cytoplasm to an anterior that lacks the anterior signal

Nusslein-Volhard et al., Science 238, 1675-1681

What you may know already

Bicoid: Anterior signal

Nanos: Posterior signal

How do we know this?

Maternal effect screens

bcd/bcd mother

Normal egg

Larva missing a head

Nusslein-Volhard et al., Science 238, 1675-1681

Three classes of maternal effect phenotypes

Nusslein-Volhard et al., Science 238, 1675-1681

The real cuticles

wt bcd oskar torso

Nusslein-Volhard et al., Science 238, 1675-1681

Mutant combinations

St Johnston and Nusslein-VolhardCell 1992

Transplantation of cytoplasm into bcd mutants

Nusslein-Volhard et al., Science 238, 1675-1681

Controlling anterior posterior polarity

Nusslein-Volhard et al., Science 238, 1675-1681

The difference between the posterior and anterior signals

Nusslein-Volhard et al., Science 238, 1675-1681

Distinct Properties of the signals

• Anterior signal-instructive. Induces polarity at site of injection.

• Posterior signal-permissive. Polarity independent of injection site.

Evidence for Bicoid

• Rescue activity spatially distributed

• Amount of rescue dependent on amount injected

• Linear relationship between gene dose and rescue activity

• mRNA asymmetrically localized and protein expressed in a gradient

Bicoid rescue assay

Nusslein-Volhard et al., Science 238, 1675-1681

% rescueactivity

100%

50%

% egg length100% 0%40%

Rescue activity is spatially distributed

% rescue activity

0 1/3 2/3 1

Amount of rescue is dependent on amount injected

100%

50%

Transplanted volume

Linear relationship between gene dose and rescue activity

100%

50%

0 1 2 3

% rescue activity

Dose of bicoid gene in mothers

mRNA assymetrically localized and protein expressed in a gradient

mRNA

Protein

Driever and Nusslein-Volhard Cell 54, 83-93

Bicoid

• Bicoid has the properties of the anterior signal

• Does Bicoid need to interact with any other anteriorly localized signals to work?

Bicoid is necessary and sufficient

Bicoid mRNA made in vitro

mRNA injected at posterior pole

Bicoid protein expression

Larva with two heads

Driever et al., 1990 Development 109, 811-820

Bicoid

• Bicoid is the anterior signal

• What does Bicoid encode?

• How does Bicoid work?

Bicoid encodes a homeodomain containing protein.

Hunchback lacks anterior segments

Nusslein-Volhard and Wieschaus 1980 Nature 287, 795

Bicoid mRNA

Bicoid protein

Hunchback mRNAexpression

Hunchback is expressed at the anterior pole

Driever 1993

Hunchback gene

Driever and Nusslein-Volhard Nature 337, 138-143

Bicoid is a transcriptional activator

Driever and Nusslein-Volhard Nature 337, 138-143

mRNA assymetrically localized and protein expressed in a gradient

mRNA

Protein

Driever and Nusslein-Volhard Cell 54, 83-93

Morphogen

• Long range action

• Affect polarity

• Concentration dependent

• Different fates at different concentrations

Morphogen

• Long range action

Bicoid mRNA

Bicoid protein

Hunchback mRNAexpression

Hunchback is expressed at the anterior pole

Driever 1993

Morphogen

• Affect polarity

Bicoid is necessary and sufficient

Bicoid mRNA made in vitro

mRNA injected at posterior pole

Bicoid protein expression

Larva with two heads

Driever et al., 1990 Development 109, 811-820

Morphogen

• Concentration dependent

Concentration dependence of Bicoid function

# of bicoid genes in the mother

1

2

0

4

6

hb-lacZ expression

Concentration dependence of Bicoid function

Struhl et al., 1989 Cell 57, 1259-1273

Morphogen

• Different fates at different concentrations

Distinct Bicoid binding sites are activated at different concentrations of Bicoid

Morphogen

• Long range action

• Affect polarity

• Concentration dependent

• Different fates at different concentrations

How is bicoid mRNA localized to the anterior pole?

Other anterior class mutants

Driever and Nusslein-Volhard Cell 54, 94-104

Bicoid gradient affected in the mutants

Driever and Nusslein-Volhard Cell 54, 94-104

The mutants affect different stages of bicoid mRNA deposition in the developing oocyte

St. Johnston et al., 1989 Development sup 13-19

Localization of bicoid mRNA: recent paper

Irion and St Johnston 2007 Nature 445, 554-558