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TIGP-MCB-Developmental Biology
Axis formation
Yi-Hsien Su
Institute of Cellular and Organismic Biology
Academia Sinica
March 1st, 2016
Three body axes of the bilaterians (animals with the bilateral
symmetry)
Anterior-posterior (AP), dorsal-ventral (DV), left-right
(LR) axes refer to body axes in bilaterians
D
A
R
L
P
V
Moroz et al., 2014, Nature
Satoh et al., 2014, Proc. R. Soc. Biol. Sci.
Most studies are focused on a few model bilaterians
35-40 animal phyla
Drosophila
Xenopus and
other
amphibians
Satoh et al., 2014, Proc. R. Soc. Biol. Sci.
Approaches to development
Experimental embryology – since the beginning of the
twentieth century; microsurgical experiments on embryos of
frogs and sea urchins; embryonic induction
Developmental genetics – genetic screens on the fruit fly
Drosophila
Molecular Biology – molecular cloning; DNA sequencing
Summary of
normal development
Male and female adults
meiosis
Male and female gametes
blastopore
Fertilization
Zygote
Cleavage
Blastula or blastoderm
Gastrulation
Gastrula
(invagination,
involution, epiboly,
convergent
extension…)
Cell differentiation, organogenesis, growth…
Anteroposterior axis
Dorsoventral axis
Left-right axis
Amphibian embryos as model for experimental embryology
- salamander, frog (Rana), clawed frog (Xenopus)
Xenopus egg is polarized before fertilization
Pigmented: animal side/little yolk
Unpigmented: vegetal side/dense yolk
“cortical rotation”
Sperm entry point (anywhere in the
animal hemisphere) becomes the future
ventral side
Pigmented
animal region
Gray crescent
(gastrulation starts
here)
Vegetal region
Cleavage of a Xenopus egg
- First cleavage divides the gray crescent in half
Gray crescent
Gray crescent
Blastula
Frog gastrulation and the dorsal lip
Blastula
Gray crescent
Dorsal lip
Hans Spemann and Hilde Mangold’s famous experiment in 1924
“Organizer center”
“Organizer”
“Spemann-Mangold organizer”
: induce the host’s ventral
tissues to change their fates and
organize host and donor tissues
into a secondary embryo
Molecular mechanisms of amphibian axis formation
Major questions:
1. How did the organizer get its properties?
1. What factors were being secreted from the organizer?
Molecular mechanisms of amphibian axis formation
Major questions:
1. How did the organizer get its properties?
Two factors: β-catenin on the dorsal side and Nodal-related
signal on the vegetal side
2. What factors were being secreted from the organizer?
1. How did the organizer get its properties?
How does the organizer form?
Fate map of the Xenopus blastula
organizer
Nieuwkoop center
Two factors in the
Nieuwkoop center: βcatenin on the dorsal
side and Nodal-related
signal on the vegetal
side
1. The dorsal signal: β-catenin
Disheveled protein stabilized β-catenin on the dorsal side
2. The vegetal Nodal-related signal
vegT maternal RNA
vg1 maternal RNA
VegT: transcription factor
Vg1: Nodal-like protein
The nuclearized β-catenin on the dorsal side and
Nodal-related signal from the Nieuwkoop center
activate several organizer genes
2. What factors were being
secreted from the organizer?
Functions of the organizer:
1. Self-differentiate into dorsal mesoderm (ex. notochord)
2. Dorsalize the surrounding mesoderm
3. Dorsalize the overlying ectoderm and induce formation of the neural tube
“Induction of neural ectoderm and dorsal mesoderm”
Searching for the organizer factor(s) (Smith and Harland, 1992)
Dorsalized gastrulae (LiCl-treated)
constructed cDNA library
mRNA synthesized from sets of plasmids
injected into ventralized embryos (UV irradiated)
look for plasmid clones whose mRNAs were able to restore dorsal tissues
Noggin
UV irradiated
(ventralized)
noggin
chordin
Noggin
mRNA
injection
(dose)
dorsalized
Sasai et al., 1994
(Yoshiki Sasai 1962-2014)
Some organizer factors are inhibitors of BMPs, which
induce epidermis formation
BMP (ventral)
BMP antagonists (dorsal)
The organizer also has the anterior-posterior temporal
specificity of inducing ability
Anterior structures
Posterior structures
The head inducers: Wnt antagonists
Injecting cerberus mRNA in the
ventral vegetal cell induces extra
head formation
In Greek mythology,
Cerberus is a monstrous
multi-headed dog
Double gradient interaction
- BMP gradient: dorsal-ventral gradient
- Wnt gradient: posterior-anterior polarity
How do embryos establish their polarity in the first place?
