Alle hoofdstukken, uitgezonderd H14: Teratogens and birth defects. Dit is een cursus voor het vak 'concepts of developmental biology' gegeven aan de 3e bachelor Biomedische Wetenschappen (KUL). Dit wordt gedoceerd door prof. Zwijsen, prof. Callaerts en prof. Tylzanowski. Dit document bevat uitgebre...
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1. History and basic concepts: Fate and potency - AZ ....................................................................................... 8
1. Cell division ..................................................................................................................................................... 9
Activation of the zygotic genome ................................................................................................................ 11
2. Differentiation .............................................................................................................................................. 12
Restriction vs potency.................................................................................................................................. 12
Waddington’s landscape .............................................................................................................................. 13
Germ layers .................................................................................................................................................. 14
Potency ........................................................................................................................................................ 14
Specification and determination ................................................................................................................. 14
3. Growth .......................................................................................................................................................... 17
4. Migration ...................................................................................................................................................... 17
5. Patterning ..................................................................................................................................................... 18
6. Morphogenesis ............................................................................................................................................. 18
7. Apoptosis ...................................................................................................................................................... 19
Cleavage stages ................................................................................................................................................ 19
Cleavage divisions: two rounds of differentiation ....................................................................................... 20
Specification of embryonic and extraembryonic lineages ........................................................................... 21
Two cell fate decisions ................................................................................................................................. 21
Wave 1: cell divisions result into ‘inside’ and ‘outside’ cells: .................................................................. 21
Wave 2: cells of ICM seggregate into: ..................................................................................................... 22
Specification of EPI and PE .......................................................................................................................... 23
TF Gatekeepers of TE, EPI and PE fate: ........................................................................................................ 23
2. Methods in Developmental Biology - PC ..................................................................................................... 24
Looking at embryos at different levels ............................................................................................................. 25
Histology ...................................................................................................................................................... 25
Reconstruction ............................................................................................................................................. 25
Microscopy .................................................................................................................................................. 25
Live imaging ................................................................................................................................................. 26
Fate mapping ............................................................................................................................................... 26
Manipulating embryos ..................................................................................................................................... 27
Ligation ........................................................................................................................................................ 28
Transplantation ............................................................................................................................................ 29
Explants ....................................................................................................................................................... 29
Ablation ....................................................................................................................................................... 29
Microinjection.............................................................................................................................................. 29
Studying cell migration ................................................................................................................................ 30
Molecules & embryos ....................................................................................................................................... 30
Transcripts (in situ hybridization)................................................................................................................. 30
Immunohistochemistry: looking at protein ................................................................................................. 31
Genes, gene networks and development.......................................................................................................... 32
Gene regulation: reporter genes, protein-DNA interaction, targeted mutagenesis .................................... 32
Gene regulation: reporter genes ................................................................................................................. 32
Single cell approaches ................................................................................................................................. 34
Gene network: systems biology approaches ............................................................................................... 34
1
, Loss of function approaches ................................................................................................................... 34
Transgenesis ............................................................................................................................................ 35
Gain-of-function approaches. ................................................................................................................. 35
Genome editing ...................................................................................................................................... 36
Development and evolution: comparative methods......................................................................................... 36
Molecular phylogeny ................................................................................................................................... 37
Gene families ............................................................................................................................................... 37
Cross-species approaches ............................................................................................................................ 37
3. Model organisms - PT ................................................................................................................................. 38
History .............................................................................................................................................................. 39
Use of animals in research ............................................................................................................................... 39
Experimental models ........................................................................................................................................ 40
Hydra ........................................................................................................................................................... 42
C.Elegans ...................................................................................................................................................... 44
Fruit fly......................................................................................................................................................... 45
Frog .............................................................................................................................................................. 48
Holt-Oram syndrome .............................................................................................................................. 48
Zebrafish ...................................................................................................................................................... 48
Chicken ........................................................................................................................................................ 50
Tissue grafts ............................................................................................................................................ 51
Mouse .......................................................................................................................................................... 52
4. Gastrulation – AZ ........................................................................................................................................ 55
