Developmental Biology The Spemann Experiment Spemann Mangold 1923

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

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 • • 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 Ventral

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/m. RNA: 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 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 • • Orientation of implant Embryo’s response to the procedure Contamination Embryo variation Back