INHOUDSTAFEL
INHOUDSTAFEL ..........................................................................................................................................1
0. INTRODUCTIE..........................................................................................................................................3
Different kind of experiments to address sufficiency versus necessity hypotheses ................................................. 3
1. HISTORY AND BASIC CONCEPTS ...........................................................................................................4
1. Fate and potency .............................................................................................................................................. 4
7 stappen van development ...................................................................................................................................... 4
1. Cell division ...................................................................................................................................................... 4
Somatic divisions .............................................................................................................................................. 4
Embryonic cleavage........................................................................................................................................... 4
Activation of the zygotic genome ........................................................................................................................ 6
2. Differentiation ................................................................................................................................................... 6
Restriction vs potency ....................................................................................................................................... 6
Waddington’s landscape ................................................................................................................................... 6
Germ layers ...................................................................................................................................................... 7
Potency ............................................................................................................................................................ 7
Specification and determination......................................................................................................................... 8
3. Growth ............................................................................................................................................................. 8
4. Migration .......................................................................................................................................................... 9
5. Patterning ......................................................................................................................................................... 9
6. Morphogenesis ............................................................................................................................................... 10
7. Apoptosis .................................................................................................................................................... 11
Cleavage stages ..................................................................................................................................................... 11
Specificatie van embryonic en extraembryonic lineages ........................................................................................ 12
1st wave of diffrentiation .................................................................................................................................. 13
2nd wave of diffrentiation .................................................................................................................................. 14
2. METHODS IN DEVELOPMENTAL BIOLOGY.......................................................................................... 20
A look throughout the animal kingdom ..................................................................................................................... 20
Looking at embryos at different levels ...................................................................................................................... 21
Histology ............................................................................................................................................................ 21
Microscopy ......................................................................................................................................................... 21
Live imaging ........................................................................................................................................................ 22
Fate mapping ...................................................................................................................................................... 22
Door Pigmentatie............................................................................................................................................. 22
Door genetically encoded tracers ..................................................................................................................... 23
Manipulating embryos ............................................................................................................................................ 24
Ligation .............................................................................................................................................................. 24
Transplantation .................................................................................................................................................. 24
Explants ............................................................................................................................................................. 24
Ablation.............................................................................................................................................................. 25
cell migration ...................................................................................................................................................... 25
Molecules & embryos ............................................................................................................................................. 26
Transcripts in situ hybridization ........................................................................................................................... 26
1
, Proteins (immunohistochemistry) ........................................................................................................................ 26
Genes, gene networks & embryos ............................................................................................................................ 27
Gene regulation .................................................................................................................................................. 28
Reporter genes ................................................................................................................................................ 28
Protein-DNA interaction ................................................................................................................................... 28
Targeted mutagenesis ..................................................................................................................................... 28
Single cell approaches ........................................................................................................................................ 29
Systems biology approaches ............................................................................................................................... 29
Deciphering gene function ...................................................................................................................................... 29
Loss-of-function approaches ............................................................................................................................... 29
Transgenesis ...................................................................................................................................................... 30
Gain-of-function approaches............................................................................................................................... 31
Genome editing .................................................................................................................................................. 31
Development and evolution .................................................................................................................................... 32
Molecular phylogeny ........................................................................................................................................... 32
Gene families ..................................................................................................................................................... 32
Cross-species approaches .................................................................................................................................. 32
2
,0. INTRODUCTIE
DIFFERENT KIND OF EXPERIMENTS TO ADDRESS SUFFICIENCY VERSUS NECESSITY
HYPOTHESES
“A cat is sufficient to cause a pit in the road”
“A cat is necessary to cause a pit in the
Take the cat and put her on a road without pits. road”
Observe what happens over time:
First remove all cats, then observe what
Is a pit formed? Yes? Then a cat is sufficient. happens over time to a road without pits.
No? Then a cat is not sufficient to form a pit.
Are pits formed over time? Yes? Then a
This would be a “move it” experiment cat is not necessary. No? Then cats are
necessary to form pits in the road.
