February 06

Developmental biology: Drosophila segmentation and repeated units

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* egg: generate the system

* larva: eat and grow

* pupa: structures inlarvae grow out to form adult fly: metamorphosis

(Drosophila is a holometabolous insect)

February 06

Developmental biology: Drosophila segmentation and repeated units

* Drosophila embryo: larva

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February 06

Developmental biology: Drosophila segmentation and repeated units

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Embryo stage: developmental patterning: most obvious characteristics in resulting larvae: segments

Segments are partially coatedwith hairs: denticles

Fate map

How do you get from “empty bag” to organized (segmented) larva?What is required? What gene Activities? Where and When?

February 06

Developmental biology: Drosophila segmentation and repeated units

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Loss of gene activity will tell us what it is required for:Mutagenize flies and screen for segmental phenotypes (Nüsslein-Volhard and Wieschaus):

Idea is to find all possible genes that when ko’d could lead toa developmental anomaly…

Approach: randomly mutagenize whole genome;Isolate single gene mutants;Study their phenotypes (in embryo)

40.000 individual mutations created:

150 very clear segmentation phenotypes….

February 06

Developmental biology: Drosophila segmentation and repeated units

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Segmental phenotypes (Nüsslein-Volhard and Wieschaus):

* found 150 mutants that disruptpatterning in embryo on basisof cuticle defects.

* amongst these, some haveremarkable phenotypes:segments disappear in blocks

gap mutants

February 06

Developmental biology: Drosophila segmentation and repeated units

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February 06

Developmental biology: Drosophila segmentation and repeated units

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mutant phenotype causedby loss of gene activity

gap gene B

In gap mutantsblocks ofconsecutivesegments aredeleted. Clone gap genes:

determine expression domain

Area of gene expression corresponds to affected body area in mutant

February 06

Developmental biology: Drosophila segmentation and repeated units

* From gradients to large blocks of expression

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February 06

Developmental biology: Drosophila segmentation and repeated units

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gap genes expressed in broad regions.

Answer comes from group of mutants found in same mutagenesis screen:

targets?

all encode transcription factors:

February 06

Developmental biology: Drosophila segmentation and repeated units

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Knirps protein

Fushi tarazu pair rule protein

knirpsknirps actioninduces expressionof one ftz stripe

Only three blocksof gap gene expression:how to get sevenstripes?

February 06

Developmental biology: Drosophila segmentation and repeated units

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And we have functional test:in knirps mutant, stripe should disappear….

in fact kruppel mutant

February 06

Developmental biology: Drosophila segmentation and repeated units

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Gap transcription factorproteins, each induce one stripe

Combined action of twogap proteins (or gap +head/tail proteins)define stripe each again.

Hunchback proteinKruppel protein

33ToTo77Stripes!Stripes!

February 06

Regulation of eve stripes 3 and 7

hb-Z kni

eve 1 to 7 eve 1 and 7

February 06

The even-skipped (eve) stripes: a model to study how transcriptional regulation establishes positional

information

• Regulatory elements are fused to a lacZ reporter gene and their expression is followed in transgenic flies. Eve has a long promoter element, which can be divided into regions that drive expression of various lacZ-transgenes (and indeed expression of eve) in different stripes (stripe-elements).

February 06

Developmental biology: Drosophila segmentation and repeated units

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Trancript is made in nucleus:translated into cytoplasm.

Transcription factor proteinsare not transported out of cell:influence transcription in own nucleus

In fly early embryo:transcription factor proteinscan influence next door nucleibecause there are no cellmembranes to stop the proteinsfrom diffusing.

In fact, the diffusion is important,creates a gradient of activity:fine patterning.

February 06

Developmental biology: Drosophila segmentation and repeated units

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coding regionof pair rule gene

Transcription regulation of typical pair rule gene:

low affinityhigh affinity

February 06

Developmental biology: Drosophila segmentation and repeated units

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pair rule genes = pair rule mutants!

* in mutant embryo,every other segment isdeleted.

* pair rule gene expressedin seven stripes. In mutant:expression domains aredeleted.

* pair rule proteins aretranscription factors (again)

February 06

Developmental biology: Drosophila segmentation and repeated units

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Targets of pair rule transcription factor proteins?

Pair rule protein

segment polarity gene

winglessexpression

wildtype

pairedmutant

February 06

Developmental biology: Drosophila segmentation and repeated units

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How:pair rule stripes

segment polarity stripes

Each stripe of pair rule protein defines a stripe of segment polarity gene expression.Each segment polarity gene is thus induced by two different sets ofpair rule proteins (7 + 7 makes 14).

Fine tuning of stripe limits (anterior) by overlap of pair rule proteins

14 stripes of segment polarity gene expression: 14 segments!14 stripes of segment polarity gene expression: 14 segments!

February 06

Developmental biology: Drosophila segmentation and repeated units

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So far, transcription factors were able to define areas surroundingthe nuclei were gene is expressed: no cell membranes.

Expression domains of genes

Expression domains of proteins

February 06

Developmental biology: Drosophila segmentation and repeated units

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QuickTime™ and aSorenson Video decompressorare needed to see this picture.

February 06

Developmental biology: Drosophila segmentation and repeated units

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Summary of early zygotic development in Drosophila embryos:

Bicoid protein hunchback RNA

Simple axis derived maternal system, divides egg into discrete units(mutants display loss of exactly the units).

Transcription factor gradients create sets of “seven” identical units.

February 06

Developmental biology: Drosophila segmentation and repeated units

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The action of the pair rule genes generates segmental stripes ofexpression of each segment polarity gene.

In fact, pair rule protein action defines segments!

Up until and including the pair rule proteins: all act within non-cellularenvironment, all are transcription factors.Segment polarity proteins act in cellular environment (not all transcriptionfactors, become expressed at time of cellularization).

February 06

Developmental biology: Drosophila segmentation and repeated units

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Further patterning within segments:next lecture