Development and Gene Regulation Flashcards
What are the stages of embryogenesis?
- Fertilisation
- Cleavage
- Gastrulation and Patterning
- Cell rearrangement
- Basic body plan established
- Organogenesis
• Cells differentiate to organs and tissue
- Metamorphosis
• Cells become progressively organised into organs and tissue of the organism
What does a cells position within the embryo determine?
• Determines specific identity, morphology, physiology of cell
What is patterning?
- Cells perceive position
- At differentiation, discrete sets of genes expressed according to position of cell
- Conveys positional information leading to differential gene expression (transcriptional changes)
How can gene activity within an embryo be detected? Which probes are used and when?
• RNA in situ hybridisation
o Probe is ss nucleic acid (RNA) with labelled nucleotide
• Immunolocalisation (protein)
o Probe is antibody
- Fixation
- Hybridisation
- Wash
- Visualisation
- Different expression in different cells = different identities
- Gene expression can be differential even before cells have differentiated
What are morphogens and how can they be studied genetically?
- Concentration varies according to distance from source
- Continuous gradient converted to discrete pattern of cell fate (uses threshold points)
- Different genes turned on in response to concentration of morphogen
- Minuscule amounts in embryo
- Hard to biochemically purify
- Studying via Genetic approach
o Easy to see mutations affecting development
o Don’t need to assume about number/nature of molecules involved
o Study developmental events lacking biochemical assay
How is positional information conveyed during embryogenesis according to Wolpert?
• Via morphogens
What are master regulators? What happens if they mutate?
- Transcription factors
- Activated specifically depending on location
- Determine specific patterns of gene expression
- Mutations significantly impact development
- Loss of function mutation
o Default gene expression (e.g. no head)
• Gain of function mutation
o Ectopic expression/miss-expression
o E.g. eyeless in drosophila
• Development not determine by one master regulator. Uses activity of multiple or redundant regulatory factors
What is eyeless in drosophila? What happens if miss expression occurs? Transcription factor/master regulator
• Ectopic expression = wrong time, wrong place (eyes everywhere)
Which types of organisms should be used for studying development?
- Easy to cultivate, rapid reproduction, small, sexual cycle
- E.g. Drosophila
o Embryo develops externally
o Rapid lifecycle
o Segmented body (easy to see mutations)
o Exoskeleton of larva has anatomical landmarks (head, dentricle belts, tail)
What are the differences between mutations in zygotic-acting genes and maternal-acting genes?
Zygotic-Acting Gene Mutation
o Phenotype reflects genotype
o E.g. w/w X W/w = 50% of each in reciprocal crosses
Maternal-Acting Gene Mutation
o Maternal genotype determines progeny phenotype (MATERNAL EFFECT)
o E.g. w/w mum leads to wingless progeny regardless of dad
o E.g. W/w mum leads to winged progeny regardless of dad
What is a zygotic acting gene?
• Gene only active in zygote
o Follows fertilisation
What kinds of zygotic mutants can be identified in drosophila larvae?
- Gap
* Missing contiguous segments along A-P axis - Pair-rule
* Missing alternating segments along A-P axis - Segment polarity
- Each segment missing from anterior or posterior region
- E.g. lose anterior part of each segment
- Dorso-ventral
- Lose or gain denticle belts
- Denticle belts on ventral side
What is a maternal acting gene? How can they be identified in drosophila larvae?
- Gene expressed in maternal tissue and active in zygote
- Genotype of mum determines phenotype of offspring
- Hetero mum = viable offspring
What are the features of bicoid, nanos and torso mutants? What do they each lack and which axis do they affect?
- Maternal Effect Mutants
- A-P axis
- Anterior – bicoid
o Mutant = no head
• Posterior – nanos
o Mutant = no abdomen
• Terminal – torso
o Mutant = no terminal structures
What are the steps in drosophila embryogenesis and what makes it unusual? What is syncytium?
- Fertilisation
- Rapid, synchronous nuclear divisions
- NOT cellular divisions (unlike frogs)
- Same cell, lots of nuclei in cytoplasm (syncytium)
- Nuclei migration to periphery (syncytial blastoderm)
- Cell membranes form around nuclei (cellular blastoderm)
What is the action of bicoid protein?
- Maternal effect gene, encodes transcription factor
- Lots of it expressed at anterior (more concentration)
- Acts like morphogen (move concentration = move development)
How can bicoid morphogen be used with zygotic hunch back mutants? What are the features of the hb protein? How is hb expression controlled?
