Pattern formation typically involves:
Some initial spatial differences in molecular (inc. mRNA)
distribution or gene expression of a few patterning molecules
is set up:
•location of maternal transcription factors in early
Drosophila
•location of maternal growth factors in the frog Xenopus
Concentration- or time-specific downstream gene responses
to these molecules:
•genes directly turned off/on (best shown in Drosophila)
•receptors engaged, signal tranduction pathways activated
and genes turned off/on (best shown in vertebrates)
But this is like the progressive drawing of more and more
detailed blueprints.
What is being spatially organized is information.
But an organism is spatially organized structures such as
organs made of collections of cells.
How is the blueprint--eg. the spatial patterns of transcription
factors-- actually turned into structures?
This process is called Morphogenesis: the process whereby new
shapes are moulded and sculpted.
The information read-out causes cells to individually alter their
behaviours. Collectively this then reorganizes cell assemblages,
which changes the size and shape of organs.
So, this is moulding and sculpting, but where there is no
sculptor; the clay forms itself.
Morphogenesis or Developmental Mechanics:
The read-out of the blueprint alters:
•Cell adhesion
•Cell shape
•Cell movement
•Cell proliferation/death
•Extracellular materials
Changes in these qualities at the single cell level alter the
form of the cell groups, the tissues, organs and the entire
embryo.
All these are governed by “motor” or “functional” molecules
encoded in specific genes.
And these genes are under the control of the “blueprint”
genes.
Establishing the Vertebrate Body Axes and
forming Three Tissue Layers (Gastrulation)
•Anterior/ Posterior (or Rostral/Caudal)
•Dorsal/Ventral
R
•Left/Right
A
L
•Example: frog Xenopus
D
V
P
How do you study this?
Classic Techniques
•Label known cells in embryos to see what they do (physical description— fate map).
•Cut out (or kill or damage) bits of embryos :
What is lost in the embryo later in development? Tests regulation. Defines fields.
What can the isolated bit develop as? Tests specification.
•Stick the bit back in at the wrong place or time:
What does the bit develop as in the abnormal tissue environment? Tests
competence, potency, determination.
Does the bit modify the development of its new neighbour tissue. Tests induction
Recombine different bits in tissue culture:
Do they modify each others development?
Molecular Techniques
•What molecules/genes are expressed around the time and position of interesting
developmental events:
random or directed mutagenesis, screen for interesting phenotype, work back to
gene. Evidence of gross gene function – direct or indirect.
in situ hybridization & antibody probes --- Guilt by association
Insertion of molecules/genes into appropriate or inappropriate cells:
active---------------finer scale, more detailed evidence of gene function
change amount
inhibitory versions
Vertebrate example…amphibian….
Xenopus egg starts with some pattern…...
animal
vegetal
•Xenopus egg (oocyte) is spherical.
•Relatively large single cell diam. >1 mm.
•Distinct thin cortical layer just under cell
membrane (cortical layer--tubulin & actinrich).
ie. central/peripheral difference
•Black pigment granules are in the upper
part of the cortical layer (upper side is
black).
•Yolk masses in cytoplasm; denser than
normal cytoplasm (underside is yellowish).
ie. upper/lower difference---by convention
upper is called “animal pole” and lower is
called “vegetal pole”.
In situ hybridization for
Vg-1 mRNA
W3
Other molecules are also unequally
positioned……..eg.
•mRNA for Vg-1 (TGF-beta family
growth factor) found in vegetal
cortex.
•Xwnt-11 (another growth factor)
mRNA also chiefly vegetal.
•mRNA for VegT (transcription
factor) in vegetal cortex.
The egg (single cell) is radially
symmetrical about the animalvegetal axis….how is this symmetry
broken?
Sperm entry breaks the radial symmetry…..
In the zygote:
•Cortex rotates with respect to internal cytoplasm (microtubuledriven)
•Differentially distributed molecules (incl. mRNA) in A-V axis of
cortex will be shifted with respect to differentially distributed
molecules in A-V axis of internal cytoplasm.
W3
Sperm entry point
1
V
2
2 cells
(now bilaterally
symmetrical)
“A”
3
Nieuwkoop centre
“P”
8 cells
Viewed from above ie
looking down on animal
pole.
D
4 cells
Viewed from the side
opposite the entry point of
the sperm.
=future dorsal side
V
2
D
4 cells
W3
There is already (4 cells) some
specification of body regions,
demonstrated by dividing the 4
cell stage embryo into dorsal 2
cells versus ventral 2 cells, and
allowing development. The 2
halves are not equal. [Divide a 2
cell embryo and each cell forms a
complete but small embryo]
But specification around all axes is NOT simultaneous…..
D
4 cells
However, division
along the D-V axis
shows no specification.
The embryo regulates
to replace the missing
left & right regions.
2 complete embryos
but smaller
Cell numbers increase quickly…..
Continued mitosis is very rapid (25 min/cycle)--- termed
CLEAVAGE.
