Molecular mechanisms in
development
Dr Megan Davey
megan.davey@roslin.ed.ac.uk
Learning Outcomes
• Describe the role of cell adhesion molecules signalling and creating
boundaries between tissues
• Understand the difference between a ‘paracrine’ interaction and a
‘juxtacrine’ interaction
• Name the major families of paracrine factors
• Give examples of when and where these factors act during
development
• Describe why Sonic Hedgehog is thought to act as a morphogen
• Explain the role of Sonic Hedgehog and TGFβ proteins in patterning
the neural tube
• Case Study:Molecular mechanisms of Limb Development
Molecular mechanisms controlling• Cell adhesion
• Cell signaling
– Paracrine
Hh, Fgf, Wnt, TGFβ
– Juxtacrine
Notch pathway, Cadherins
• Extracellular matrix (ECM)
• Cell migration
1.Cell Adhesion
-Are cells in an embryo all the same?
Xenopus laevis
Cell Adhesion
-Are cells in an embryo all the same?
6 hours of development
gastrulation
neuralation
These are Xenopus laevis embryos from soon after fertilisation until neuralation
Cell Adhesion
-Are cells in an embryo all the same?
Cadherins
-Ca-dependent adhesion molecules
Extracellular domains adhere cells together
Participate in intracellular signalling
Link and help assemble the cytoskeleton via
actin or intermediate filaments
Cadherins
-mediate binding between cells by homophilic mechanism
•Cadherins are calcium-dependent cell–cell adhesion glycoproteins
composed of five extracellular cadherin repeats, a transmembrane region
and a highly conserved cytoplasmic tail.
•They are often concentrated at intercellular junctions e.g. adherens
junctions
•Classic cadherin function allows cells to adhere in a homophilic manner
•Cadherins can be classified into four groups•Classical-E-cadherin (epithelial), N-cadherin (neural), N-cadherin2, Pcadherin (placenta)
•Desmosomal cadherins (via intermediate filaments)
•Protocadherins
•Unconventional- R-cadherin (retinal), VE-cadherin (vasculature)
Cadherins
-transmit signals to the cytoskeleton via catenins
Cadherins in action-neuralation
Specific tissue expression=specific tissue cohesion
If the N-cadherins are inactivated in the cells of the presumptive neural tube,
the neural tube will not form
2.Cell Signalling
• Paracrine or Juxtacrine?
The FGF signalling pathway
•22 FGF ligands
•4 FGF receptors
•Via receptor tyrosine kinases
•Expressed in•Limb, face, somites, midbrain
development, blood vessels
•Actions•Cell proliferation
•‘Organiser’ ability
Mutations cause•Achondroplasia=FGFR3
•Apert’s Syndrome=FGFR2
FGF8
The TGFβ superfamily
•TGFβs, BMPs (Bmp2-7), GDFs, others
(e.g.nodal)
•TGFβ-receptors
•Inhibitors-follistatin, chordin, noggin etc
Expressed in•Neural tube, limb, vasculature, lung…
Mutations causeLoeys–Dietz syndrome (TGFβR1, 2)
Marfan syndrome (?)
Bmp7
The Wnt signalling pathway
•~19 ligands
•Fz, LRP receptors
•‘Canonical’, non-canonical signalling (PCP
pathway)
Expressed in•Neural, kidney, teeth, hair, bone, limb
•Mutations cause•Wilms tumour, Split hand/Foot Syndrome,
Skeletal dysplasia, tooth agenesis
The Hedgehog signalling pathway
•3 ligands (Shh, Dhh, Ihh)
•2 receptors (Ptc1, Ptc2)
•Expressed in
•Limb, neural tube, brain, bone,
vasculature, lung gut and many more
Actions
•Morphogen, Cell proliferation, cell,
survival, chemotaxis
Mutations cause•Cyclopia (loss), polydactyly (too much)
Shh
The Neural Tube
Morphogens in the Neural TubeShh
V1
V2
MN
V3
FP
Shh
Shh
Shh
Shh
Shh
FP
V3
MN
V1
V2
MN
V3
FP
Shh
BMP4+7
Morphogens in the Neural Tube
Shh and TGFβ
TGFβ
BMP4
Shh
TGFβ Inhibitors
RP
D1
D2
D3
V1
V2
MN
V3
FP
Morphogens in the Neural Tube
Shh and TGFβ
RP
D1
D2
D3
V1
V2
MN
V3
FP
3. The Extracellular Matrix
•ECM consists of macromolecules secreted by cells into their
immediate environment e.g. proteoglycans, collagen, fibronectin
and laminin
•ECM can be a permissive substrate for adhesion/cell migration
•ECM can acts as directional guide during cell migration
•ECM can act as a source of signalling factors
Fibronectin
•
•
•
•
