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Genetics > Development and Gene Regulation > Flashcards

Development and Gene Regulation Flashcards

1
Q

What are the stages of embryogenesis?

A
  1. Fertilisation
  2. Cleavage
  3. Gastrulation and Patterning
  • Cell rearrangement
  • Basic body plan established
  1. Organogenesis

• Cells differentiate to organs and tissue

  1. Metamorphosis

• Cells become progressively organised into organs and tissue of the organism

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2
Q

What does a cells position within the embryo determine?

A

• Determines specific identity, morphology, physiology of cell

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3
Q

What is patterning?

A
  • 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)
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4
Q

How can gene activity within an embryo be detected? Which probes are used and when?

A

• RNA in situ hybridisation

o Probe is ss nucleic acid (RNA) with labelled nucleotide

• Immunolocalisation (protein)

o Probe is antibody

    1. Fixation
    1. Hybridisation
    1. Wash
    1. Visualisation
  • Different expression in different cells = different identities
  • Gene expression can be differential even before cells have differentiated
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5
Q

What are morphogens and how can they be studied genetically?

A
  • 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

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6
Q

How is positional information conveyed during embryogenesis according to Wolpert?

A

• Via morphogens

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7
Q

What are master regulators? What happens if they mutate?

A
  • 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

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8
Q

What is eyeless in drosophila? What happens if miss expression occurs? Transcription factor/master regulator

A

• Ectopic expression = wrong time, wrong place (eyes everywhere)

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9
Q

Which types of organisms should be used for studying development?

A
  • 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)

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10
Q

What are the differences between mutations in zygotic-acting genes and maternal-acting genes?

A

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

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11
Q

What is a zygotic acting gene?

A

• Gene only active in zygote

o Follows fertilisation

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12
Q

What kinds of zygotic mutants can be identified in drosophila larvae?

A
  1. Gap
    * Missing contiguous segments along A-P axis
  2. Pair-rule
    * Missing alternating segments along A-P axis
  3. Segment polarity
  • Each segment missing from anterior or posterior region
  • E.g. lose anterior part of each segment
  1. Dorso-ventral
  • Lose or gain denticle belts
  • Denticle belts on ventral side
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13
Q

What is a maternal acting gene? How can they be identified in drosophila larvae?

A
  • Gene expressed in maternal tissue and active in zygote
  • Genotype of mum determines phenotype of offspring
  • Hetero mum = viable offspring
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14
Q

What are the features of bicoid, nanos and torso mutants? What do they each lack and which axis do they affect?

A
  • 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

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15
Q

What are the steps in drosophila embryogenesis and what makes it unusual? What is syncytium?

A
  1. Fertilisation
  2. Rapid, synchronous nuclear divisions
  • NOT cellular divisions (unlike frogs)
  • Same cell, lots of nuclei in cytoplasm (syncytium)
  1. Nuclei migration to periphery (syncytial blastoderm)
  2. Cell membranes form around nuclei (cellular blastoderm)
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16
Q

What is the action of bicoid protein?

A
  • Maternal effect gene, encodes transcription factor
  • Lots of it expressed at anterior (more concentration)
  • Acts like morphogen (move concentration = move development)
17
Q

How can bicoid morphogen be used with zygotic hunch back mutants? What are the features of the hb protein? How is hb expression controlled?

A
  • 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

18
Q

What does D-V patterning involve? What do spatzle, toll and dorsal encode? How do they interact?

A
  • Maternal effect mutations
  • Cell signalling pathway
  • Spatzle

o Secreted ligand

• Toll

o Transmembrane spanning receptor

• Dorsal

o Transcription factor

    1. Spatzle ligand forms concentration gradient (high at ventral, low at dorsal)
    1. Forms gradient of activated Toll receptors
    1. Forms gradient of intracellular signalling
    1. Forms differential gene activation
  • Level of signalling determines pattern of gene regulation (lots of expression ventrally, that’s where denticles form)
19
Q

What are the links between morphogens and pre-axial polydactyl?

A
  • Morphogen makes digits grow from posterior part of limb bud
  • Sonic hedgehog (shh) = morphogen for A-P polarity of limb and digits
20
Q

What is ectopic expression?

A

• 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

21
Q

What are pair rule genes? What happens to mutants?

A
  • Formation of alternating segments
  • Mutants have lost alternating segments (odd or even skipped)
  • Expressed in periodic pattern (every second segment, 7 alternating stripes)
22
Q

What is eve? What does it encode and how does it relate to stripe regulation?

A
  • 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
23
Q

What transcription factors activate and repress gene expression for stripes?

A
  • Activators : bicoid and hunchback
  • Repressors: Gap genes (Kruppel and giant)
24
Q

What controls stripe expression?

A
  • 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

25
Q

What is homeosis? What are the features of homeotic genes?

A
  • 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
26
Q

What are the homeotic genes for segment identify in drosophila and what mutations are possible?

A

• Bithorax cluster

o Posterior to anterior changes

• Antennapedia cluster

o Anterior to posterior changes

27
Q

What are hox genes? What structure do they have?

A
  • 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

28
Q

What factors establish segment and segment identity (patterning)?

A
  • Maternal morphogens (Broad regions)
  • Gap Genes (smaller regions)
  • Pair-rule genes (individual segments)
  • Hox genes (segment identify)
29
Q

What are the features of vertebrate hox genes?

A
  • 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

Genetics (3 decks)

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