Introduction of gastrulation ............................................................................................................................. 55
Themes/questions ............................................................................................................................................ 55
Germ layers, epithelium and mesenchyme EMT .............................................................................................. 56
EMT .............................................................................................................................................................. 57
MET .............................................................................................................................................................. 57
Gastrulation in: ................................................................................................................................................. 58
Xenopus ....................................................................................................................................................... 58
Epiboly .................................................................................................................................................... 59
Involution ................................................................................................................................................ 59
From blastula to gastrula: cell migration ................................................................................................ 60
Who are the players that make this process happen? ............................................................................ 61
Microdissection experiments ............................................................................................................. 61
Transplantation experiments ............................................................................................................. 62
Recombination experiments .............................................................................................................. 63
Role of β-catenin in dorsal-ventral axis specification? ....................................................................... 65
Zebrafish ...................................................................................................................................................... 68
Cell movement ........................................................................................................................................ 70
Stretching the zebrafish epiblast cells generates mesoderm .................................................................. 71
The embryonic shield = zebrafish organizer ............................................................................................ 71
Chicken ........................................................................................................................................................ 72
What are the cues for mesoderm patterning?........................................................................................ 74
Mouse .......................................................................................................................................................... 74
Mouse gastrulation initiates at E6.5 ....................................................................................................... 76
1st molecular appearance of A-P axis precedes gastrulation ................................................................. 77
Human ......................................................................................................................................................... 78
Cell movements during human gastrulation ........................................................................................... 78
Example Modeling gastrulation ....................................................................................................................... 80
‘Embryo’ development in a dish: embryoids ............................................................................................... 80
5. Positional information – PC ........................................................................................................................ 84
Inspirational experiments ................................................................................................................................. 84
Early sea urchin development ..................................................................................................................... 84
Hydra head formation – an inhibitory gradient ........................................................................................... 84
Chicken limb development .......................................................................................................................... 85
Competence of cells: the ability of cells to respond to an instructive signal ............................................... 86
Lewis Wolpert – definition of positional information ....................................................................................... 86
The French flag model – a conceptional framework to generate hypotheses .................................................. 86
Morphogen: definition...................................................................................................................................... 87
Positional information and Drosophila ............................................................................................................. 87
Early embryonic development ..................................................................................................................... 87
Cellular blastoderm: fatemap ...................................................................................................................... 89
From an early embryo to a segmented larva ............................................................................................... 89
Maternal genes ............................................................................................................................................ 91
Gradients ..................................................................................................................................................... 93
Bicoid: anterior determinant .................................................................................................................. 93
Bicoid: localization of mRNA ................................................................................................................... 94
Maternal genes: more than one gradient ............................................................................................... 95
Segmentation genes - converting gradients into stripes ............................................................................. 96
Bicoid encodes a homeodomain protein that can act as transcription factor or translational repressor
................................................................................................................................................................ 96
Bicoid: anterior determinant .................................................................................................................. 97
Gap and pair-rule genes .......................................................................................................................... 98
Even-skipped regulation: a tale of many enhancers ............................................................................... 98
Even-skipped stripe 2: a model enhancer ............................................................................................... 98
Pair-rule and segment polarity genes ..................................................................................................... 99
Putting it all together ................................................................................................................................. 100
Implications in biomedicine ............................................................................................................................ 100
Discovery of signaling pathways ................................................................................................................ 100
Understanding teratogens ......................................................................................................................... 101
6. Signaling pathways in developmental biology – PT ................................................................................... 101
Inductive signaling .......................................................................................................................................... 101
History of TGF beta .................................................................................................................................... 105
BMP ........................................................................................................................................................... 107
BMP function in limb patterning ........................................................................................................... 108
Extracellular antagonists............................................................................................................................ 109
Noggin ....................................................................................................................................................... 109
Receptors ........................................................................................................................................................ 110
Receptor KO ............................................................................................................................................... 111
TGF- signaling ......................................................................................................................................... 111
Smad .......................................................................................................................................................... 114
Structure of Smads ............................................................................................................................... 114
