Transcript The Spemann experiment

Developmental Biology
The Spemann Experiment
Spemann & Mangold, 1923
Danny Ben-Zvi
Overview
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Developmental Biology
Embryological Vocabulary
The Spemann experiment
Prospects
Developmental Biology
• “The study of the process by which organisms
grow and develop” wikipedia
• Grow: From a single cell to a multicellular,
specialized organism. A process repeated
successfully time after time.
• Develop: developmental processes take place
throughout life
– Progenitor cells: muscle, bone marrow, neurons, skin
– Tumors
Developmental Biology
• Paradox: There are not enough genes to encode
the organism’s complexity
• Genes are “re-used”
– Timing, localization, combinations, dosage
– The concept of “One gene – one character” is
generally wrong
• Self organization
– Intercellular communication
– Formation of complex structures
Developmental Biology
Model Organisms
• Why use model organisms?
– Ethical reasons
– Grow faster
– Rapid reproduction, many embryos
– Extra-organism development
• Fish, amphibians, insects…
• Development is a highly conserved
evolutionary process
Model Organisms
• Vertebrates, athropods, mollusks, and even worms
have many similar proteins and DNA sequences
• Genes and proteins from one organism can be
used in other organisms
• Genomes of many model organisms were
sequenced
Model Organisms
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Mouse - Mus musculus
Chicken - Gallus gallus
Zebrafish - Danio rerio
Black Toed Frog – Xenopus leavis
Salamander – Triton cristatus/teaniatus
Sea Urchin – Strongylocentrotus purpuratus
Round Worm - Caernohabitis elegans
Fruit Fly – Drosophila Melanogaster
• Various plants
Vocabulary - Axes
animal
Animal-Vegetal
vegetal
Dorsal
Anterior
Dorsal-Ventral
Anterior Posterior
Posterior
Ventral
First Stages of Embryonic
Development
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Fertilization/Oogenesis
Cleavage
Gastrulation
neurulation
Fertilization/Oogenesis
• Egg activation
– Formation of the zygotic DNA
– Initiation of the developmental processes
– Symmetry breaking event
Cleavage
• Embryo divides in an extraordinary fast rate –
mitosis every 30-40 minutes
• Virtually no growth in size
• Controlled by pre-existing maternal
proteins/mRNA: no time for transcription and
translation new zygote genes
• Creation of the blastocoel - cleavage cavity and
blastula - sphere of cells surrounding it
• movie
Animal pole (top) view
Blastula
Blastocoel
Gastrulation
• Formation of three germ layers:
– Ectoderm (outer layer) - skin
– Mesoderm (middle layer) – muscles, bones
– Endodern (inner layer) – digestive track
• Formation of embryonic axes
• Activation of zygote genes
• Considerable movement of cells - without growth
Vegetal
(bottom)
view
movie
Vegetal
(bottom) view
Blastopore lip
Blastopore
Gastrulation
• Movie
Gastrulation
Anterior
Head
Skin (ectoderm)
Dorsal
Trunk
Ventral
Tail
Anus
Gut (endoderm)
Posterior
Fate Map
Neurulation
• Elongation of the embryo
• Formation of the neural tube and
notochord (in chordates), somites, and
early organ predecessors: kidney, heart
Dorsal view
Neural
Tube
Neurulation
• movie
Spemann Experiment
• Outline: Graft a tissue from one embryo into another
embryo, and see what happens – Cut and Paste
• Main observation: A graft of a specific tissue (the
organizer) to a specific location can induce
Siamese twins connected at the belly.
• Conclusion: The embryo’s cells are not committed
to a certain fate.
Spemann Experiment
• movie
• Experimental details
Summary of Results
“A piece taken from the upper blastopore lip of a
gastrulating amphibian embryo exerts an
organizing effect on its environment ... Such
a piece can therefore be designated as an
organizer”
Orginial blastopore lip
Graft
Vegetal view
Summary of Results
“These secondary embryonic primordia are
always of mixed origin.”
“…an organizer of another species is used
for induction, then the chimeric
composition can be established with
certainty and great accuracy”
Controls? Statistics?
• Control:
– The authors do not present a control experiment:
grafting to other locations, at other times, etc.
– They do state however, that development is impeded
after the grafting procedure
• Statistics
– From H. Mangold’s lab notebooks we can learn that
only 15% of the embryos survived the graft
– Spontaneous Siamese twin may occur “naturally” at a
lower rate
Spemann’s innovation
• Until 1923, an embryo had
a predefined “fate map”.
Spemann proved that this
was not the case
• Cells up to a certain stage
are pluripotent – can have
many developmental fates
• Spemann established the concept of organizer
• Stem cells and control over cell fate
Prospects
• Spemann won the Nobel prize in 1935
• Hilde Mangold died in 1926…
• “Spemann Organizer” was found in all
vertebrates, including human
• Dorsal-Ventral patterning has become the model
system for embryonic patterning
• Stem cells are the promise for many future
therapies
• summary
Molecular Basis of the Organizer
• A gradient of morphogens determines the
fate of the cells in the embryo.
Morphogen
concentration
– Morphpgen: A polypeptide that governs the
development of a tissue
– Morphogens are produced from a defined
heart
source
– Their concentration provides positional
kidney
information regarding the distance from the
muscle
source
• The organizer secretes both morphogens
nerves
and their inhibitors which diffuse
Ventral the embryo
Lateral
Dorsal
throughout
(Organizer)
Molecular Basis of the Organizer
Morphogen
concentration
• There are about 5 main families of Vertebrates
Drosophila
morphogens used in all the developmental
heart
processes
kidney all
• These families are shared by almost
multi-cellular organisms
muscle
neural chord
• Drosophila uses the same morphogen as
Ventral
Lateral
Dorsal
vertebrates to pattern its dorsal ventral axis
• But in the opposite direction
Summary
• Vocabulary
• Embryo development is highly conserved in
evolution
• Cells are not committed to a certain fate
(pluripotent). They interact and influence each
other and then specialize
• Dorsal ventral axis formation is a central model
system for pattern formation
Questions?
Cycle Length During Cleavage
Back
Choice of Model Organism
• Extra-cellular development
• T. teaniatus survives the grafting
procedure
• T.cristatus has less pigmentation than
T.teaniatus
• Similar species
– Though grafting between evolutionary distant
organisms works as well
Experimental Procedures:
1. Fertilization:
a) Exert
sperm (testes) and eggs.
Medium
b) Manually fertilize in dish
Sterility
2. Graft
“handle
with
care”
a) Peel
the Chorion
off the
two embryos
b) Cut the receiving embryo where the graft will
be inserted
c) Excise the graft from the donating embryo
d) Put the graft on the receiving embryo
Grafting Experiments
• Graft region near the dorsal lip of T.cristatus
(light) at gastrula and implant it in animal side
of T.teaniatus/alpestris (dark) at gastrula.
• Fix the embryo at neurula, make cross
sections, and characterize the resulting
chimera
Controlled Experimental Variables
• Size of graft
• Exact location of graft from donor embryo
• Exact developmental stage of each
embryo
• Graft location in the receiving embryo
Uncontrolled Experimental
Variables
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Orientation of implant
Embryo’s response to the procedure
Contamination
Embryo variation
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