Developmental Revision Flashcards
4 developmental problems
- Regional specification - generation of a pattern
- Cell differentiation
- Morphogenesis - movement of cells and tissues
- Growth
Mutation in single copy of PAX6?
Mutation in both copies of PAX6?
- Aniridia (loss of iris)
- Eyes are completely absent with severe brain defects (lethal)
Is PAX6 function sufficient and required fro eye development?
Yes, PAX6 is both sufficient and required for eye development
What is both sufficient and required for egg activation after fertilisation?
Increase in Ca2+
Organisms that undergo holoblastic cleavage?
- 4 organsims
- C. Elegans
- Xenopus
- Mouse
- Humans
Organisms that undergo meroblastic cleavage?
- 2 organsims
- Chick
- Zebrafish
Organism that undergoes superficial cleavage?
Drosophila
4 things that drive tissue rearrangements?
- Cell shape changes
- Localised cell proliferation
- Localised cell death
- Change in expression of cell-surface receptors
8 types of tissue rearrangements (explain them)
- Condensation
- Epithelium to mesenchymal transition
- Mesenchymal to epithelium transition
- Involution
- Invagination
- Cavitation
- Epiboly
- Convergent extension
Useful techniques of C. Elegans
- Cell ablation
- Mutagenesis
Cell ablation in C. Elegans
- 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
Useful techniques of Zebrafish
- Mutagenesis
- Cell transplantation
- Injections
- Transgenesis
- Targeted knockouts
What is a transgenic organism?
- Unstable transgenic
- Stable transgenic
- An organism carrying a gene that has been incorporated into its genome using recombinant DNA
- Unstable - extrachromosomal expression
- Stable - permanently integrated into genome
Useful techniques of mouse?
- Mutagenesis
- Targeted knockouts via ES cells
Useful techniques of Drosophila?
- Mutagenesis
- Transgenesis
- Clonal analysis
Pole cell transplantation in Drosophila
- 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
2 types of genes that affect development of Drosophila egg (imaginal discs)
- Maternal effect genes - determine polarity of egg (bicoid)
- Zygotic genes - responsible for body plan of embryo
Useful techniques of Xenopus
- Injections
- Transgenesis
- Tissue transplantation
Useful techniques of Gallus Gallus?
- Tissue transplantation
- Retroviral injection
- Electroporation
Cell fate
Is what a cell will normally become in development
Fate maps are used to…
Identify the normal fates of cells
4 methods of producing a fate map?
- 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
Commitment - Specification is…
Reversible
Commitment - Differentiation is…
Irreversible
Comparison of fate map with specification map shows that…
Cells need to receive signals as they are not specified at the blastula stage
Mosaic development
Cells are determined early
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
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
What is induction?
Signalling from one cell type to another, with a change in specification of the receiving cell
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
What is competence?
- The ability to respond to a signal
- More cells are competent to respond than actually do so
Xenopus Animal Cap Assay
- Animal cap cells are induced by vegetal tissue
- Animal cap is competent to respond to this induction and become mesoderm
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
Bicoid gradient in drosophila embryo?
- Highest at anterior end and decreases towards posterior end
Bicoid codes for transcription factor that activates…
What does this activate
- Hunchback transcription
- Activates cascade of of gene expression and leads to segmentation
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
What is redundancy?
When one gene takes over from dominant gene to undertake same job
Forward genetic analysis
- Starts with a mutant phenotype to identify the mutated gene
- In homozygous recessive
Reverse genetic analysis
- Starts with a gene sequence to identify what phenotype arises
- In homozygous recessive
Semi-dominant
An intermediate phenotype between dominant and recessive phenotypes seen in a heterozygote
Haplosufficient
One wild type allele is sufficient for normal development
Haploinsufficient
One wild type allele is insufficient for normal development
Embryonic lethal
- Causes death in the homozygote
- Phenotype common in homozygous knockout mice
Null (amorph) mutation
Complete loss of protein function
Hypomorph mutation
Partial loss of protein function
Hypermorph mutation
- Overexpression of gene
- Gain in protein function
Dominant negative (antimorph) mutation
Mutant copy interferes with function of wild type protein function
Neomorph mutation
Protein acquires new function
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
Complementing mutations are in…
Different genes
Non-complementing mutations are in…
The same gene
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
Steel mutant
No steel factor produced = no pigment cell produced
Kit mutant
No steel factor receptor produced = no pigment cell produced
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
Phenotype of
- Hh mutants
- Smoothened mutants
- Wingless mutants
All loss of naked cuticles
Phenotype of
- Patched mutants
Excess naked cuticles
Cell fates in Drosophila
- Dorsal ectoderm express
dpp gene
Cell fates in drosophila
- Neuroectoderm express
rhomboid gene
Cell fates in drosophila
- Mesoderm express
Twist and Snail
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
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
Dorsal
Transcription factor
- Activates transcription of mesodermal genes (Twist, Snail and Rhomboid)
- Represses transcription of zygotic genes (dpp)
- Acts a morphogen
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
Autonomous function
Mutations that affect the cells in which it is present
Non-autonomous function
Mutations that affect other cells
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
Homeobox
Codes for a homeodomain which is a sequence specific DNA binding motif
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
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
HOX genes
- Specify segment identity in animals with segmented body plan
- E.g. determine site of limb bud formation
Site of limb bud determined by…
HOX genes
- HoxB5 knockout mouse forelimbs develop at a more anterior level
HOX genes restrict expression of…
Where is expression restricted to?
- Fibroblast Growth Factors (FGFs)
- Limb forming regions
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
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
Manipulation of AER
- Removal of AER results in truncated limbs
- AER can be replaced with beads soaked in FGFs to give normal development
Manipulation of PZ
- Length of time spent in PZ determines cell fate
- Cells that are first to leave PZ form proximal parts of limb
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
Proneural cluster cell fates
- One cell becomes a neuroblast (primary cell fate)
- Other cells become epidermal cells (secondary cell fate)
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
Recessive mutations in Delta or Notch result in…
Lots of neuroblasts and no epidermis
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
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
4 cells of sensory bristle
- Hair cell
- Sheath cell
- Socket cell
- Neurone
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
How does numb and notch interact?
Numb inhibits notch function
Loss of Notch function results in…
4 neurone cells
Loss of Numb function results in…
4 socket cells
