Molecular Basis of Development
and Molecular Embryology
Lecture 2 of 2
Human Developmental Biology
module
Morphogenesis
Morphogenesis or “creation of form”. How the
organism takes on a three dimensional shape
with all the cell types in the right place to form
structures and carry out functions
Morphogensis is the outcome of correct pattern
f
formation.
ti
P. Murphy
•The important task of laying down the
body plan- the molecular basis of positional
information- gene transcription and cell
communication.
Pattern formation (the arrangement of cellular
events in space) in animals begins in the very
early embryo when the body plan is laid down,
when the head end is distinguished from the
tail, the dorsal from ventral etc. In mammals
this occurs during gastrulation.
•Morphogenesis:
•The limb as an example of morphogenesis
•The importance of induction and cell
signalling in the limb.
•Clinical relevance of known regulatory
genes in limb development
The molecular signals and cues experienced by
the cells that tell them their relative positions
within the embryo are called positional
information.
Positional information.
Morphogenesis is the outcome of correct
pattern formation
But how are the cells arranged in a pattern?
By receiving information (molecular signals
and cues) that indicates their relative
positions within the embryo:
this is called positional information.
How can we define position in a 3D object?
By co-ordinates along 3 axes.
right
ventral
dorsal
left
Morphogenesis could be organised by
cells within the embryo receiving
information according to such coordinates.
1
Positional information.
When do cells first start to become different
to each other?
If cells could acquire some kind of
positional address, could that lead to the
generation of a pattern?
The flash card
analogy (Lewis
Wolpert).
At the molecular level, how could this
information be g
generated?
What is the molecular basis of positional
information?
two ways in which cells can receive
information:
1 Localisation of cytoplasmic
1.
determinants
2. Induction
A pattern could emerge in the embryo by
cells receiving a positional addressPerhaps through a set of molecular
signals unique to that position.
1. The localisation of cytoplasmic
determinants or cell polarisation
This type of information is the basis
of morphogenesis.
2. Induction
•One source of information is RNA and
protein molecules in the cytoplasm that
could
cou
d influence
ue ce the
t e future
utu e regulation
egu at o of
o the
t e
Where one group of cells influences the
development of a neighbouring group of
cells.
ll
cell.
These might include transcription factors
The phenomenon of induction was first
demonstrated by Hans Spemann and
Hilde Mangold in 1924.
2
Experiment to demonstrate the inductive
action of the “organiser” in the Newt
gastrula.
Hans Spemann and Hilde Mangold 1924.
Fig 47.24
Important features of the organiser
experiment:
1. organiser cells from the donor could
change the fate of the recipient cells.
2. the organiser cells can set in motion a
chain of events leading to the production
of a new body plan.
So Spemann called these cells the primary
organiser.
How can induction be achieved at the
molecular level?
By cells sending molecular signals to
each other.
A gene can be turned on and off in
response to signals that a cell
receives.
Induction by neighbouring cells
⇒ Cell signalling.
A gene can be turned on and off in
response to signals that a cell receives.
Fig 18.15b
3
So cell signalling is the basis of
induction.
Signalling molecules
The developing limb as a
model system for studying
morphogenesis
→ produced by the inducing cells
→ detected by the responding cells,
The changing morphology of the
developing limb
(in the mouse)
lead to a series of changes inside the
responding cell
ultimately a change in the gene
expression pattern within.
Time - the 4th dimension
In studying limb
morphogenesis,
the 4th dimension
is also
particularly
important.
Limb
development is
very dynamic.
The same
molecule at
two
different
time points
may be
doing two
different
things.
Positional information in the
developing limbHow are the cells patterned?
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Organiser regions in the developing
limb
Apical Ectodermal Ridge (AER).
Organiser: (e.g. Spemann’s organiser)
A group of cells that influences the
development of surrounding tissue.
If the AER is removed
the limb ceases to grow.
How is this achieved at the molecular
level?
By
y induction:- by
y the secretion of
signalling molecules that communicate
with surrounding cells.
•
Two important “organisers” have been
defined in the limb.
Removal of the AER at
different stages leads to
truncation of the limb at
different points along
the P/D axis
Fig.
47.23a
1. The Apical Ectodermal Ridge (AER)
2. The Zone of Polarising Activity (ZPA)
The AER is a major signalling centre in
the limb.
The AER secretes FGF8 (fibroblast
growth factor 8).
Fgf8 gene
expression in the
AER
The major active signal produced by the AER
is FGF 8
If the AER is removed but beads containing
p
FGF 8 are added, limb development
can
continue, near to normal, if enough FGF 8 is
added.