Mechanisms underlying development can only be studied by disturbing the normal developmental process in specific
ways and observing what happens
→ model organisms, explant, organoids, etc.
→ block-it/lose-it (loss-of-function or LOF) experiments address necessity
→ move-it (gain-of-function or GOF) experiments address sufficiency
→ show-it/find-it experiments: correlative evidence: base to formulate a hypothesis Test a hypothesis by disturbance:
Bv immunofluorescentie
• A section of a neural tube
• Immunofluorescent staining shows presence of 3 different proteins (antigens) using 3
different antibodies that are labelled with different fluophores (red, green, blue)
• This is a show-it experiment
• Presence of different colors red – orange – yellow – green – clear blue – dark blue
suggests that there are 6 different classes of neuronal cells that express different
combinations of the 3 antigens
3
, 1. HISTORY AND BASIC CONCEPTS
1. FATE AND POTENCY
GOALS:
• Comprehension of concepts of e.g. fate, potency, genomic equivalence and reprogramming
• Understanding how to assess the specification and determination status of cells
• Comprehension of features of cleavage stages of embryos
• Understanding mechanisms of lineage decisions in early mammalian cleavage embryos
• Interpreting consequences of gain- and loss-of-function of lineage gatekeepers
• Later: making links to other lectures
7 STAPPEN VAN DEVELOPMENT
1. CELL DIVISION
Zygote → Blastula/Blastocyst → Gastrula → Neurula
SOMATIC DIVISIONS
= Normale mitotische delingen van lichaamscellen (groei, onderhoud, vervanging).
EMBRYONIC CLEAVAGE
= De zeer vroege, snelle delingen van het bevruchte eicel (zygote) die leiden tot de vorming van de blastula/blastomeren.
• Gap phases (G1 and G2) are omitted
• Cell cycles rapidly between M and S phases: extremely short cell cycle
Bv:
- frog: 1-37,000 cells (43 hrs)
- fruitfly: 1-50,000 cells (12 hrs)
- … mouse and human: 1-4 cells (~ 48 hrs)
→ Direction of cell divisions
→ Symmetric versus asymmetric divisions
4
INHOUDSTAFEL ..........................................................................................................................................1
0. INTRODUCTIE..........................................................................................................................................3
Different kind of experiments to address sufficiency versus necessity hypotheses ................................................. 3
1. HISTORY AND BASIC CONCEPTS ...........................................................................................................4
1. Fate and potency .............................................................................................................................................. 4
7 stappen van development ...................................................................................................................................... 4
1. Cell division ...................................................................................................................................................... 4
Somatic divisions .............................................................................................................................................. 4
Embryonic cleavage........................................................................................................................................... 4
Activation of the zygotic genome ........................................................................................................................ 6
2. Differentiation ................................................................................................................................................... 6
Restriction vs potency ....................................................................................................................................... 6
Waddington’s landscape ................................................................................................................................... 6
Germ layers ...................................................................................................................................................... 7
Potency ............................................................................................................................................................ 7
Specification and determination......................................................................................................................... 8
3. Growth ............................................................................................................................................................. 8
4. Migration .......................................................................................................................................................... 9
5. Patterning ......................................................................................................................................................... 9
6. Morphogenesis ............................................................................................................................................... 10
7. Apoptosis .................................................................................................................................................... 11
Cleavage stages ..................................................................................................................................................... 11
Specificatie van embryonic en extraembryonic lineages ........................................................................................ 12
1st wave of diffrentiation .................................................................................................................................. 13
2nd wave of diffrentiation .................................................................................................................................. 14
2. METHODS IN DEVELOPMENTAL BIOLOGY.......................................................................................... 20
A look throughout the animal kingdom ..................................................................................................................... 20
Looking at embryos at different levels ...................................................................................................................... 21
Histology ............................................................................................................................................................ 21
Microscopy ......................................................................................................................................................... 21
Live imaging ........................................................................................................................................................ 22
Fate mapping ...................................................................................................................................................... 22
