Transcript DV axis HT12

Animal Development
Drosophila axis formation
Part 2: D-V patterning
In this lecture
 The origin of D-V asymmetry
 Maternal effect genes
 Dorsal/Ventral patterning
 Multiple signalling pathways are involved in setting up D-V
patterning. These pathways are used in different contexts during
development and are highly conserved in different organisms.
The egg production line
• Anterior
• Young
Posterior
Old
As the ovarioles develop, follicle cells migrate over the oocyte
and the nurse cells pump their contents into the oocyte,
leaving only a remnant of the nurse cells behind at the anterior
end.
A Drosophila egg is 400 mm long, 160 mm in diameter.
Early development in Drosophila
After thirteen divisions membranes
ingress from the cortex to enclose each
nucleus, to form the cellular blastoderm.
Dorso-ventral axis formation
• The egg is polarised on the DV axis
(compare with animals where DV polarity
is defined by the sperm entry point).
• The ovary is not polarised on the DV axis,
so how is the asymmetry established?
gurken (a TGFb family protein) produced by the
oocyte induces dorsal follicle cell fate, the first sign of
D-V axis formation.
Dorsal follicle cells
grk mRNA
Grk protein
stage 8
Grk protein
stage 10
Ventral follicle cells
 Step 1: Microtubule re-organisation.
 Oocyte nucleus moves to an anterior position near the oocyte cell
membrane.
 grk mRNA is made in the nurse cells, transported to the anterior of the
oocyte, then to the cortex overlying the oocyte nucleus, and anchored there.
 Grk protein is synthesised, has limited diffusion, and signals to the follicle
cells migrating overhead, which take on dorsal fates.
Mirr represses pipe expression in dorsal follicle cells.
Andreu M J et al. Development 2012;139:1110-1114
Mirr binds to a conserved regulatory element in pipe.
Andreu M J et al. Development 2012;139:1110-1114
D/V patterning involves a Serine
protease cascade
 Share homology with trypsin-like
family of extracellular serine
proteases.
 Typically secreted as inactive
zymogen forms that are activated by
proteolytic cleavage between N and
C terminal domains.
Pre-activated forms of Snake and
Easter lacking N-terminal sequences
have been used to order
Gastrulation defective, Snake and
Easter in a cascade
 In vitro Easter can cleave Spatzle
to create active form in embryos
Snake and Easter zymogens are
freely diffusible, therefore local
activation is critical
Problem:
Dorsal protein is the transcription factor that interprets the DV information
in the egg. Dorsal as well as Toll (the receptor of the pathway) and its
ligand Spatzle are found throughout the syncytial blastoderm, not just the
ventral or dorsal side! How can Dorsal act as a morphogen and Toll
signalling generate its gradient only in the ventral side?
Solution:
The critical step is the generation of the ventral signal by the only ventralspecific gene: Pipe. Translocation of Dorsal from the cytoplasm to the
nuclei of the ventral cells occurs during the 14th cycle of cell division.
Nuclei that take up Dorsal express ventralising genes and repress
dorsalising genes.
Question:
What is the asymmetrical cue that leads to translocation of Dorsal into the
nuclei of only ventral cells (Pipe target)?
Controlling the nuclear
translocation of Dorsal
Spn27A
CHORION
Easter
Spaetzle
Snake
Windbeutel
And
pipe
PERIVITELLINE
SPACE
EMBRYO
Gastrulation
Tube
Defective
Cactus
Toll
Pelle
MyD88
Nudel
FOLLICULAR CELLS
Plasma
membrane
Nuclear
membrane
Dorsal
Organisational similarities of proteolytic
cascades in development, coagulation and
immunity
Generation of dorsal-ventral polarity
In egg, after fertilisation.
7. Nudel and the Pipe target (factor x)
interact to split the Gastrulation-deficient
(Gd) protein. Nudel may determine the
timing of this signal.
8. The activated Gd protein splits the
Snake (Snk) protein, and activated Snk
cleaves the Easter (Ea) protein.
