1
Q
4 developmental problems
A
- Regional specification - generation of a pattern
- Cell differentiation
- Morphogenesis - movement of cells and tissues
- Growth
2
Q
Mutation in single copy of PAX6?
Mutation in both copies of PAX6?
A
- Aniridia (loss of iris)
- Eyes are completely absent with severe brain defects (lethal)
3
Q
Is PAX6 function sufficient and required fro eye development?
A
Yes, PAX6 is both sufficient and required for eye development
4
Q
What is both sufficient and required for egg activation after fertilisation?
A
Increase in Ca2+
5
Q
Organisms that undergo holoblastic cleavage?
- 4 organsims
A
- C. Elegans
- Xenopus
- Mouse
- Humans
6
Q
Organisms that undergo meroblastic cleavage?
- 2 organsims
A
- Chick
- Zebrafish
7
Q
Organism that undergoes superficial cleavage?
A
Drosophila
8
Q
4 things that drive tissue rearrangements?
A
- Cell shape changes
- Localised cell proliferation
- Localised cell death
- Change in expression of cell-surface receptors
9
Q
8 types of tissue rearrangements (explain them)
A
- Condensation
- Epithelium to mesenchymal transition
- Mesenchymal to epithelium transition
- Involution
- Invagination
- Cavitation
- Epiboly
- Convergent extension
10
Q
Useful techniques of C. Elegans
A
- Cell ablation
- Mutagenesis
11
Q
Cell ablation in C. Elegans
A
- If anchor cell is removed there is no vulva formation
- If anchor cell is mutated there is no vulva or multivulva formation
- Shows anchor cell is involved in vulva formation
12
Q
Useful techniques of Zebrafish
A
- Mutagenesis
- Cell transplantation
- Injections
- Transgenesis
- Targeted knockouts
13
Q
What is a transgenic organism?
- Unstable transgenic
- Stable transgenic
A
- An organism carrying a gene that has been incorporated into its genome using recombinant DNA
- Unstable - extrachromosomal expression
- Stable - permanently integrated into genome
14
Q
Useful techniques of mouse?
A
- Mutagenesis
- Targeted knockouts via ES cells
15
Q
Useful techniques of Drosophila?
A
- Mutagenesis
- Transgenesis
- Clonal analysis
16
Q
Pole cell transplantation in Drosophila
A
- Cells taken from posterior end of one embryo and transplanted to anterior end of second embryo
- Cells retained pole cell function
- Cells then transplanted into posterior end of third embryo and adult fly develops with germ cells with genotype of second embryo amongst its own
17
Q
2 types of genes that affect development of Drosophila egg (imaginal discs)
A
- Maternal effect genes - determine polarity of egg (bicoid)
- Zygotic genes - responsible for body plan of embryo
18
Q
Useful techniques of Xenopus
A
- Injections
- Transgenesis
- Tissue transplantation
19
Q
Useful techniques of Gallus Gallus?
A
- Tissue transplantation
- Retroviral injection
- Electroporation
20
Q
Cell fate
A
Is what a cell will normally become in development
21
Q
Fate maps are used to…
A
Identify the normal fates of cells
22
Q
4 methods of producing a fate map?
A
- Observation
- Natural pigmentation
- Label/Marker - look for progeny of original cell that have label/marker
- Orthotopic transplantation - cells taken from labelled organism and implanted in similar place in another embryo
23
Q
Commitment - Specification is…
A
Reversible
24
Q
Commitment - Differentiation is…
A
Irreversible
25
Comparison of fate map with specification map shows that...
Cells need to receive signals as they are not specified at the blastula stage
26
Mosaic development
Cells are determined early
27
Autonomous and Non-autonomous mechanism of Regulative development
- Autonomous mechanism - intrinsic factors make cells different e.g. segregation of cytoplasmic contents
- Non-autonomous mechanism - extrinsic factors make cells different e.g. signalling molecules
28
How does the french flag model account for regulative development?
- If length of line varies (more or less cells) the pattern will regulate
- If the line is cut in half the system will regenerate
29
What is induction?
Signalling from one cell type to another, with a change in specification of the receiving cell
30
What was the Amphibian Organiser experiment?