How do we detect a Smad activation? .................................................................................................. 115
Smad KO ................................................................................................................................................ 115
Smad interaction factors ....................................................................................................................... 117
WNTs .......................................................................................................................................................... 117
3
, Wnt signaling cascades ......................................................................................................................... 118
What are the components of the Wnt pathway? ........................................................................................... 120
Function of sFRPs ....................................................................................................................................... 120
In vivo assays ............................................................................................................................................. 120
Receptors ................................................................................................................................................... 121
Frizzleds ................................................................................................................................................ 121
Arrow/LRP ............................................................................................................................................. 121
LRP6 function ................................................................................................................................... 122
Another Wnt receptor- Ryk ................................................................................................................... 123
Yet another receptor- Ror2 ................................................................................................................... 123
Conclusion ................................................................................................................................................. 123
So how do the Wnts manage to act as morphogens? .................................................................................... 124
Dally and dally-like ..................................................................................................................................... 124
Another way is through Shibire ................................................................................................................. 125
Intracellular players ........................................................................................................................................ 125
Dsh ............................................................................................................................................................. 125
-catenin .................................................................................................................................................... 126
GSK-3 kinase .............................................................................................................................................. 126
Axin ............................................................................................................................................................ 126
APC ............................................................................................................................................................ 127
Lef/Tcf ........................................................................................................................................................ 127
Wnt crosstalk ............................................................................................................................................. 131
Hedgehog (HH) → zie ook limb development! .......................................................................................... 133
Noncanonical hh signaling .................................................................................................................... 134
FGF ............................................................................................................................................................. 135
Outline of the Fgf signaling pathway .................................................................................................... 135
Conclusion ...................................................................................................................................................... 136
7. Anteroposterior patterning – PC ............................................................................................................... 137
Goals ............................................................................................................................................................... 137
Maternal and segmentation genes revisited .................................................................................................. 137
The body plan and homeotic genes ................................................................................................................ 137
Homeotic transformations: change of developmental fate ............................................................................ 137
The Lewis model ............................................................................................................................................. 138
Cloning Antennapedia: understanding the molecular basis ........................................................................... 139
Drosophila Hox genes are organized in two clusters ...................................................................................... 139
The homeobox encodes the homeodomain .................................................................................................... 139
The homeodomain: an evolutionarily ancient and highly conserved DNA-binding domain ........................... 140
Colinear Hox gene expression ......................................................................................................................... 140
Hox genes in other species – vertebrates and mammals ............................................................................... 141
Homeotic transformation in the mammalian embryo............................................................................... 142
Hox genes: an ancient system for body patterning ................................................................................... 142
Hox genes: limb patterning – medical relevance ............................................................................................ 142
Hox genes: a model to study evolution ........................................................................................................... 144
A case study: changing limb numbers in Arthropods ..................................................................................... 144
Evolving: through changes in Hox protein function ................................................................................... 144
Evolving: through changes in Hox gene expression ................................................................................... 145
Evolving: through changes in Hox target genes ......................................................................................... 145
4
,8. Patterning: mesoderm and neuroectoderm patterning - AZ...................................................................... 145
Positional information Recapitulation mesoderm induction .......................................................................... 146
Mesoderm patterning in frog and mouse ....................................................................................................... 147
Xenopus ..................................................................................................................................................... 147
Organizerconcept ....................................................................................................................................... 147
Differences between show-it, move-it and block-it experiment ............................................................... 149
Opposing gradients Different types of mesoderm .................................................................................... 149
Exercise ...................................................................................................................................................... 153
Mouse ........................................................................................................................................................ 154
Ectoderm development................................................................................................................................... 157
Neuroectoderm induction and patterning ................................................................................................ 157
Organizerconcept .................................................................................................................................. 157
Neural plate/groove/tube closure ............................................................................................................. 158
Co-linearity (A-P) between neural tissue and mesoderm .......................................................................... 160
Neural tube defects ................................................................................................................................... 161
Exercise ...................................................................................................................................................... 162
Dorso-ventral patterning of the neural tube ............................................................................................. 163
Neural crest cell ......................................................................................................................................... 166
Neural crest lineage, fate map, plasticity ....................................................................................................... 166
Neural crest cell fate map .......................................................................................................................... 167
