Programmed cell death and embryonic development

Dept. of Histology & Embryology

Xiangya School of Medicine, CSU

Definition of Programmed Cell Death

programmed cell death - llec fo epyt a

dezilaiceps sesu llec eht hcihw ni htaed

edicius llec a ;flesti llik ot yrenihcam ralullec

lortnoc ot snaozatem selbane taht msinahcem

netaerht taht sllec etanimile dna rebmun llec

lavivrus s'lamina eht

1885 Flemming first mentioned cell death in rabbit ovarian follicle (chromatolysis) is physiological, and described chromatin was condensed into half-moon shape (crescent);

1914 Gräper: Chromatolysis of all cells eliminated from organs; phagocytosis of neighboring cells; exploring the phenomenon the organ construction;

1951 Glucksmann reviewed cell death in ontogenesis; a normal part of animal development, and detailed the process of chromatolysis；

1964 Lockshin & Williams proposed the concept of "programmed cell-death" in their research thesis related to insect (silkworm moth) metamorphosis

Important events in the history of cell death

1966 Saunders: “The death clock is ticking;” cell death is a suicide

1972 Kerr, Wyllie, Currie: Apoptosis is general in morphology and among animals

Important events in the history of cell death

Sydney Brenner of the Salk Institute used the nematode Caenorhabditis elegans, which became a multicellular model experimental system, to follow cell division and differentiation from the fertilized egg to the adult via microscopic observation. He demonstrated that a specific gene mutation, induced by ethyl methane sulfonate, could be linked to a specific effect in nematode organ development. His work on nematodes created an experimental system that laid the foundation for the study of apoptosis.

John Sulston of the Wellcome Trust Institute in England mapped cell lineages, where every cell division and differentiation could be followed in the development of C. elegans. There are only 959 cells in an adult nematode (1090 to 959 cells, 131 cell lost). He showed that specific cells lineages (nerves) undergo programmed cell death, as an integral part of the normal differentiation process.

Robert Horvitz of MIT discovered and characterized key genes controlling cell death in C. elegans. He identified the first two bona fide "death genes", ced-3 and ced-4. Functional ced-3 & ced-4 genes are a prerequisite for cell death to be executed. Another gene, ced-9, protects against cell death by interacting with ced-3and ced-4. He has shown how these genes interact with each other in the cell death process and that corresponding genes (a ced-3-like gene) exists in humans.

The Nobel Prize in Physiology or Medicine 2002

Sydney Brenner H. Robert Horvitz John Sulston

THE 2002 NOBEL PRIZE WINNER

PCD has been the subject of increasing attention and research

efforts. This trend has been highlighted with the award of the

2002 Nobel Prize in Physiology or Medicine to Sydney Brenner

(United Kingdom), H. Robert Horvitz (US) and John E. Sulston

(UK)

The creation of the conception of PCD

Carroll Williams was always known for his colorful phraseology, and as graduate students we always tried to emulate him. Because computers were just beginning to be talked about at the time, programmed cell death seemed to be a particularly modern and colorful way of describing what we saw. It was a metaphor stating what I thought was pretty obvious — if a biological process occurs at a defined location and time, then it must in some fashion be programmed or written into the genetics of the organism-----from Williams’ student .

PCD and APOPTOSIS

Apoptosis is used as a synonym for PCD

Apo: apart

Ptosis: fallen

Shedding of leaves from trees

During embriogenesis ------ occurs as PCD

Post-embrional life------- as apoptosis

PCD and its significance during embryonic development

Optimization of system matching

To regulate the cell number by eliminating wasted, useless,

unwanted, or crippled cells!

For example, 80% nerve cells, 70-95% oocyte died in this way.

Sculpting of tissues

Such as the formation of the fingers and toes of

the fetus in chicken, mouse or human.