(one part/side is different from the other)
1. Internal factors: unequal distribution of maternal mRNA/protein in the egg
during oogenesis
Xenopus
fly
sea urchin
ascidian
1. External cues: result from sperm entry or other localized stimuli that
originate externally to the egg surface
amphibian
fly
Fertilization
Cortical rotation
Drosophila larval and adult
Genes involved in shaping the larval and
adult fly were identified in the early 1990’s
using “forward genetics”:
• randomly mutagenize flies
• screen for mutations
• genes cloned and characterized
Anterior-posterior and dorsal-ventral polarity of the Drosophila oocyte
Gurken mRNA
Bicoid mRNA passing
into the oocyte from the
nurse cells
Gurken protein
9.9 Expression of the gurken message and protein between the oocyte
nucleus and the dorsal anterior cell membrane
Gurken mRNA
Gurken protein (more mature oocyte)
Gurken protein (younger stage)
Gurken signals those follicle cells to
become the more columnar dorsal
follicle cells
Establishes dorsal-ventral polarity in
the follicle cell layer that surrounds the
growing oocyte
Dorsal-ventral patterning of the Drosophila embryo
Nuclearization of the Dorsal
protein on the ventral side
Conserved signaling system involved in dorsal-ventral
patterning between arthropods and vertebrates
Anterior-posterior patterning of the Drosophila embryo
Maternal effect genes
Bicoid protein
gradient
Gap gene protein
Hunchback protein
Kruppel protein
(First zygotic genes to be
expressed in broad domains)
Pair-rule gene
product
fushi- tarazu
(not enough
segments)
(Seven strips)
Segment polarity
gene product
engrailed
(14-segment-wide units)
(determines the fate of each segment)
Homeotic mutations – convert one body part into another (along the AP axis)
Antennapedia
mutant
Bithorax
mutant
These Hox genes contain a common DNA motif called homeobox
(encodes 60 amino acid homeodomain for DNA binding)
Hox genes form clusters and are responsible for specifying
anteroposterior identity to body levels
Spatial order of their expression is often the
same as their order on the chromosome
Left-right asymmetry in Bilaterians
Right
Left
Left-Right (LR) asymmetry is a common feature in “bilateria”
vertebrate
mouse
invertebrates
ascidian
sea urchin
fiddler crab
Wikipedia
Snail
rudiment
Nishide et al., 2012,
Development
Ibanez-Tallon et al., 2003,
Human Mol. Genet.
Su Lab
Grande and Patel, 2009,
Nature
Conserved nodal expression in the left lateral plate mesoderm
of vertebrate embryos
Mouse
Asymmetric
morphogenesis
Mouse embryo
Chicken
Xenopus
Zebrafish
Speder et al., 2007, Curr. Opinion Genet. Dev.
Nodal signaling cascade
Norris, 2012, BMC Biol.
Hamada et al., 2002, Nat. Rev. Genet.
Nodal signaling cascade is important for LR asymmetric
development in vertebrate embryos
Wild-type
Xenopus tadpole
Right
Pitx2 injection
on the right side
Left
Pitx2 knockout mice
Lin et al., 1999, Nature
Chick embryo expressed
pitx2 on the right side
Logan et al., 1998, Cell
Nodal signaling cascade is also important for LR asymmetric
development in invertebrate embryos - Ascidian
Nodal expression
on the left side
Nodal inhibitor affects tail bending and position of the brain vesicle
Nishide et al., 2012, Development
Nodal signaling cascade is also important for LR asymmetric
development in invertebrate embryos – Sea urchin
Left
control
Right
BMP
inhibition
Left
Right
Nodal expression
on the right side
Right
Left
Duboc et al., 2005, Dev. Cell
BMP signaling
(pSmad1/5/8)
Luo and Su, 2012, PLoS Biol.
Molina et al., 2013, Curr. Opinion Genet. Dev.