Different types of neural crest cells ........................................................................................................... 168
Neural crest syndromes ............................................................................................................................. 170
Piebaldism: neural crest cell syndrome (c-Kit mutation) ...................................................................... 170
Waardenburg syndrome ....................................................................................................................... 171
DiGeorge syndrome .............................................................................................................................. 171
Cleft lip/palate ...................................................................................................................................... 171
Neural crest cells and evolution ................................................................................................................ 171
Conclusion ...................................................................................................................................................... 172
9. Somitogenesis and limb development – PT .............................................................................................. 174
Somitogenesis................................................................................................................................................. 174
Somites ...................................................................................................................................................... 175
What happens in the somites? ............................................................................................................. 176
Somites form at the junction of retinoic acid (anterior) and FGF (posterior domains). ............................ 176
FGF antagonists ..................................................................................................................................... 178
Primaxial and abaxial domains of vertebrate mesoderm .......................................................................... 181
Limb development .......................................................................................................................................... 181
Limb patterning ......................................................................................................................................... 182
Limb induction ........................................................................................................................................... 183
HOX = homeobox gene ......................................................................................................................... 185
Digit length is additively determined by Hoxd and Hoxa gene products.......................................... 185
The apical ectodermal ridge (AER) induces progress zone ................................................................... 187
Limb identity .............................................................................................................................................. 190
AP axis ................................................................................................................................................... 192
DV axis................................................................................................................................................... 193
Digits ..................................................................................................................................................... 194
The role of BMP signaling in determination of digit identities ............................................................. 195
10. Folding morphogenesis – AZ ................................................................................................................... 195
From 3-laminar to tube-in-a-tube embryo ..................................................................................................... 195
From a flat-disc to a shrimp-shaped chick embryo .................................................................................... 196
What is folding morphogenesis? What are the consequences? ..................................................................... 196
5
, Ventro-lateral morphogenesis in the mouse ............................................................................................. 197
Gut internalization and body wall closure ............................................................................................ 200
Ventral folding morphogenesis + axial rotation (in the mouse) → multiple tissue rearrangements +
encasement in extraembryonic membranes ........................................................................................ 201
Gut regionalization and gut-associated organs ..................................................................................... 201
Endoderm becomes regionalized ..................................................................................................... 201
Regional specification of gut endoderm and splanchnic mesoderm .................................................... 202
Gastrointestinal tract and associated organs ........................................................................................ 202
Ventro-lateral morphogenesis in human ................................................................................................... 203
Intra-embryonic coelomic cavities: 3 body cavities .............................................................................. 206
Drivers of folding morphogenesis ................................................................................................................... 206
Folding disorders/defects ............................................................................................................................... 208
Gastroschizis .............................................................................................................................................. 208
Hernia diaphragmatica .............................................................................................................................. 208
11. Branching morphogenesis - PC ............................................................................................................... 209
Branching morphogenesis: goals ................................................................................................................... 209
Branched structures: where and why? ........................................................................................................... 209
Nervous system ......................................................................................................................................... 209
Blood vessels ............................................................................................................................................. 210
Tracheal system ......................................................................................................................................... 210
Lung ........................................................................................................................................................... 210
Kidney ........................................................................................................................................................ 210
Mammary gland ........................................................................................................................................ 211
Branched structures: function and structure .................................................................................................. 211
Branching morphogenesis: ............................................................................................................................. 211
Definition ................................................................................................................................................... 211
Approaches to study .................................................................................................................................. 211
Modes of branching (EXAM) ...................................................................................................................... 212
Events and regulation ................................................................................................................................ 212
5 cases ............................................................................................................................................................ 212
Vasculature ................................................................................................................................................ 212
Vasculature development: vasculogenesis and angiogenesis ............................................................... 212
Vasculogenic hotspots .......................................................................................................................... 213
Studying angiogenesis........................................................................................................................... 213
Stages of vasculature development ...................................................................................................... 213
Tracheal system ......................................................................................................................................... 214
Different stages of tracheal development: embryogenesis & metamorphosis .................................... 215
Cellular basis ......................................................................................................................................... 215
Genetics ................................................................................................................................................ 216
Molecular mechanisms ......................................................................................................................... 216
Tracheal and vascular development: parallels ...................................................................................... 217
Lung ........................................................................................................................................................... 218
