LSM3233; lecture 1, 16 Aug 2013

Developmental Biology of Animals: An introduction

1. History and Tradition in Developmental Biology 2. The first steps into development: Fertilization and cleavage
A/P Christoph Winkler Dept. of Biological Sciences dbswcw@nus.edu.sg S1A, level 06-07
http://www.sgul.ac.uk/depts/immunology/~dash/

Human embryo at blastocyst stage (64 cells)

Lecture outline
Animal Development: A/P Winkler 16 Aug: The beginnings of development 23 Aug: Gastrulation and axis formation 30 Aug: Development of the nervous system 06 Sep: Segmentation Development of muscles and bone 13 Sep: E-learning: Re-cap fertilization and cleavage (IVLE multimedia) 20 Sep: Development and regeneration of limbs 04 Oct: Sex determination and differentiation Plant Development: 11 Oct 15 Nov A/P Loh

Important note: The Animal Developmental Biology Practical will be held on: Sept 3rd, 2-6pm, LSLAB 3 (attendance mandatory) Sept 17th, 2-6pm, LSLAB 3 (attendance mandatory) Sept 18th to 20th, LSLAB 3, Open Lab whole day (attendance flexible according to experimental schedule)

Please mark your calenders!

1. Honesty in academic communication

In academia, we pursue truth and knowledge founded on trust that the work is one's own and is accurate, reproducible, and truthful. This trust and the reputation of the individual and NUS are destroyed by dishonest behavior.
Fabrication making up data or information (lying) synthesizing or creating data for unperformed experiments, constructing graphs and figures unsupported by data or information Falsification deliberately manipulating or altering data or academic/professional credentials (cheating), doctoring photos and graphs,
altering data in tables, using data from one experiment for a different experiment
*http://nus.edu.sg/osa/so/guidelines/student-conduct

1. Honesty in academic communication

Plagiarism submission of ideas, phrases, paragraphs or figures of others as your own (stealing)
Others include books, journals, internet sources and classmates they must be acknowledged reusing your own work without attribution The university considers plagiarism an offence and will subject students to disciplinary action

What is plagiarism and how to avoid it?

Short note
http://www.cdtl.nus.edu.sg/success/sl7.htm http://emodule.nus.edu.sg/ac https://connect.le.ac.uk/p72155629/

E-tutorials on plagiarism

2. Respecting the rights of others

Not infringing the learning process of fellow students
Distractions, e.g. talking, mobile phone browsing, arriving late Uncooperative or failure to listen to others during group work Improper attire for field/lab work resulting in others having to work alone Allowing ones work to be copied depriving others opportunity to learn

Teaching, Training and Reinforcing Ethical Conduct in the Classroom

The mystery of developmental biology

animal left vegetal

dorsal anterior ventral right posterior

0 hours after fertilization

Kane & Karlstrom, 1996

16 hours after fertilization

48 hours after fertilization - hatching Zebrafish embryo 0.5 hrs 20 hrs

Developmental biology: A historical perspective

Probably one of the first developmental biologist: Aristotle (384-322 B.C.) He analyzed the development of chicken embryos by cracking open

eggs on each successive day of its 3week incubation

Aristotle was the first to formulate the idea that an embryo develops gradually from a single undifferentiated egg cell (the concept of epigenesis).
http://clendening.kumc.edu/dc/pc/Aristotle.jpg

However, Aristotle also noted that

-the male embryo develops a human soul about 40 days after conception, whereas a female embryo acquires its soul 80 days after conception. -the heart is the organ of the human mind or consciousness (cardiac or cardiocentric hypothesis) - in humans, menstrual fluid forms the material of the embryo, semen gives it form and animation

? ? ? ? ?

The next 2000 years:

Nothing much happened (at least in developmental biology)
1651 - William Harvey: On the generation of living creatures All animals, including humans, originate from eggs first description of the chicken blastoderm, heart formation 1672 Marcello Malpighi: First microscopic analysis of chicken development first description of neural groove, somites, arterial and vein circulation

www.kambeck.com/kap8.html

Gilbert SF, 2006 Fig. 1.2

17th 18th century:

Preformation versus Epigenesis

Preformation: embryo is pre-formed and then simply grows in size Epigenesis: complexity of growing embryo is progressive
Gilbert SF, 2002 Fig. 7.1

Homunculus Nature works as small as it wishes

Support of epigenesis came from:

Careful analysis of early chicken development

(e.g. de-novo formation of heart)

Regeneration phenomena (Hydra) Improved microscopes, new staining techniques: The end of the preformation theory in the 1820s

Mid-1800s: Emergence of the cell theory

all organisms are made from (typically) large numbers of cells egg and sperm are single cells

organisms emerge from a progressive series of cell divisions and cell differentiation (specialization)
An unknown organizing force is responsible and this force is a property of the organism itself (contained already in the egg; transmitted by germ cells)
www.kambeck.com/kap8.html

Christian Pander Heinrich Rathke Karl Ernst von Baer

Developmental biology: The anatomical area

http://8e.devbio.com/images/ch10/regu11.GIF

Comparative embryology Evolutionary embryology Teratology (birth defects)

Karl Ernst von Baer

The four von Baers laws (1828):
1. General features appear earlier in development than specialized features (e.g. notochord, spinal cord) Less general (more specialized) characters develop from the more general characters (e.g. skin feathers, scales, hairs, nails) The embryo of a given species , instead of passing through the adult stages of lower animals, departs more and more from them. (e.g. visceral clefts gill slits, eustachian tube) The early embryo of a higher animal is never like a lower animal, but only like its early embryos
Gilbert SF, 2006 Fig. 1.5

Comparative Embryology

2.