•This produces a solid ball of cells ---MORULA
•With even more cells, the ball becomes hollow due to inward
pumping of water----BLASTULA
•This is all done on maternal gene products
•At the end of blastula stage (~4-5000 cells):
cell division slows suddenly
embryonic gene transcription begins
beginning of GASTRULATION, a stage of rearrangement
What ends cleavage phase?
•In frogs the embryo is the same size as the egg i.e. more and
more cells of smaller and smaller size.
•The cytoplasm: nucleus ratio falls.
•Adding more DNA to the oocyte leads to premature embryonic
gene transcription.
•Model: there may be a general maternally derived transcriptional
repressor in the oocyte cytoplasm, whose amount per nucleus
drops below some threshold, so the repression is lifted (Just like
Drosophila, except not in a syncytium).
As cleavage procedes, not all cells in the blastula are the same…..
•Cleavage parcels up the forming nuclei with increasingly small
sub-divisions of pre-existing cytoplasm.
•Since molecules (incl. mRNA) are differentially distributed, new
cells will “inherit” cytoplasm with differing amounts of these
molecules.
•These cytoplasmic determinants could control how one cell
could differentially signal to other cells (eg. if they are growth
factors) or how other genes inside the same cell could be
controlled (eg. if for transcription factors).
“Anterior and dorsal”-ness is related to the
amount of displacement of the cortex.
W3
The Nieuwkoop centre* cells can induce
changes in neighbouring cells…….
W3
*Identified by doing many grafts of various cell groups from many regions into many regions at many stages.
And some molecules can duplicate the
function of the Nieuwkoop centre
W3
Fates and Specifications in the Blastula.
Mapping fate by cell
labelling
Fate:What cells (ie.
their descendents) do...
W3
Specification: What
cells can do when
separated from other
tissues and grown in
a “neutral”
environment.
Interactions between cells generate new cell/tissue types
Separating cell groups
and growing in isolation
reveals specifications.
Separating cell groups
then recombining them
reveals INDUCTIONS.
How could this action at
distance occur?
W3
Embryonic Induction…..
•Responding tissue needs to be exposed to inducer for several
hours.
•Result of induction eg. new genes expressed, may occur many
hours after the induction has ceased.
•Cells to be induced will not wait forever to be induced; they soon
lose competence to respond. How might this occur?
W3
Embryonic Induction…..
•Responding tissue show
different response(eg. different
genes expressed) to different
concentration of inducer.
•Gradient of response extending
away from source could be
established (morphogenetic
gradient).
•Cell response can be promoted
up a gradient by higher inducer
concentration, but cannot be
demoted down a response
gradient by drop in inducer
concentration.
W3
Animal Cap Assay
(Activin=
TGF-beta
family member)
organizer
A series of inductions pattern the future mesoderm
The experiments….
How is crude specification
converted to fate?
The interpretation….3 or 4 inductive interactions
Spemann organiser
Nieuwkoop
centre
W3
Some of the inducing molecules are known….
W3
Some apparent inducing molecules act by inhibition……..
Some inducers act by binding to and inhibiting other inducers
•Secreted factors noggin and chordin both bind to and inhibit the TGF-beta
type growth factor BMP-4.
•The secreted factor Frizbee is related to Frizzled, the cell membrane receptor
for Wnt-family growth factors. It acts as a competitive inhibitor of
Wnt/Frizzled signalling.
W3
Noggin mRNA
(in situ hybridization)
This first induction sets up a new Induction Centre
…….the Spemann Organizer
………this signals to neighbouring cells
………...controls organization of the Blastula
…………. instigates & orchestrates cell re-arrangements
…………….to give a 3 layered, patterned embryo
General principle: Any initial variation between 2
zones can, if there is signalling between zones and
the ability to respond to signals, self-generate finer
and finer patterns.
Other vertebrates show similarA-P and D-V pattern
generation systems, after allowing for gross differences in
size and shape of the group of cells giving rise to the embryo.
sphere of cells
W3.20
sphere of cells
Flat plate of
cells on top
of immense
yolk mass
Plate of
cells folded
into cupshape
This first induction sets up a new Induction Centre
…….the Spemann Organizer
………this signals to neighbouring cells
………...controls organization of the Blastula
…………. instigates & orchestrates cell re-arrangements
…………….to give a 3 layered, patterned embryo
………………gastrulation
Wolpert: “It’s not birth, death or marriage that is the
most important moment of your life, it’s gastrulation.”
What forms are vertebrates generating?
A tube with 3 basic layers
several subdivisions
Ectoderm =outer layer
epidermis (skin)
neural (CNS & PNS)
Endoderm=inner layer
absorptive cells
secretory cells
Mesoderm=middle layer
notochord
muscle
skeleton
connective
kidney
blood
How does this 3 layered structure physically
come into being? What is its morphogenesis?
(original German term coined by Wilhelm
Roux for this expresses the idea well:
Entwicklungsmechanik = developmental
mechanics
G10
G10
G10
End vertebrate pattern formation to gastrulation #1