- Blastopore lip was grafter onto the ventral side of another embryo
- This induced formation of a second axis in the embryo and the tissue was derived from the host not the graft
31
What is competence?
- The ability to respond to a signal
| - More cells are competent to respond than actually do so
32
Xenopus Animal Cap Assay
- Animal cap cells are induced by vegetal tissue
| - Animal cap is competent to respond to this induction and become mesoderm
33
Cap cells are
- Fated to become...
- Specified to become...
- Induced to become...
- Fated to become neural cells
- Specified to become epidermis
- Induced to become muscle cells
34
Bicoid gradient in drosophila embryo?
- Highest at anterior end and decreases towards posterior end
35
Bicoid codes for transcription factor that activates...
| What does this activate
- Hunchback transcription
| - Activates cascade of of gene expression and leads to segmentation
36
Is MyoD sufficient and required to direct muscle fate?
- MyoD is sufficient to direct muscle fate - fibroblasts differentiate into muscle when transfected with MyoD
- MoD is NOT required to direct muscle fate - mice lacking MyoD still develop skeletal muscle, Myf5 acts redundantly with MyoD
37
What is redundancy?
When one gene takes over from dominant gene to undertake same job
38
Forward genetic analysis
- Starts with a mutant phenotype to identify the mutated gene
- In homozygous recessive
39
Reverse genetic analysis
- Starts with a gene sequence to identify what phenotype arises
- In homozygous recessive
40
Semi-dominant
An intermediate phenotype between dominant and recessive phenotypes seen in a heterozygote
41
Haplosufficient
One wild type allele is sufficient for normal development
42
Haploinsufficient
One wild type allele is insufficient for normal development
43
Embryonic lethal
- Causes death in the homozygote
| - Phenotype common in homozygous knockout mice
44
Null (amorph) mutation
Complete loss of protein function
45
Hypomorph mutation
Partial loss of protein function
46
Hypermorph mutation
- Overexpression of gene
| - Gain in protein function
47
Dominant negative (antimorph) mutation
Mutant copy interferes with function of wild type protein function
48
Neomorph mutation
Protein acquires new function
49
Process of Mutagenesis
- Expose adult male to mutagen
- Breed mutations through homozygosity
- Screen for interesting phenotypes
- Complementation testing to assign mutations to loci
- Maintain stock of heterozygous carriers
50
Complementing mutations are in...
Different genes
51
Non-complementing mutations are in...
The same gene
52
In a complementation test, when 2 different mutants are crossed:
- If no mutant phenotypes are seen, the genes...
- If 25% show the mutant phenotype, the genes...
- No mutant phenotype = Complement each other
| - 25% mutant phenotype = Do not complement each other
53
Steel mutant
No steel factor produced = no pigment cell produced
54
Kit mutant
No steel factor receptor produced = no pigment cell produced
55
Epistasis analysis
- Cross between heterozygote mutants to examine the double mutant
- Phenotype of one mutant masked by the other = recessive epistasis
- Can be used to determine the order in which genes act
- Epistatic genes usually act downstream
56
Phenotype of
- Hh mutants
- Smoothened mutants
- Wingless mutants
All loss of naked cuticles
57
Phenotype of
| - Patched mutants
Excess naked cuticles
58
Cell fates in Drosophila
| - Dorsal ectoderm express
dpp gene
59
Cell fates in drosophila
| - Neuroectoderm express
rhomboid gene
60
Cell fates in drosophila
| - Mesoderm express
Twist and Snail
61
Dorsalised mutants
- Excess of dorsalised material
- Decreased ventralised material
- Toll, Spätzle and Dorsal mutants
- Dorsal cannot enter nucleus to activate mesodermal genes (twist, snail and rhomboid)
- Zygotic gene (dpp) is expressed throughout embryo and all cells adopt a dorsal fate
62
Ventralised mutant
- Decreased dorsalised material
- Excess of ventralised material
- Cactus mutant
- Dorsal enters nucleus of all cells across DV axis to activate mesodermal genes (twist, snail and rhomboid) throughout the embryo