Lung development ................................................................................................................................ 218
Mechanism ........................................................................................................................................... 219
Different solutions ................................................................................................................................ 219
Kidney ........................................................................................................................................................ 219
Kidney development ............................................................................................................................. 220
Molecular mechanism .......................................................................................................................... 220
Branching pattern regulation ................................................................................................................ 220
Mammary gland ........................................................................................................................................ 221
Stages of mammary gland development .............................................................................................. 221
Molecular mechanisms ......................................................................................................................... 222
6
, Branching morphogenesis: common themes ................................................................................................. 222
Branching morphogenesis and disease - EXAM ............................................................................................. 223
12. Development in a dish – PT .................................................................................................................... 224
Embryonic stem cells (ESC) ............................................................................................................................. 224
History ....................................................................................................................................................... 224
Mechanism ................................................................................................................................................ 225
Artificial vs natural mouse embryo ............................................................................................................ 226
Mesenchymal stem cells (MSC) ...................................................................................................................... 228
What can we do with stem cells? ................................................................................................................... 229
Human epidermal epithelial cell cultures with different density and different duration in culture. ......... 229
3D printing with live cells................................................................................................................................ 231
There are also negative sides to stem cells ..................................................................................................... 231
Multipotent stem cells .................................................................................................................................... 232
Induced pluripotent stem cells (iPSCs) ............................................................................................................ 232
Organoids ....................................................................................................................................................... 233
Chimeras ......................................................................................................................................................... 235
Cloning ............................................................................................................................................................ 236
13. Eye development – PC ............................................................................................................................ 237
Vision: solving a problem in different ways .................................................................................................... 237
The adult mammalian eye .............................................................................................................................. 238
The adult mammalian retina ..................................................................................................................... 238
The adult Drosophila eye ................................................................................................................................ 239
Eye development in mammals ........................................................................................................................ 239
Eye development in mammals: embryonic lineages that contribute to the eye ....................................... 240
Eye development in Drosophila ...................................................................................................................... 240
Eye development in Drosophila: adult structures derive from imaginal discs and histoblasts.................. 240
Eye development in Drosophila: the eye and head capsule derive from the eye-antennal imaginal disc 240
Eye development in Drosophila: precursors and differentiating photoreceptors...................................... 241
Eye development: inspirational experiments .................................................................................................. 242
Eye development: deciphering molecular mechanisms – PAX6 ...................................................................... 242
PAX6 – a highly conserved transcription factor ......................................................................................... 243
PAX6 – human Aniridia .............................................................................................................................. 243
PAX6 – mouse and rat Small eye ................................................................................................................ 244
PAX6 in mammalian eye development ...................................................................................................... 244
PAX6 in other tissues: development and adult .......................................................................................... 244
Deciphering early eye development – from Drosophila to mammals ............................................................ 244
Analysis of gene function: overexpression studies .................................................................................... 245
PAX6 – a ‘master regulator’ of eye development ...................................................................................... 246
Gene networks: Eyeless is expressed in undifferentiated precursors in a pattern partially overlapping with
So and Eya.................................................................................................................................................. 246
Sine oculis and eyes absent act downstream of eyeless ............................................................................ 247
Gene networks: the relationship between Eyeless and Twin-of-eyeless ................................................... 247
Eye development in mammals: network of transcription factors that establish the eye field .................. 248
Eye development in mammals: signaling networks establish boundaries in the optic vesicle .................. 249
Gene networks in various stages of eye development .................................................................................... 249
7
, Eye development in mammals: network of transcription factors and signaling molecules............................ 249
Eye development in mammals: axes in the optic cup ................................................................................ 250
Eye development in mammals: temporal progression of retinogenesis in the mouse ............................. 250
Implications in biomedicine ............................................................................................................................ 251
Congenital eye defects............................................................................................................................... 251
Mutation identified in OTX2.................................................................................................................. 251
Mutation in SOX2 .................................................................................................................................. 252
Mutations in RAX .................................................................................................................................. 252
Cone-rod dystrophy .............................................................................................................................. 252
Mutation in ATOH7 ............................................................................................................................... 253
Understanding teratogens ......................................................................................................................... 253
Evolution of the eye ........................................................................................................................................ 254
Flies and bees ............................................................................................................................................ 254
Mutant in flies: spacemaker ...................................................................................................................... 254
0. Introduction
1. History and basic concepts: Fate and potency - AZ
Watch the movies of the earliest stages of development of Xenopus, Zebrafish and Human
Write down 3 remarkable observations for each of the movies (ahead of class):
• Xenopus:
• Zebrafish:
• Human
Algemeen: cell division. The cells don’t grow, they become smaller = cell cleavage (de cellen
in de eenheid worden kleiner, maar de eenheid zelf niet). Initially you see the individual cells,
but then it becomes fuzzy (unclear), the cell borders disappear. A cavity starts to form.