Gd, Snk and Ea are serine proteases
9. The activated Easter protein splits
Spatzle; activated Spatzle binds to Toll
receptor protein.
10. Toll activation activates Tube and Pelle,
which phosphorylate the Cactus protein.
Cactus is degraded, releasing it from
Dorsal.
11. Dorsal protein enters the nucleus and
ventralises the cell.
Searching for the target of
Pipe
Zhang et al (2009), Curr Biol 19, 1200-1205
Vitelline
Membrane Like
(VML) is a Pipe
target and is
localised at the
anterior-lateral
side of the
oocyte
Genetic Interactions of vml
with pipe
Maternal Mutant Background
+/+; +/+; pipe7/pipe2 (n = 1681)
Proportion of Embryos Exhibiting Denoted DV Phenotypes
D0
D1
D2
D3
15.7% ± 0.9%
40.7% ± 1.2% 31.5% ± 1.1
12.1% ± 0.8%
Vml/Vml; +/+; pipe7/pipe2 (n = 554)
89.5% ± 1.3%
10.3% ± 1.3%
0.2% ± 0.2%
0
Vml/+; +/+; pipe7/pipe2 (n = 327)
48.0% ± 2.8%
39.1% ± 2.7% 11.6% ± 1.8%
1.2% ± 0.6%
gdVM90/+; +/+; pipe7/pipe2 (n = 208)
78.8% ± 2.8%
20.2% ± 2.8%
0.8%
1.0% ± 0.7%
Toll signalling
Domain structure of Toll
signal peptide
(locates
protein to
transmembrane
membrane)
domain
intra-cellular
domain (26%
amino-acid
identity with
human
interleukin-1
receptor
 Toll is a transmembrane protein found throughout the cell membrane of the egg
that acts as a receptor for a localised external signal (Spatzle)
 Toll membrane protein is activated by Spatzle on the ventral side of the embryo
 In wild type embryos, amount of ligand is limited; in wild-type embryos Toll limits
the diffusion of its own ligand by sequestering it as soon as it is produced.
Separation of dorsal and cactus
proteins
• Cactus binding to Dorsal protein inhibits Dorsal’s nuclear
entry sequence, and thus Cactus sequesters Dorsal in
the cytoplasm. Dorsal is a transcription factor
Cactus
binds via
ankyrin
repeats
Pelle protein
kinase, probably
through an
intermediate,
phosphorylates
Cactus protein.
P
Cactus
degrades
Dorsal
protein can
enter
nucleus
D-V patterning so far…
 D-V patterning is initially set up during oogenesis, via asymmetric
mRNA localisation.
 Extracellular proteolysis provides ligand ventrally for the ubiquitous
receptor encoded by Toll.
 Cactus proteolysis, releasing the morphogen Dorsal into ventral
embryonic nuclei.
 D-V patterning depends on cell-cell signalling rather than on localised
determinants in the egg.
 The signals controlling Dorsal access to nuclei use exclusively
maternal products until the receptor coded by Toll is activated.
 Dorsal and cactus then have both maternal and zygotic contributions
 Transcriptional targets of Dorsal are zygotic.
Members of the Toll-like Receptor family
TLRs are an evolutionary ancient and well conserved family of proteins
Human TLR family consists of 10 members: TLR1-TLR10
Conserved pathway for regulating
transcription factors in both Drosophila and
mammals
Spatzle
IL-1
Toll
IL-1R
Tube
MyD88
Pelle
IRAK
?
TRAF-6
Cactus
IKB
Dorsal
NFKB
Toll and its ligand are
also involved in the
Drosophila immune
reponse against
fungal infections.
Several toll-related
genes have been
discovered in
humans, which may
be involved in both
the immune response
and early
development.
Conserved pathway for regulation of
nuclear localisation
 Phosphorylated IKB is
ubiquinated and subsequently
degraded by the proteosome.
