Transcript AP axis-biochemists MT11

Animal Development
Drosophila axis formation
Part 1: A-P patterning
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In this lecture:
The origin of Anterior-Posterior Axis
Mutant screens to isolate segmentation genes
Genetic analysis of early acting determinants
Important roles of post-transcriptional regulation and
mRNA/protein localisation
Methods of dissecting enhancers
Dosage-dependent activation of zygotic genes
Hierarchical organisation of segmentation genes
THEY LIVE….
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)
1
The early embryo
is a syncitium
The unusual feature of
the Drosophila early
embryo is that the first
13 mitoses are nuclear
divisions
without
concomitant cytoplasmic
division, making the
embryo a syncitium-a
multinucleated cell. After
division 9, the plasma
membrane of the oocyte
evaginates
at
the
posterior
pole
to
surround each nucleus
thus creating the pole
cells, which will form the
fly’s germ line.
Segments in embryos are maintained throughout development
Forming complex pattern:
establishing positional
information
The Hunt for Mutants
30,000 independently-derived mutants in genes required for
survival.
8,000 mutants define genes required for embryonic survival
(these became the focus the study).
750 mutants have specific effects on A/P or D/V patterning.
150 genes with specific effects on A/P or D/V patterning
identified by the 750 mutants (average of ~ 5 alleles per
gene).
A Detour Into Embryonic Anatomy – Denticle Bands
Denticle bands on a 1st instar larva.
Denticle bands are hair-like projections on the ventral
cuticle of an embryo.
Denticle bands provide an easily visualized marker of
embryonic/larval pattern.
Maternal effect genes
• Phenotype of the embryo is determined by the genotype of the mother.
• The polarity and spatial coordinates of the embryo are initially set by
the products of these genes (therefore, sometimes called “coordinate
genes”).
• The gene products, either mRNA transcripts, proteins, or cell surface
ligands are contributed by the nurse cells or follicle cells as the egg is
constructed.
• The dorsal-ventral axis (1 gene-system, 12 genes) and anteriorposterior axis (3 gene-systems; anterior, 4 genes, posterior, 11 genes,
and terminal, 6 genes) determined by maternal effect genes.
• Originally isolated as homozygous mutant, adult females that lay
normal looking eggs that do not develop at all, regardless of the genetic
contribution of the male.
Four Independent Genetic Regulatory Systems Specify
the Anteroposterior and Dorsoventral Axes
Maternal effect genes
• All four systems share several properties:
(i) the product of (at least) one gene is localized in a
specific region of the egg,
(ii) this spatial information results (directly or indirectly) in
an asymmetrical distribution of a transcription factor,
(iii) the transcription factor is distributed in a
concentration gradient that defines the limits of
expression of one or more zygotic target genes, such
as segmentation genes.
Bicoid mutant embryos lack head and thorax
structures
+/+
mother
bicoid/bicoid
mother
Establishment of AP
axis in oogenesis and
bicoid localisation by
Gurken signalling.
 In early stage egg chambers MTOC
is in the oocyte, and gurken mRNA is
localised at posterior.
 Translation and limited diffusion
means signal sent to overlying
posterior follicle cells (received via
torpedo receptor).
 A signal is sent back which activates
protein kinase A in the egg
 oocyte cytoskeleton is re-organised
and directs the localisation of bicoid
and oskar, defining the A-P axis.
EGF signalling between the oocyte nucleus and follicle cells
Effect of replacement of the 3’ UTR
of the nos mRNA with the 3’ UTR
of bcd mRNA
• The nos-bcd
transgene is able to
localise at the
anterior pole and as
a consequence
NOS protein will
inhibit translation of
the hb and bcd
mRNAs.
The Bcd gradient
mRNA (in situ)
Protein (Ab staining)
Concentration gradients of BCD
and HB-M establish A-P axis
• Positional information along
the A-P axis of the syncitial
embryo is initially established
through
the
creation
of
concentration gradients of two
transcription factors: Bicoid
(BCD) and Hunchback (HB-M).
These are products of two
maternal effect genes their
mRNAs provided by the
mother and stored in the
embryo
until
translation
initiates. These factors interact
to generate different patterns
of gene expression along the
axis.
Bicoid is an
Anterior
Morphogen
Note that bicoid
(and other
maternal effect
gene products)
diffuse in the
shared cytoplasm
of the syncytial
blastoderm.
This is a unique
feature of insect
embryogenesis.
BCD acts as a concentration-dependent manner
Thresholds can turn gradients
into sharp boundaries
– Bicoid protein
required for early
activation of zygotic
hunchback.
– Bicoid contains
homeobox
– Mutations in
homeobox results in
failure of Bicoid
protein to interact with
hunchback target
sequences.
bicoid protein gradient
– gradient is interpreted at least at four different
levels (thresholds).
bicoid as a repressor of posterior fates
Bicoid binds the 3’ UTR of
caudal mRNA and
suppresses translation.
Caudal protein
Caudal protein enters the
nuclei at the posterior end of
the syncytial blastoderm and
helps specify posterior fates
At the Posterior: nanos
localisation by Gurken
signalling
 oocyte cytoskeleton is reorganised and directs the
localisation of bicoid and oskar,
defining the A-P axis.
 oskar mRNA binds Kinesin I and
Staufen proteins.
 Kinesin I localises oskar mRNA
to posterior
 Staufen allows translation of
oskar mRNA
 Oskar protein binds nanos
Effect of posterior group genes on
hunchback.
Posterior group genes:
• primary gene is nanos.
• nanos mRNA is tightly
localised to the
posterior pole of the
egg.
Effect of posterior group genes on hunchback.
The role of nanos is to disable hb maternal mRNA at the
posterior end of the egg.
Four Independent Genetic Regulatory Systems Specify the
Anteroposterior and Dorsoventral Axes
Terminal group genes
Torso: TRK signalling via MAPK
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.
SUMMARY
Nurse cells surrounding the oocyte in the ovarian
follicle provide it with large amounts of mRNAs and
proteins, some of which become localised in particular
sites. The oocyte produces a local signal, which induces
follicle cells at one end to become posterior follicle cells.
The posterior follicle cells cause a re-organisation of the
oocyte cytoskeleton that localises bicoid and hunchback
mRNA to the anterior end and other mRNAs such as oskar
and nanos to the posterior end of the oocyte. Following
fertilisation, development starts and these mRNAs are
translated. Subsequently, gradients of the BCD and HB
proteins define the anterior nuclei-the embryo is still a
syncytial blastoderm, while inhibition of translation of their
mRNAs by Nanos define the posterior cells. Nuclei in
between receive a variable amount of BCD and HB
resulting in differential activation or repression of target
genes and finally in different developmental cell fates.