Arrowheads indicate PCD cells. PCD in duck or tortoise

Indicated by arrow

PCD and its significance during embryonic development

Shortening and rotation

of the myocardial portion

of the OFT(outflow tract

(OFT)) coincident with

cardiomyocyte apoptosis

Sculpting of tissues

Extensive postnatal apoptosis occurs invessels that regress after birth, ie, ductus arteriosus and the umbilical arteries

Removal of transitional or

useless structures

Deletion of

transitional

structures

Evolution of aortic arches

Deletion of transitional structures

Pronephros-

pronephric tubule and

duct

Mesonephros-

mesonephric tubule

and duct

Tadpole tail removed by apoptosis during development

Deletion of transitional structures

•Autoreactive T cells with the

potential to attack "self" are

removed by apoptosis

Self antigenrecognizing cell

Essential for the removal of

cells that threaten homeostasis

Morphological changes during apoptosis

Cell shrinkage

Apoptotic B Cells

Hoechst stainingHE staining

Morphological features of Morphological features of

apoptosisapoptosis

Membrane blebbing

macrophage

Apoptoti

c bodies

Biochemical changes during apoptosis

Phosphatidylserine inside outannexin V, a Ca+ dependent PS binding protein

Annexin V staining

flow cytometry

Activation of Caspase dependent endonuclease (CAD).

Biochemical changes during apoptosis

180-200bp

DNA “ladder”

Biochemical changes during apoptosis

Terminal deoxynucleotidyl transferase (TDT) mediated dUTP nick end labeling(TUNEL)Principle: TdT mediates incorporation of biotinylated dUTP into 3’ OH ends of fragmented DNA

Biochemical changes during apoptosis

Proteins in favor for apoptosis ↑

Death receptor superfamily, such as TNFR1

, Fas/APO-1, DR,3,4 and 5, and so on

Caspase family (cysteine-containing aspartate-specific

protease): Caspase 1-10,13. 11 and 12 species specific

Apaf-1 (apoptotic protease activating factor-1)

Mitochondrial membrane releasing factors: Smac/

DIABLO

Proteins against apoptosis ↑Bcl-2 family (B cell lymphoma/Leukemia-2, Bcl-2), but BAX

subfamily in favor of apoptosis

Cell Death Pathways

Death

Execution

IAPs

Smac/

DIABLO

AIF

Endonuclease

activation

DNA broken

Death

Harmful factor→ changes of ER Ca2+ stable status and accumulation of incorrect folding proteins→pro-caspase-12→ caspase-12→BAX activated→mitochondria →Cyt C and Apaf1 release→ caspase9 activated→caspase-3 activated→apoptosis

Endoplasmic reticulum (ER) pathway

Hormones regulate PCD during embryonic development

The loss of tadpole tail is controlled by thyroxine

The PCD of thymocyte is regulated by Glucocorticoid

The regression of paramesonephric duct/ Muller duct is controlled by

Mullerian inhibiting substance

Estrogen inhibits PCD of Granulosa cells

FSH/LH inhibit PCD of ovarian follicle

Insulin inhibits PCD of lens epithelial cells

Growth factors/cytokins regulate PCD during embryonic development

Lack of NGF，FGF ，TGF，G-CSF leads to

PCD

Necrosis

Necrosis vs. Apoptosis

Cellular condensation

Membranes remain intact

Requires ATP

Cell is phagocytosed, no

tissue reaction

Ladder-like DNA

fragmentation

In vivo, individual cells

appear affected

Active death

• Cellular swelling

• Membranes are broken

• ATP is depleted

• Cell lyses, eliciting an

inflammatory reaction

• DNA fragmentation is

random

• In vivo, whole areas of

the tissue are affected

• Passive death

Necrosis Apoptosis

Questions

Can you imagine how it likes about DNA gel

electrophoresis from extracts of necrotic tissue?

TUNEL also labels necrotic cells?

Autophagy, 3rd cell death

31

Receptor pathway (physiological):

Death receptors:

(FAS, TNF-R, etc)

FAS ligand TNF

Deathdomains

Adaptor proteins

Pro-caspase 8 (inactive) Caspase 8 (active)

Pro-execution caspase (inactive)Execution caspase (active)

DeathMITOCHONDRIA

zymogen enzyme

H2O2

Growth factor

receptors

casp9Bcl2

PI3KAkt

BAD

Apaf1

Cyt.CATP

The mitochondrial pathway

casp3

casp3

IAPs

Smac/

DIABLO

AIF

Bax

Bax

p53

Fas

Casp8

Bid

Bid

Bid

DNA

damage