Transcript A-P axis 2010

Drosophila anterior-posterior
axis formation during early
embryogenesis
Genetics Unit Department of Biochemistry
[email protected]
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
THEY LIVE….
The origins of Anterior-Posterior
polarity
• The anterior-posterior polarity
of the embryo, larva and adult
has it’s origin in the anterior
posterior polarity of the egg.
The maternal effect genes
expressed in the mothers
ovaries produce mRNAs that
are placed in different regions
of the egg. These messages
encode transcriptional and
translational regulatory proteins
that diffuse through the
syncytial blastoderm and
activate or repress the
expression of certain zygotic
genes
Axes in the Drosophila embryo
are specified in the egg
• Cooperation between nurse cells, which make the
maternal mRNAs and follicle cells that signal to
the oocyte and reorganise its cytoskeleton, is
essential for localisation of bicoid and nanos
Movement of the oocyte nucleus is directed my microtubules
EGF signalling between the oocyte nucleus and follicle cells
At the end of
signalling posterior and
dorsal
follicle cells have obtained
a different identity
Different cytoskeletal systems in the same cell
Intermediate filaments (vimentin)
Microtubules (tubulin)
Microfilaments (actin)
The cytoskeleton performs several tasks that are importa. nt for the formation of assymetries: control of the
location of the mitotic cleavage plane within the cell, control of cell shape and directed transport of
molecules and organelles within the cell. All of these tasks depend on the fact that these rods are polar
structures exhibiting directionality. That is one end of the cytoskeletal rod can be chemically distinguished
from the other
Polarity of subunits in an actin microfilament
Distribution of tubulin in animal cells
Microtubules radiate out from a microtubule organising centre.
The negative (minus) ends of the microtubules are in the centre
and the positive (plus) ends are at the periphery of the cell.
Motors and cargoes
Different motors are used for travelling to different directions carrying different
cargoes. For example dynein can travel in the opposite direction than kinesin. What’s
the value of having multiple independent trafficking systems? A part of the answer is
division of labour.
Nuclear divisions in the syncytial
embryo
•
It is important to note that in the
early
stages
in
Drosophila
embryonic development there are
nuclei divisions inside the embryonic
cytoplasm (the syncytium). This is
important in order to understand how
in these stages transcription factors
are used to drive the gene regulation
cascade, which will lead to the final
patterning of the embryo after 24h
D
P
A
V
Drosophila embryonic
development in real time
• Most insect eggs undergo
superficial cleavage, where in a
large number of mass of
centrally located yolk confines
cleavage to the cytoplasmic rim
of the egg. During these early
stages of division there are no
cells formed but the embryo is a
syncytial blastoderm meaning
that the egg is a large cell
containing within a common
cytoplasm the dividing nuclei.
Following division cycle 13 the
oocyte plasma membrane folds
inward between the nuclei and
cellularisation begins.
D
A
P
V
Breaking symmetry: the way to
establish positional information
• A) Create a population of developmentally
identical cells
• B) Create an asymmetry within that population
• C) Exploit the asymmetry to set up a chemical
concentration gradient within the population of
cells
• D) Different cells will mount different responses
to local concentrations of these gradients
• E) Different responses will create different states
of developmental potential
Pattern formation in Drosophila
• A) the cytoskeleton imposes an asymmetry on the egg.
• B) Gradients of soluble proteins are laid down along the
A-P and the D-V axis, stimulating gene action and
• C) leading to broad regions of gene expression.
The Heidelberg screen
• Christiane Nüsslein-Volhard,
Eric Wieschaus and colleagues
performed extensive mutational
screens, essentially saturating
the Drosophila genome for
mutations that alter the A-P and
D-V patterns of the larva
exoskeleton with the
assumption that such alterations
would reflect changes in the
basic body plan.
Mutational analysis of
Drosophila early embryogenesis
•
Two broad classes
of genes affecting
the body pattern:
zygotic genes,
which are expressed
exclusively in the
zygote being part of
its DNA and
maternal genes,
which are
contributed by the
mother and the
phenotype of the
offspring depends
on the phenotype of
the mother.
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 (HBM). 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.
Localisation of A-P determinants
in the early embryo
In situ hybridisation (RNA)
Antibody detection (protein)
Nanos mRNA and protein localisation
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.
MESSAGE: Positional information in Drosophila embryonic A-P axis
is generated by protein gradients. The gradients ultimately depend
on diffusion of newly translated protein from a localised source of
mRNAs anchored by their 3’ UTR to ends of cytoskeleton filaments
Studying the BCD gradient I
• In embryos derived
from bcd mothers the
anterior (head and
thorax) part of the
body is missing (a).
This can be restored
by injection of either
anterior cytoplasm (b)
or the actual wild type
mRNA (c).
Studying the BCD gradient II
• The amount of BCD
protein can be changed
by varying copies of
bcd+ in the mother.
This
changes
the
position
of
the
gradient
to
more
posterior regions.
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.
READING LIST
Wolpert et al, Principles of Development
St Johnston D and Nusslein-Volhard C (1992) The origin of
pattern and polarity in the Drosophila embryo Cell 68, 201219
St Johnston D (2005) Moving messages: the intracellular
localisation of mRNAs Nat Rev Mol Cell Biol 6, 363-375.
Further reading: Zimyanin et al, 2008 In vivo imaging of
oskar mRNA transport reveals the mechanism of posterior
localisation Cell 134, 843-853