Nodal signaling cascade is also important for LR asymmetric
development in invertebrate embryos – Snails
Snail
Sinistral
control
Dextral
Dextral shell
Sinistral shell
Grande and Patel, 2009, Nature
Nodal
inhibition
Nodal signaling is required for left-right asymmetric
development.
What controls the initial left-right asymmetric expression of
nodal?
Dynamic nodal expression in the mouse embryo
(Lateral
plate
mesoderm)
Node
Node
Shiratori and Hamada, 2006, Development
Leftward fluid flow generated by rotational movement of node cilia
Intrinsic nodal flow
Artificial nodal flow
Nonaka et al.,
2002, Nature
Shiratori and Hamada, 2006,
Development
Pitx2-lacZ
Nodal
Polarization of node cells and model for the sensing of nodal flow
by immotile cilia
Planar cell polarity (PCP) core proteins:
Prickle2 and Vangl1: anterior side
Dvl: posterior side
Pkd2-Pkd1l1 complex: Ca2+ channel activity
Cerl2: Nodal antagonist
Immotile cilia detect the leftward flow
Nodal mRNA
(pSmad2)
Distribution of the PCP proteins determines
the positions of the basal bodies, from
which the cilia grow
Yoshiba and Hamada, 2013, Trends in Genetics
The left-right asymmetry pathway in the mouse embryo
(lateral plate mesoderm)
(paraxial mesoderm)
Nodal
Norris, 2012, BMC Biol.
Shiratori and Hamada, 2006, Development
Left-right organizers and the flow model of symmetry
breakage
Blum et al., 2014, Development
Axial patterning in non-bilateria
D
A
R
L
P
V
Moroz et al., 2014, Nature
Satoh et al., 2014, Proc. R. Soc. Biol. Sci.
Cnidarian model systems used in developmental biology
oral
aboral
mouth oral
Planula
larva
aboral
Technau and Steele, 2011, Development
How do the axes of cnidarians relate to the AP and DV axes
of bilaterians?
Double gradients in vertebrate embryos
- BMP gradient: dorsal-ventral gradient
- Wnt gradient: posterior-anterior polarity
Wnt signaling in Cnidaria
Clytia Wnt3 RNA
Momose et al., 2008, Development
Nematostella planula larva
Kusserow et al., 2005, Nature
Wnt expression in Hydra
Hobmayer et al., 2000, Nature
Evolution of Wnt signaling and axis formation during
animal evolution
B: Posterior Wnt
stripe in Drosophila
C: sponge WntA
D: Xenopus Wnt8
E: Hydra Wnt3
(no Wnt gene was found)
Holstein, 2012, CSH Perspect. Biol.
In all studied animals, a posterior Wnt signaling center defines the posterior pole of
the body axis.
Asymmetric expression of BMP-like genes and BMP antagonists
in Cnidaria
Nematostella
* blastopore
mid
gastrula
Directive axis
(bmp)
(bmp)
oral
(bmp)
planula
aboral
Acropora
blastopore
(bmp)
(bmp)
Technau and Steele, 2011, Development
blastopore
bmp2/4
Hayward et al., 2002, PNAS
Spemann-Mangold organizer expresses chordin and after
gastrulation the dorsal blastopore lip has high Wnt activity
Xenopus
Nematostella
chordin
Sasai et al., 1994
Technau and Steele, 2011, Development
Kusserow et al., 2005, Nature
Cnidarian blastopore = organizer???
Organizer activity of the blastopore lip of a Nematostella gastrula
Xenopus
Nematostella
*Oral ends of the embryos
Kraus et al., 2007, Curr. Biol.
Nodal signaling sets up axial asymmetry for polyp budding
Nodal
Watanabe et al., 2014, Nature
Wnt/β-Catenin signaling is essential for Nodal expression
and bud formation
Wnt expression in Hydra
Hobmayer et al., 2000, Nature
Watanabe et al., 2014, Nature
Nodal signaling and body axes during animal evolution
BMP/Chordin
Nodal/Pitx2
Wnt/β-catenin
Primary axis:
anterior-posterior or
oral-aboral axis
Watanabe et al., 2014, Nature
Axis Formation
1. AP and DV axis determinations in vertebrates
2. AP and DV axis determinations in fruit fly
3. LR asymmetry in bilaterians
4. Axial patterning in non-bilaterians