Door Pigmentatie............................................................................................................................................. 22
Door genetically encoded tracers ..................................................................................................................... 23
Manipulating embryos ............................................................................................................................................ 24
Ligation .............................................................................................................................................................. 24
Transplantation .................................................................................................................................................. 24
Explants ............................................................................................................................................................. 24
Ablation.............................................................................................................................................................. 25
cell migration ...................................................................................................................................................... 25
Molecules & embryos ............................................................................................................................................. 26
Transcripts in situ hybridization ........................................................................................................................... 26
1
, Proteins (immunohistochemistry) ........................................................................................................................ 26
Genes, gene networks & embryos ............................................................................................................................ 27
Gene regulation .................................................................................................................................................. 28
Reporter genes ................................................................................................................................................ 28
Protein-DNA interaction ................................................................................................................................... 28
Targeted mutagenesis ..................................................................................................................................... 28
Single cell approaches ........................................................................................................................................ 29
Systems biology approaches ............................................................................................................................... 29
Deciphering gene function ...................................................................................................................................... 29
Loss-of-function approaches ............................................................................................................................... 29
Transgenesis ...................................................................................................................................................... 30
Gain-of-function approaches............................................................................................................................... 31
Genome editing .................................................................................................................................................. 31
Development and evolution .................................................................................................................................... 32
Molecular phylogeny ........................................................................................................................................... 32
Gene families ..................................................................................................................................................... 32
Cross-species approaches .................................................................................................................................. 32
2
,0. INTRODUCTIE
DIFFERENT KIND OF EXPERIMENTS TO ADDRESS SUFFICIENCY VERSUS NECESSITY
HYPOTHESES
“A cat is sufficient to cause a pit in the road”
“A cat is necessary to cause a pit in the
Take the cat and put her on a road without pits. road”
Observe what happens over time:
First remove all cats, then observe what
Is a pit formed? Yes? Then a cat is sufficient. happens over time to a road without pits.
No? Then a cat is not sufficient to form a pit.
Are pits formed over time? Yes? Then a
This would be a “move it” experiment cat is not necessary. No? Then cats are
necessary to form pits in the road.
Mechanisms underlying development can only be studied by disturbing the normal developmental process in specific
ways and observing what happens
→ model organisms, explant, organoids, etc.
→ block-it/lose-it (loss-of-function or LOF) experiments address necessity
→ move-it (gain-of-function or GOF) experiments address sufficiency
→ show-it/find-it experiments: correlative evidence: base to formulate a hypothesis Test a hypothesis by disturbance:
Bv immunofluorescentie
• A section of a neural tube
• Immunofluorescent staining shows presence of 3 different proteins (antigens) using 3
different antibodies that are labelled with different fluophores (red, green, blue)
• This is a show-it experiment
• Presence of different colors red – orange – yellow – green – clear blue – dark blue
suggests that there are 6 different classes of neuronal cells that express different
combinations of the 3 antigens
3
, 1. HISTORY AND BASIC CONCEPTS
1. FATE AND POTENCY
GOALS:
• Comprehension of concepts of e.g. fate, potency, genomic equivalence and reprogramming
• Understanding how to assess the specification and determination status of cells
• Comprehension of features of cleavage stages of embryos
• Understanding mechanisms of lineage decisions in early mammalian cleavage embryos
• Interpreting consequences of gain- and loss-of-function of lineage gatekeepers
• Later: making links to other lectures
7 STAPPEN VAN DEVELOPMENT
1. CELL DIVISION
Zygote → Blastula/Blastocyst → Gastrula → Neurula
SOMATIC DIVISIONS
= Normale mitotische delingen van lichaamscellen (groei, onderhoud, vervanging).
EMBRYONIC CLEAVAGE
= De zeer vroege, snelle delingen van het bevruchte eicel (zygote) die leiden tot de vorming van de blastula/blastomeren.
• Gap phases (G1 and G2) are omitted
• Cell cycles rapidly between M and S phases: extremely short cell cycle
Bv:
- frog: 1-37,000 cells (43 hrs)
- fruitfly: 1-50,000 cells (12 hrs)
- … mouse and human: 1-4 cells (~ 48 hrs)
→ Direction of cell divisions
→ Symmetric versus asymmetric divisions
4