- Gap gene: Hunchback (Hb) = loss of anterior segments
- More hb expressed anterior (follows bicoid gradient)
- Bicoid binds hb promoter
- 5’ cis-acting regulatory element with 3 bicoid binding sites
- Regulates hb expression
- Amount of expression determined by occupancy within cic-acting regulatory site
- More sites bound with bicoid (3 max) = more expression
- Bicoid concentration below threshold = no hb expression
- Cis-acting regulatory element functions as switch responding to certain threshold concentrations of bicoid What allows distinct gap gene activation?
- Multiple gap genes which are expressed in different areas
o Cis-acting regulatory sequences control expression
o Different bind site arrangements
o different bicoid protein affinities
o Gap genes activated at different threshold concentrations
• Bicoid activates zygotic gap genes in distinct domains along A-P axis
What does D-V patterning involve? What do spatzle, toll and dorsal encode? How do they interact?
- Maternal effect mutations
- Cell signalling pathway
- Spatzle
o Secreted ligand
• Toll
o Transmembrane spanning receptor
• Dorsal
o Transcription factor
- Spatzle ligand forms concentration gradient (high at ventral, low at dorsal)
- Forms gradient of activated Toll receptors
- Forms gradient of intracellular signalling
- Forms differential gene activation
- Level of signalling determines pattern of gene regulation (lots of expression ventrally, that’s where denticles form)
What are the links between morphogens and pre-axial polydactyl?
- Morphogen makes digits grow from posterior part of limb bud
- Sonic hedgehog (shh) = morphogen for A-P polarity of limb and digits
What is ectopic expression?
• Ectopic shh expression
o Wrong place
o Wrong time
o Anterior
o Mutation in cis-acting regulatory element 1mb from shh
• Ectopic expression often dominant mutation
o Single nucleotide change, disrupt gene expression
What are pair rule genes? What happens to mutants?
- Formation of alternating segments
- Mutants have lost alternating segments (odd or even skipped)
- Expressed in periodic pattern (every second segment, 7 alternating stripes)
What is eve? What does it encode and how does it relate to stripe regulation?
- eve= even skipped (mutant loses odd stripes)
- eve promoter
- Expression of stripes determined by cis-acting regulatory elements
- Each stripe independently regulated (turned on one by one)
- Cis-acting regulatory elements binding sites for multiple transcription factors
What transcription factors activate and repress gene expression for stripes?
- Activators : bicoid and hunchback
- Repressors: Gap genes (Kruppel and giant)
What controls stripe expression?
- Combinatorial control
- Stripe expression determined by combinatorial control
- Combination of activators and repressors
- E.g. stripe 2 because either side have lots of gap gene (repressor) expression
- Pair rule enhancers = molecular switches active at certain positions along A-P axis
- Independent regulation
o Enhancers respond to different sets of transcription factors along axis
• Combinatorial control
o Combination of activators and repressors determine transcriptional activity of each enhancer
What is homeosis? What are the features of homeotic genes?
- Replacement of one body part/segment with another
- Homeotic mutants
- Dominant mutation
- Single mutation, drastic effect on development
- Affect repeated structures
- Does not affect segment number
What are the homeotic genes for segment identify in drosophila and what mutations are possible?
• Bithorax cluster
o Posterior to anterior changes
• Antennapedia cluster
o Anterior to posterior changes
What are hox genes? What structure do they have?
- Homeotic Genes (Hox genes)
- Master regulators of segment identity
- Same chromosome
o Clusters
o Bithorax cluster
o Antennapedia cluster
• Gene order = body region order affected by each mutation
o 5’ anterior
o 3’ posterior
• Hox genes as transcription factors
o Conserved region in both clusters (180 bp HOMEOBOX)
o Homeobox has 60AA DNA-binding homeo domain (binds DNA)
o Transcriptional regulation for segment identity
o Expressed in imaginal discs
o Regulate larval segment identity
o Expression = co linear with gene arrangement on chromosome
o Each segment/imaginal disc expresses specific combination of hox genes
What factors establish segment and segment identity (patterning)?
- Maternal morphogens (Broad regions)
- Gap Genes (smaller regions)
- Pair-rule genes (individual segments)
- Hox genes (segment identify)
What are the features of vertebrate hox genes?
- Almost identical AA in homeodomain
- Present in clusters
- Clusters may be duplicated
- Similar genes in clusters have same function
- Pattern of expression = co-linear
- Specify identity of structures along A-P axis
