Transcript segmentation

Developmental biology:
Drosophila segmentation and repeated units
* egg: generate the system
* larva: eat and grow
* pupa: structures in
larvae grow out to form
adult fly: metamorphosis
(Drosophila is a
holometabolous insect)
February 06
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Segmentation pattern
 Obvious segmentation begins to develop by
germ band extension stage.
 The embryonic segmentation pattern has
direct analogs to the final segments of the
adult.
 Segmentation pattern can be thought of as
classical segments or midsegment-tomidsegment intervals called parasegments.
 Some early embryonic segments become
incorporated into the complex structures of
the head and mouth.
Developmental biology:
Drosophila segmentation and repeated units
* Drosophila embryo: larva
February 06
3
Developmental biology:
Drosophila segmentation and repeated units
Embryo stage: developmental patterning:
most obvious characteristics in resulting larvae: segments
Segments are partially coated
with hairs: denticles
How do you get from “empty bag”
to organized (segmented) larva?
What is required? What gene
Activities? Where and When?
Fate map
February 06
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Developmental biology:
Drosophila segmentation and repeated units
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 to
a 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
5
Developmental biology:
Drosophila segmentation and repeated units
Segmental phenotypes
(Nüsslein-Volhard and Wieschaus):
* found 150 mutants that disrupt
patterning in embryo on basis
of cuticle defects.
* amongst these, some have
remarkable phenotypes:
segments disappear in blocks
gap mutants
February 06
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Developmental biology:
Drosophila segmentation and repeated units
February 06
6
Developmental biology:
Drosophila segmentation and repeated units
In gap mutants
blocks of
consecutive
segments are
deleted.
gap gene B
February 06
mutant phenotype caused
by loss of gene activity
Clone gap genes:
determine expression
domain
Area of gene expression
corresponds to affected
body area in mutant
7
Developmental biology:
Drosophila segmentation and repeated units
* From gradients to large blocks of expression
February 06
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Developmental biology:
Drosophila segmentation and repeated units
gap genes expressed in broad regions.
all encode transcription factors:
targets?
Answer comes from group of mutants found in
same mutagenesis screen:
February 06
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Developmental biology:
Drosophila segmentation and repeated units
Knirps protein
Fushi tarazu pair rule protein
Only three
blocks
knirps
action
of gap gene
expression:
induces expression
how to get
of seven
one ftz stripe
stripes?
February 06
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Developmental biology:
Drosophila segmentation and repeated units
And we have functional test:
in knirps mutant, stripe
should disappear….
in fact kruppel mutant
February 06
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Developmental biology:
Drosophila segmentation and repeated units
Gap transcription factor
proteins, each induce one
stripe
Combined action of two
gap proteins (or gap +
head/tail proteins)
define stripe each again.
3
To
7
Stripes!
February 06
Hunchback protein
Kruppel protein
12
Regulation of eve stripes 3 and 7
February 06
eve 1 to 7
eve 1 and 7
hb-Z
kni
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
Trancript is made in nucleus:
translated into cytoplasm.
Transcription factor proteins
are not transported out of cell:
influence transcription in
own nucleus
February 06
In fly early embryo:
transcription factor proteins
can influence next door nuclei
because there are no cell
membranes to stop the proteins
from diffusing.
In fact, the diffusion is important,
creates a gradient of activity:
fine patterning.
13
Developmental biology:
Drosophila segmentation and repeated units
Transcription regulation of typical pair rule gene:
high
affinity
low affinity
February 06
coding region
of pair rule gene
14
Developmental biology:
Drosophila segmentation and repeated units
pair rule genes =
pair rule mutants!
* in mutant embryo,
every other segment is
deleted.
* pair rule gene expressed
in seven stripes. In mutant:
expression domains are
deleted.
* pair rule proteins are
transcription factors (again)
February 06
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Developmental biology:
Drosophila segmentation and repeated units
Targets of pair rule transcription factor proteins?
Pair rule protein
wildtype
wingless
expression
paired
mutant
February 06
segment polarity gene
17
Developmental biology:
Drosophila segmentation and repeated units
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 of
pair 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!
February 06
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Developmental biology:
Drosophila segmentation and repeated units
So far, transcription factors were able to define areas surrounding
the nuclei were gene is expressed: no cell membranes.
Expression domains of proteins
Expression domains of genes
February 06
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Developmental biology:
Drosophila segmentation and repeated units
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
February 06
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Developmental biology:
Drosophila segmentation and repeated units
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
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Developmental biology:
Drosophila segmentation and repeated units
The action of the pair rule genes generates segmental stripes of
expression 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-cellular
environment, all are transcription factors.
Segment polarity proteins act in cellular environment (not all transcription
factors, become expressed at time of cellularization).
February 06
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Developmental biology:
Drosophila segmentation and repeated units
Further patterning within segments:
next lecture
February 06
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Developmental biology:
segment polarity genes and cell signalling
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* transcription factors expressed in large blocks
* induce expression of downstream transcription factors in smaller blocks
* generates segmental expression (14) of segment polarity genes.
February 06
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