FN functions as an adhesion molecule linking cells to one another
or to other molecules (collagen and proteoglycans)
FN has an important role in cell migration
The roads over which migrating cells travel are paved with FN
FN leads germs cells to the gonads and heart cells to the midline
of the embryo
Proteoglycans
Can bind and concentrate growth factors using sulphated side chains
e.g. kidney development
Credit Ian Smyth, Monash University, Wellcome Images
Proteoglycans
Can bind and concentrate growth factors using sulphated side chains
e.g. kidney development
Branching of ureteric bud
to form collection ducts
Credit Ian Smyth, Monash University, Wellcome Images
Proteoglycans
Kidney mesechyme
Branching of ureteric bud
to form collection ducts
Ureteric bud
cytokines
proteogylcans
Ureteric bud cell
Proteoglycans
Can bind and concentrate growth factors using sulphated side chains
e.g. kidney development
Ureteric bud cell
+chlorate ions
+chlorate ions +HGF
Ureteric bud cell
Integrins
On the extracellular side integrins bind
to the sequence Arg-Gly-Asp found in
adhesion
molecules
including
fibronectins
On the intracellular side they bind
Vinculin and a-Actinin, these proteins
bind to Actin filaments
This dual binding allow cells to move by
contracting Actin filaments against the
EM
The Limb Bud: Case Study
Anatomy:Mouse Limbs
Forelimb
Hindlimb
Anatomy: Chicken Limbs
Wing
2
4
1
3
2
Leg
3
4
Limb anatomy review
V
A
D
Pr
2
4
D
P
3
Anatomy
Radius
2
4
Ulna
3
Anatomy: 2, 3, 4 or I, II, III???
Anatomy:Limb Bud
Areas of Cell Death
Anterior necrotic zone
Opaque patch
Posterior necrotic zone
Apical Ectodermal Ridge
(AER)
How a limb grows
Anatomy: Proximal-Distal
-Progress Zone Model
(Summerbell, Lewis, Wolpert 1973)
19HH
21HH
23HH
Stylopod
Zeugopod
Autopod
Anatomy: Proximal-Distal
The limb elements are formed in a proximo-distal progression
-Progress Zone Model (Summerbell, Lewis,
Wolpert 1973)
The time spent in the Progress Zone determines
the position of the cell on the proximodistal axis
Stylopod
Zeugopod
Signal from AER
Progress zone
Autopod
FGF beads can perform the functions of the AER.
Martin G R Genes Dev. 1998;12:1571-1586
©1998 by Cold Spring Harbor Laboratory Press
FGF is the functions of the AER.
FGF8
FGF4
FGF beads can induce and pattern a limb
Anatomy: Anteroposterior Digit Identity
Wing
2
4
1
3
2
Leg
3
4
Anteroposterior Digit Identity
normal wing
‘Duplicated’ wing
Host limb
Donor limb
(Saunders & Gasseling, 1968)
-transplanting areas of programmed cell death, found that posterior limb
tissue caused the induction of ‘mirror image duplications’
Anteroposterior Digit Identity
normal wing
(Saunders & Gasseling, 1968)
-transplanting areas of programmed cell death, found that posterior limb
tissue caused the induction of ‘mirror image duplications’
‘Duplicated’ wing
Induced extra digits
Anteroposterior Digit Identity
normal wing
(Saunders & Gasseling, 1968)
-transplanting areas of programmed cell death, found that posterior limb
tissue caused the induction of ‘mirror image duplications’
2
3
4
‘Duplicated’ wing
3
Reverse AP polarity
4
2
3
4
Anteroposterior Digit Identity
normal wing
‘Duplicated’ wing
Host limb
Donor limb
(Saunders & Gasseling, 1968)
-The posterior mesenchyme was the called thhe Zone of Polarising Activity (ZPA)
-The ZPA is an organiser
ZPA organiser function is conserved between chicken
and mouse
Retinoic Acid: The limb morphogen?
-Shh expressing cells induced mirror image duplications
-Retinoic acid induces Shh
Morphogens
• A morphogen is a potent signal which specifies different
cell types at different concentrations e.g. Shh in the limb
and neural tube
2
3
4
Shh
Shh
•The ‘French Flag’ model of morphogen action
Morphogens
-Testing the morphogen theory
normal
2
4
3
3
‘duplication’
4
2
3
4
Morphogens
-Testing the morphogen theory
3
4
2
4
Shh
Shh
3
FGF beads can perform the functions of the AER.
Martin G R Genes Dev. 1998;12:1571-1586
©1998 by Cold Spring Harbor Laboratory Press
Polydactyly: Extra digits are very common
Naturally occuring polydactyly -extra Shh