3.

4.

Charles Darwin (1859): On the origin of species

Different early vertebrate embryos show remarkable similarities (but also important differences)

Developmental biology: The experimental area

Experimental embryology
Wilhelm Roux (1850-1924) pioneered experimental embryology to understand causative factors in development by posing hypothesis and testing them. One of his classic experiments involved using a hot needle on a two-cell frog embryo (in 1888). Conclusion: Early frog embryo is a mosaic of self-differentiating parts (with individual sets of determinants)

www1.medizin.uni-halle.de

Defect experiments

Gilbert SF, 2006 Fig. 3.16

Isolation experiments
Gilbert SF, 2006 Fig. 3.17

Hans Drieschs experiments with sea urchin larvae (1893): Each individual blastomere can build a smaller, but otherwise normal sea urchin pluteus larvae.

Regulative development

Embryologists versus Geneticists

Clash of the Titans: In the 1930s, genetics became its own discipline and geneticists thought embryologists were old-fashioned and development could be explained by gene expression. Conversely, embryologists regarded the geneticists as uninformed and saw genetics as irrelevant to embryological questions.
Nucleus or cytoplasm: Which compartment of the fertilized egg controls inheritance?

E.B. Wilson
Gilbert SF, 2006, Figs. 4.1/4.2

T.H. Morgan

T. Boveri

N.M. Stevens

And the Nobel Prize went to

Together with his student Hilde Mangold, Hans Spemann perfected transplantation experiments in the early 1920s. Spemann won the Nobel Prize for medicine and physiology in 1935, on the discovery of the organizer.

http://www.ucalgary.ca/UofC/eduweb/virtualembryo/dev_biol.html

www.nobelprize.org

www.ssl-id.de/ulula-online.de

Hilde Mangold died of a tragic accident at the age of 29, in the year of her publication; Nobel prizes are not given posthumously.

Transplantation experiments: The Mangold-Spemann Organizer

www.nature.com/nrm/journal/v7/n4/images

Wolpert L. et al., 2002 2nd ed

Spemanns and Mangolds experiments started the hunt for the organizer molecule

The hunt for the organizer, part I:

The era of Biochemistry

In 1930s-40s, the emergence of
chemical and biochemical techniques lead to the establishment of chemical embryology that aimed to investigate chemical and physiological events in embryonic development.

Explants, extracts and candidates: The Xenopus animal cap assay

Gilbert SF, 2006, Fig. 3.20

The hunt for the organizer, part II

The era of Molecular Biology: Developmental Genetics

From the late 1970s with the advent of cell and molecular biology, developmental biology has matured into an exciting field of discipline. It transcends and integrates most aspects of biology and is now one of the hottest fields in modern life sciences.

Turner et al., 1979; Dev.Biol. 68: 96-109

The antennapedia mutation

Developmental Genetics

http://nobelprize.org

Model organisms in Developmental Biology

C. elegans, nematodes

Mus musculus, mammals

Danio rerio, fish

Xenopus laevis, amphibia Drosophila, insects Tunicates

Gallus gallus, birds Sea urchin/starfish Molluscs

Criteria for choosing the right model organism to address a particular experimental question
Xenopus

- Accessibility of embryos (egg-laying vs. live bearing)? - Size of the embryo?

C. elegans

zebrafish
600mm

- Optical transparency? - Number of embryos? - Genome sequences available? - Genome size?

Drosophila

- Cell lines available?

- Cost of animal housing?

- Relevance to human?

www.biken.osaka-u.ac.jp/

Key questions in Developmental Biology

How many genes do we need to build an embryo? Which gene/gene regulatory network controls formation of a particular organ? What is the normal role of a gene that leads to disease when mutated?

Cancer

Bone defects

The different stages of embryonic development

Fertilization

Growth and maturation Birth Organogenesis Neurulation

10,000 cells 30,000 cells

Cleavage period

Gastrulation

Morphogenesis

Fertilization and cleavage

the first cell divisions into embryo formation

www.ohsu.edu

Two-cell stage Xenopus embryo Fertilization of an oocyte

The beginning of a new life:

From One (Zygote) to Many (Blastomeres)

How does fertilization unite sperm and egg to form the zygote ? What are cytoplasmic determinants and how are they re-distributed after fertilization? How do cells acquire their fates in early cleavage stage?

How are gamete cells specialized for their functions?

