Developmental Biology

The Spemann Experiment

Spemann & Mangold, 1923

Danny Ben-Zvi

Overview

• 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

• 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

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

• 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-Vegetal

Dorsal

Ventral

Anterior

PosteriorDorsal-VentralAnterior Posterior

animal

vegetal

First Stages of Embryonic Development

• 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

Blastocoel

Blastula

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

movie

Vegetal (bottom) view

Vegetal (bottom) view

Blastopore lip

Blastopore

• Movie

Gastrulation

Gastrulation

Ventral

Posterior

Dorsal

Anterior

Anus

Tail

Trunk

Head

Skin (ectoderm)

Gut (endoderm)

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

• movie

Neurulation

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

GraftVegetal 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”

• 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?

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

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.– 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)

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

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.

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”

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

• Orientation of implant

• Embryo’s response to the procedure

• Contamination

• Embryo variation

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