Removal of IKB unmasks the
nuclear localisation sites in both
the p50 and p65 subunits of
NFKB. NFKB enters the
nucleus, binds to specific
sequences in DNA and
regulates transcription.
IKB similar to Cactus
NFKB similar to Dorsal
Degradation of IKB allows NFKB to
enter the nucleus
Dorsal-ventral polarity
 D-V axis is initially set up by maternal genes and depends on cellcell signalling rather than on localised determinants in the egg.
The role of Dorsal:
 dorsal, which encodes a transcription factor that can both activate
and repress gene expression, is the morphogenetic agent for D-V
polarity.
 Loss-of-function mutations in dorsal give rise to dorsalised embryos
(the product of dorsal is needed for differentiation of ventral cells).
 Different concentrations of Dorsal specify different patterns of gene
transcription and consequently different fates for cells.
Studying the Dorsal gradient
Differential nuclear localisation of
dorsal protein regulates zygotic genes
•
Dorsal activates specific
genes to give the mesodermal
phenotype (nervecord,
muscles etc.) and proper
gastrulation.
– twist, snail and rhomboid
•
Dorsal represses dorsalising
genes
– decapentaplegic (dpp) and
zerknullt (zen)
How does Dorsal act as both a
transcriptional activator and repressor?
Dorsal
twist
(low affinity
binding
site)
DSP1
Dorsal
(HMG-box
binding protein)
(high
affinity
binding
site)
TATA
binding
protein
zen
groucho
(transcriptional repressor)
• two types of complex – activation
– repression
The gradient of the Dorsal
protein and its interpretation
(A) The concentration
gradient of Dorsal protein in
the nuclei of the blastoderm,
as revealed by an antibody.
(B) The interpretation of the Dorsal gradient by genes that demarcate the different dorsoventral
territories; for simplicity, only two representative genes are shown. Subsequent processes will
further subdivide these territories. The decapentaplegic (dpp) gene in particular codes for a
secreted factor that will act as a local morphogen to control the detailed patterning of the
ectoderm.
The gradient of the Dorsal
protein and its interpretation
(A) The concentration
gradient of Dorsal protein in
the nuclei of the blastoderm,
as revealed by an antibody.
(B) The interpretation of the Dorsal gradient by genes that demarcate the different dorsoventral
territories; for simplicity, only two representative genes are shown. Subsequent processes will
further subdivide these territories. The decapentaplegic (dpp) gene in particular codes for a
secreted factor that will act as a local morphogen to control the detailed patterning of the
ectoderm.
A dorsal-ventral gradient in Dpp is produced by the antagonistic activity of the short
gastrulation protein (Sog).
Dpp
•
The maternal gradient of dorsal protein in
the nuclei represses dpp transcription
ventrally but not dorsally. Sog is
expresses in the ventral region of the
embryo. Sog protein diffuses into the
dorsal region and antagonizes the activity
of Dpp protein, providing positional
information in the dorsal region.
Sog
Dorso-ventral patterning is controlled by the
same genes in flies and frogs
Flies and frogs
• Dorsal neural tube of the vertebrate and ventral
nerve cord of the fly appear to be generated by
the same set of instructions.
fly
frog
Tolloid Xolloid
Sog
Chordin
Dpp
BMP-4
Can the fly sog gene rescue ventralised
Xenopus embryos?
Embryos
completely
ventralised by UV
irradiation
Ventralised
embryos partially
rescued by injection
of sog mRNA
Ventralised
embryos completely
rescued by injection
of sog mRNA
• sog and chordin can substitute for each
other!
Summary
Similar signal transduction pathways in all multicellular
organisms.
Homologous pathways form basic infrastructure, but
targets may vary.
Molecular pathways are “tool-kits” comprising versatile
ligands and receptors and molecular switches including
proteolysis and reversible protein phosphorylation.
Signalling pathways can be recruited for different
purposes.
Drosophila development shows significant similarities
with vertebrate developmental systems.