Zygote
Blastula/Blastocyst
Gastrula ➔ Ectoderm, mesoderm and endoderm are formed during gastrulation.
Neurula
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
8
, • Later: making links to other lectures
1. Cell division
Welke features zie je in frog embryo?
- The embryo didn’t grow, it kept the same size.
- Lots of cell divisions
- The cells were pigmented. ➔ we kijken van bovenaf dus we zagen de animal pole
(AP). OPGELET: AP kan ook anterior posterior betekenen. Onderaan heb je vegetal
pole (VP).
- Initially very fast differentiation
- From 1 to 37.000 cells (43h) (mouse and human: 1 to 4 cells in 48 hours)
- Frogs have to be independent very quickly. Eggs in the water, they have enemies and
can be eaten→ a lot of pressure to develop fast. In the frog embryo, the frog genome
becomes activated much later. The first stages the embryo functions on the maternal
transcripts and proteins. That’s the case for frogs, reptiles, etc. In a short time period
these eggs need to become larvae.
The somatic cell cycle is around 16 hours:
- G1
- S
- G2
- M
The cleavage cycle: early in the cell cycle (30 minutes) it only consists of a S and M phase.
There is no time to synthetize proteins. Every 30 minutes a cell divides. Later on, this
lengthens and slows down. The cleavage cycle is specific to early embryonic development,
especially during the first few divisions after fertilization. It's a rapid process, with short
intervals between rounds of cell division.
Cleavage cycle:
- Gap phases (G1 and G2) are omitted
- Cell cycles rapidly between M and S phases: extremely short cell cycle.
Complete cleavages (cells divide completely), upper part is less heavy so the cleavage
proceeds faster→size of the cells is not equal anymore = MESOLECITHAL CLEAVAGE
(holoblastic= complete)
In just 12 hours you end up with 50.000 cells for a fruit fly. There are two types of divisions:
- Symmetric: cells stay within same cell fate = two cells of the same kind
- Asymmetric divisions: you usually get two different cell types.
9
, Animal pole is on the left side. The yolk = bag filled with nutrients = to feed the developing
embryo. The animal cells develop at one side, no division takes place. The animal cells are
pushed to one side in the zebrafish embryo.
There are also different types of cleavages:
- Holoblastic = complete cleavages.
- Meroblastic = The eggs are stiffed with proteins etc. and the cleavages are thereby
stalled and thus incomplete.
ZEBRAFISH
- Embryo develops really as a disc on top of the yolk = discoidal cleavage ( Telolecithal )
- Incomplete cleavage = MEROBLASTIC CLEAVAGE
FRUITFLY
- Syncytium: nuclei will divide but membrane will not divide→lots of nuclei in 1 cell
that will be organized in the outer area of the embryo
- Yolk in the center of egg = CENTROLECITHAL CLEAVAGE
Cells in the cleavage stage are called blastomeres. Cleavage is controlled by maternal factors
and cytoplasmic volume does not increase. Rather, the zygote cytoplasm is divided into even
smaller cells. The embryo accomplishes cleavage by abolishing the gap periods of the cell
cycle (G1 and G2 phase). The pattern of embryonic cleavage is determined by 2 major
parameters:
1. 1) The amount and distribution of yolk protein within the cytoplasm, which
determine where cleavage can occur and the relative sizes of the blastomeres
2. 2) Factors in the egg cytoplasm that influence the angle of the mitotic spindle and
the timing of its formation
When one pole of the egg is relatively yolk-free, cellular divisions occur there at a faster rate
than at the opposite pole. The yolk-rich pole = the vegetal pole. The yolk concentration in
the animal pole is relatively low. The zygote nucleus is frequently displaced toward the
animal pole.
10
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