So Fgf8 expression in the AER is necessary
for the outgrowth of the limb
And for patterning of the limb along the
proximo-distal (P/D) axis
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Zone of Polarizing Activity (ZPA).
The ZPA is not a morphologically distinct
structure but is located in the posterior
mesoderm of the developing limb bud.
It was defined by its activity when
transplanted to another region:
When transplanted to the anterior, it
induces a duplication of the A/P pattern
of digits that will form
The ZPA is defined by Sonic Hedgehog
(Shh) expression.
(Shh is
another type
of signalling
molecule,
related to a
Drosophila
gene product
called
Hedgehog)
Transplanting cells that express Shh to the
anterior limb bud mimicks ZPA transplantation.
Normal wing
Thus Shh is a central active agent of the ZPA.
This is confirmed by the mouse mutations extratoes and Sasquatch (Ssq) where there is anterior
expression of Shh and additional toe(s).
This has thrown light on a number of human
limb malformations.
Sasquatch (Ssq)
mutant mice
Human preaxial
polydactyly (PPD)
Taken from Hill
et al (2003) J.
Anat 202,13
The mouse mutation is caused by an
alteration in expression of the Shh
gene: Shh is now expressed in an
ectopic anterior site.
The human mutation maps to the syntenic
region of the human genome (7q36) and is
believed to arise from a similar alteration in
SHH expression.
Inactivation of the Shh gene in the mouse leads to
a phenotype very similar to the human
malformation Acheiropodia.
Taken from Ianakiev et al (2001),
(2001)
Am J Hum Genet 68, 38
The mutation that causes Acheiropodia maps to
the same region of the human genome: 7q36.
This region is now recognised as a major site for
human limb malformations - implicating Shh in
the origin of many.
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What lies downstream of P/D and A/P
signalling in limb patterning?
Hox genes pattern both the P/D and the A/P
axes.
Hox patterning is combinatorial and
complex but particular Hox genes play
major roles at different levels along the P/D
axis. ⇒Colinearity
A mutation in
human HOXD13
causes
synpolydactyly.
(Muragaki et al
al.
(1996) Science
272, 548)
Genes of the Hox a cluster are expressed in
overlapping domains along the P/D axis
Genes of the Hox d cluster are expressed in
overlapping domains along an axis between P/D
and A/P.
Clinical relevance: Human Hox gene mutations.
Hox genes play roles in development of:
• CNS
• axial skeleton
• limb
• gut
• urogenital tract • external genitalia.
Known mutations in Human Hox genes:
HOXD13 → Synpolydactyly (Muragaki et al.
(1996) Science 272, 548)
HOXA13 → Hand-Foot-Genital Syndrome
(HFGS) (Mortlock + Innes (1997) Nat
Genet 15;179)
most cases are dominant mutations with
trinucleotide repeat expansions.
In one case, a HOXA deletion → HFGS, facial
dysmorphism, mental retardation, patent ductus
arteriosus, velopharangeal insufficiency.
Hox genes play a role in specifying the
identity of regions of the limb, as well as
the body as a whole.
A Hox code also operates in the limb
Stem cell therapies hold great promise for
regenerative therapies – including skeletal
regeneration
Our work on morphogenesis of the musculoskeletal
system in the limb has shown that
•Muscle contractions are required for embryos to form
proper bones and joints
•This
This is because the contractions generate mechanical
stimuli that are needed for cell differentiation, cell
division and morphogenesis.
Change in
skeletal
rudiments 2
days of
development
apart
Alterations to joint
formation and bone
formation with no muscle
contractions
We are now using this knowledge to improve how
stem cells are turned into cartilage and bone tissue in
vitro prior to transplantation
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Lecture 2
•Morphogenesis is brought about by cells
receiving molecular positional information
and behaving appropriately according to
time and space.
•To understand how cells become different
we consider them responding to two main
sources of information:
1. The unequal inheritance of
cytoplasmic determinants (assymetric cell
division)
2. Induction: cells influencing each
others fate through cell to cell
communication.
•Cell signalling and transcriptional
regulation form the molecular basis of
development control
•The limb is a very good model to study
morphogenesis.
•The outgrowth and shape of the limb are
controlled by at least two signalling centres/
organisers. These produce signalling
molecules that influence the development of
adjacent structures.
•The AER is necessary for the outgrowth of
the limb bud and patterning along the P/D
axis. A major signalling component of the
AER is FGF8.
•The ZPA produces signals, including Shh,
that pattern the shape and structure of digits
along the A/P axis
axis. The role of Shh in limb
patterning has explained the molecular basis
of some human limb malformations.
•We are studying how progenitor cells in the
limb are guided differently to form cartilage
and bone tissue in order to improve methods
of regenerating bone and cartilage tissue for
therapies.
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