developmental biology the spemann experiment spemann & mangold, 1923 danny ben-zvi
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Developmental Biology
The Spemann Experiment
Spemann & Mangold, 1923
Danny Ben-Zvi
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Overview
• Developmental Biology
• Embryological Vocabulary
• The Spemann experiment
• Prospects
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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
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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
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• 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
Developmental BiologyModel Organisms
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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
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Model Organisms
• 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
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Vocabulary - Axes
Animal-Vegetal
Dorsal
Ventral
Anterior
PosteriorDorsal-VentralAnterior Posterior
animal
vegetal
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First Stages of Embryonic Development
• Fertilization/Oogenesis
• Cleavage
• Gastrulation
• neurulation
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Fertilization/Oogenesis
• Egg activation– Formation of the zygotic DNA– Initiation of the developmental processes– Symmetry breaking event
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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
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Animal pole (top) view
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Blastocoel
Blastula
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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
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movie
Vegetal (bottom) view
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Vegetal (bottom) view
Blastopore lip
Blastopore
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• Movie
Gastrulation
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Gastrulation
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Ventral
Posterior
Dorsal
Anterior
Anus
Tail
Trunk
Head
Skin (ectoderm)
Gut (endoderm)
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Fate Map
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Neurulation
• Elongation of the embryo
• Formation of the neural tube and notochord (in chordates), somites, and early organ predecessors: kidney, heart
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Dorsal view
Neural Tube
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• movie
Neurulation
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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.
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Spemann Experiment
• movie
• Experimental details
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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
GraftVegetal view
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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”
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• 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
Controls? Statistics?
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Spemann’s innovation
• Spemann established the concept of organizer
• 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
• Stem cells and control over cell fate
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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
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Molecular Basis of the Organizer• A gradient of morphogens determines the
fate of the cells in the embryo.– Morphpgen: A polypeptide that governs the
development of a tissue– Morphogens are produced from a defined
source– Their concentration provides positional
information regarding the distance from the source
• The organizer secretes both morphogens and their inhibitors which diffuse throughout the embryo
Mo
rph
og
en c
on
cen
trat
ion
heart
kidney
muscle
nerves
Ventral Lateral Dorsal(Organizer)
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Molecular Basis of the Organizer
• There are about 5 main families of morphogens used in all the developmental processes
• These families are shared by almost all multi-cellular organisms
• Drosophila uses the same morphogen as vertebrates to pattern its dorsal ventral axis
• But in the opposite direction
Mo
rph
og
en c
on
cen
trat
ion
heart
kidney
muscle
neural chord
Ventral Lateral Dorsal
Vertebrates
Drosophila
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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
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Questions?
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Cycle Length During Cleavage
Back
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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
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Experimental Procedures:1. Fertilization:
a) Exert sperm (testes) and eggs.
b) Manually fertilize in dish
2. Grafta) 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
Medium
Sterility
“handle with care”
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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
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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
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Uncontrolled Experimental Variables
• Orientation of implant
• Embryo’s response to the procedure
• Contamination
• Embryo variation
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