- Zygotic gene (dpp) is repressed throughout embryo and all cells adopt a ventral fate
63
Dorsal
Transcription factor
- Activates transcription of mesodermal genes (Twist, Snail and Rhomboid)
- Represses transcription of zygotic genes (dpp)
- Acts a morphogen
64
Homeotic transformation (2 examples)
One structure is replace by another
- Recessive mutations in Ubx gene cause transformation of 3rd Thoracic segment (haltere) into 2nd Thoracic segment (wings)
- Recessive mutations in Abd-B gene cause transformation of abdominal segments A4, A5, A6 into A4, A4, A5
65
Autonomous function
Mutations that affect the cells in which it is present
66
Non-autonomous function
Mutations that affect other cells
67
Selector genes
- Cells in different segments interpret positional information differently due to activity of selector genes
- Ubx acts as selector gene to direct cells down 3rd thoracic segment pathway
68
Homeobox
Codes for a homeodomain which is a sequence specific DNA binding motif
69
Bithorax Complex (BX-C)
- Consists of Ubx, Abd-A and Abd-B
- Arrangement of genes reflects order in which they function along AP axis
- Contain homeobox (highly conserved DNA sequence)
- Expression of Bithorax is spatially restricted
70
Antennapaedia Complex (ANT-C)
- Contains homeobox genes that control development of head and thorax
- Antennapedia gene is expressed in T2 segment
- Dominant mutation in Antennapedia gene causes transformation of antenna to legs
71
HOX genes
- Specify segment identity in animals with segmented body plan
- E.g. determine site of limb bud formation
72
Site of limb bud determined by...
HOX genes
| - HoxB5 knockout mouse forelimbs develop at a more anterior level
73
HOX genes restrict expression of...
| Where is expression restricted to?
- Fibroblast Growth Factors (FGFs)
| - Limb forming regions
74
Is FGF sufficient and required for limb bud formation?
- FGF signalling is required - FGFR knockout mice embryos lack limb buds
- FGF signalling is sufficient - Local application of FGF4 protein results in ectopic limb buds
75
3 regions of limb bud and function
- Apical Ectodermal Ridge (AER) - maintains the progress zone
- Progress Zone (PZ) - length of time spent in PZ determines cell fate
- Zone of Polarising Activity (ZPA) - patterns AP axis of limb
76
Manipulation of AER
- Removal of AER results in truncated limbs
| - AER can be replaced with beads soaked in FGFs to give normal development
77
Manipulation of PZ
- Length of time spent in PZ determines cell fate
| - Cells that are first to leave PZ form proximal parts of limb
78
Manipulation of ZPA
- When transplanted from posterior limb bud to under anterior AER, results in symmetrical duplications of normal limb
- Shh is expressed in ZPA
- ZPA can be replaced with beads soaked in Shh to give normal development
79
Proneural cluster cell fates
- One cell becomes a neuroblast (primary cell fate)
| - Other cells become epidermal cells (secondary cell fate)
80
In a pro neural cluster:
- Cell displaying lots of Delta becomes...
- Cells displaying lots of Notch become...
- One cells displays lots of Delta -> Neuroblast
| - Other cells display lots of Notch -> Epidermis
81
Recessive mutations in Delta or Notch result in...
Lots of neuroblasts and no epidermis
82
How do neuroblasts form neurones?
- Neuroblasts delaminate from neuroectoderm and undergo asymmetrical division to form another neuroblast and a ganglion mother cell
- Ganglion mother cell divides again to form cells that differentiate into neurones
83
Short range cues for axon growth cone...
| Long range cues for axon growth cone...
- Short range - cell-cell contacts (surface markers)
- Long range - diffusible molecules
Can be attractive or repulsive signals
84
4 cells of sensory bristle
- Hair cell
- Sheath cell
- Socket cell
- Neurone
85
What cell gives rise to cells of sensory bristle?
| Where do they arise from?
- Sensory Organ Precursor (SOP) cells
| - SOP cells are singled out in pro neural clusters
86
How does numb and notch interact?
Numb inhibits notch function
87
Loss of Notch function results in...
4 neurone cells
88
Loss of Numb function results in...
4 socket cells
BMS 109
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