Size, number and content? Special structures and modification? Special physiology? Recognition and attraction mechanisms? Species specificity? QC test?

www.zeiss.de

The transformation of one cell into a highly specialized structure: Differentiation of a sperm
Dramatic re-organizations: Golgi forms into acrosomal vesicle (contains lysosomal enzymes: enzymatic knife) centriol forms flagellum chromatin condenses and gives nucleus characteristic shape superfluous cytoplasm is shed to give torpedolike structure

mitochondria produce energy for propulsion

No transcription!
Gilbert SF, 2006 Fig 7.2

600 sperm produced in one sec per gram testis!

A primary oocyte in the mammalian ovary

and after ovulation

Alberts et al., 2002 Fig. 20-24 http://en.wikipedia.org/wiki/Folliculogenesis

http://bakerinstitute.vet.cornell.edu/

- All primary oocytes are already formed at birth (pre-natal arrest in 1st meiotic division; 4n) - With onset of puberty: cyclic maturation of individual oocytes/follicles (arrest in metaphase II (2n) BEFORE ovulation)

- Completion of second meiotic division (1n) AFTER fertilization

Fertilization (from an oocytes point of view)

The interaction between sperm and egg stimulates the egg to resume the cell cycle and begin development, a process referred to as egg activation. A large increase in intracellular calcium is the primary trigger for egg activation. In addition, the increase in intracellular calcium is responsible for the exocytosis of cortical granules and the prevention of polyspermy.

How is this achieved?

Capacitation: The preparation of a sperm

Attainment of full fertilizing activity: Spermatozoa are activated by secretions of the female reproductive tract (usually uterus) Capacitation results in spermatozoa (i) becoming significantly more motile (ii) being able to respond to signals from immediate surrounding (iii) being able to undergo acrosome reaction

These changes largely result from increased entry of calcium which also increases cAMP levels. Its own enzymes also help it penetrate the granulosa cells surrounding the oocyte to get to the zona pellucida (ZP)

Essential Reproduction by M.H. Johnson Fig 9.4

Capacitation prepares for acrosome reaction

As a result of capacitation, the acrosome swells and fuses with the plasma membrane. The inner acrosomal membrane (with ZP3 receptors) is now exposed. This swelling involves further increases in intracellular calcium and cAMP and a rise in pH.

Kalthoff Fig 4.9

The acrosome reaction

Capacitation: The exposure of the inner membrane reveals binding sites for ZP3. Interaction with ZP3 leads to acrosome reaction: Proteolytic enzymes are released from the acrosome; allows digestion through the ZP, aided by hyperactive sperm.

Kalthoff Fig 4.9

Sperm/egg contact triggers acrosomal reaction, zona pellucida penetration, membrane fusion and introduction of male nucleus

- acrosomal reaction: release of proteolytic enzymes from acrosomal vesicles

- ZP penetration takes 5-20 min

- sperm after acrosomal reaction are very short lived; oocyte contact (i.e. membrane fusion)

has to happen soon!!

Alberts et al., Fig 20-31

Enzymatic Knife

The acrosome reaction is induced by binding of the sperm to the egg zona pellucida glycoprotein ZP3

- ZP3 receptor binding leads to sustained Ca2+ influx into

sperm head
- Ca2+ / H+ antiport pH - increases sperm mobility - induces fusion of acrosomal vesicles release of proteolytic

enzymes (which will digest the zona pellucida)

From: Evans & Florman, Nature Cell Biology 2002

After ZP penetration: More sperm/egg interactions

Receptor ligand communications allow membrane fusion and trigger calcium waves in the oocyte

From: Evans & Florman, Nature Cell Biology 2002

Calcium waves result in opening of Cortical granules: Cortical reaction

After fusion, sperm stops motility; nucleus passes into ooplasm; dramatic calcium increase (release from internal stores), wave from point of sperm entry for 2-3 min, several spikes follow (1-2 min each; every 3-15 min, for several hours).

Ca increase leads to fusion of cortical granules with membrane: release of enzymes (proteases, hexoaminidases) digest ZP3 and change ZP structure prevent further binding and penetration of spermatozoa (zona reaction)

Taken from Inoue N. et al., Nature, 2005

Calcium increase/ Calcium waves lead to: 1. Prevention of polyspermy (by altering ZP sructure) 2. Completion of second meiotic division (expulsion of 2nd polar body) 3. Establishment of female and male pronuclei

4. Modifications to the vitelline envelope, permitting the reorientation of the eggs via gravity (rotation)

Kalthoff Fig 4.16

Cortical reaction in sea urchin eggs

Gilbert S.F., 2006 Fig 7.23

uwnews.org

Fertilization accomplishes at least four things:

1. Restoration of a diploid genome by uniting the two haploid genomes of the oocyte and sperm; 2. Species-specific sperm-egg binding; 3. Egg activation, beginning with the cortical reaction, a transient calcium wave that passes across the egg, and the onset of nucleic acid and protein synthesis. 4. Contribution of paternal centrosomal material by the sperm, required for proper mitosis.

Fertilization triggers initiation of embryonic development by:

Increasing protein synthesis using preexisting maternal mRNAs and ribosomes Re-organizaton of the egg cytoplasm: Cortical Rotation