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Dev Bio Flashcards
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- 1AniridiaAutosomal dominant
Lack of iris, decreased vision, cataracts
PAX 6 gene mutation
Homozygous perinatal lethal - 2Fertilisation1) Sperm binds to zona pellucida
2) Triggers acrosomal reaction
3) Sperm penetrates z.p. & binds to egg plasma membrane - causes release of cortical granules
4) Sperm nucleus enters egg cytoplasm - 3Blocks to polyspermy in sea urchin1) Change in membrane potential (-70mV - +20mV) so sperm can no longer bind to egg membrane
2) Cortical reaction - formation of fertilisation and hyaline layer that stops more sperm entering - 4Egg ActivationCa2+ release triggered by sperm entry
Egg completes meiosis, pronuclei fuse
Ca2+ acts on proteins that control cell cycle and initiate cleavage
Increase in Ca2+ both necessary and sufficient for egg activation - 5Cell cycle in early embryoCleavage divisions rapid and synchronous
S and M phases only, no growth phases
Zygotic genome transcriptionally silent
Maternal stores of mRNA and proteins run out
Zygotic genome activation (ZGA) occurs
Cell cycle slows, asynchronous, movement begins
If maternal mRNA has mutation, egg development is impacted - 6How do cells become different?1) Segregation of cytoplasmic components (eg. setting aside of germline - P granules)
2) Cell-cell signalling - 7EctodermNeural plate
Neural crest
Placodes (sensory ganglia)
Epidermis - 8MesodermMuscle
Cartilage
Bone
Kidneys
Heart and Blood
Dermis - 9EndodermGut
Pancreas
Lungs (& airways)
Liver
Salivary Glands - 10GastrulationMain principle:
1) Mesoderm and endoderm move inside embryo
2) Formation of a gut
3) Establishment of anteroposterior and dorsoventral axes - 11Forces that drive cell and tissue rearrangementsCell shape changes caused by cytoskeletal rearrangements
Changes in expression of cell surface proteins (cell adhesion molecules)
Localised cell proliferation and cell death - 12NeurulationOccurs in vertebrates after gastrulation
Formation of neural tube (will become spinal cord) - 13Ideal characteristics for genetic analysisSmall organism
Large numbers of embryos
Short generation time
Easy to breed
Sequenced genome
Easy scoring of phenotypes (know whether they are developing properly) - 14Drosophila: Transplantation of pole plasmDonor posterior pole plasm into anterior of second embryo
Anterior cells transplanted into posterior region of third embryo
Adult fly of 3rd embryo has some germ cells with same genotypes as 1st embryo - 15C. elegansInvariant lineage - cell division patterns and fates same in all individuals
Can be manipulated genetically and physically (laser ablation) - 16Laser ablation of anchor cell in C. elegansNo vulva tissue, all cells epidermis (default?)
Mutations:
1) No vulva
2) Mutlivulva - shows all epidermal cells are capable of making vulva cells - 17cls mutant of zebrafishAll three pigment types of neural crest are missing:
Ear defects
Swim bladder uninflated
Enteric neurones missing
Colourless
Model for Waardenburg's Syndrome - 18Waardenburg's SyndromeHearing loss
Pigmentary abnormalities
Hirschprung's Disease (constipation in babies)
Mutations in human SOX 10 gene
Due to problems in neural crest - 19Physical manipulation of embryoSea urchin, xenopus and chick
Embryo must be:
large,
robust,
resistant to infection - 20Isolating blastomere in sea urchin (Driesch, 1892)2 cell embryo separated
1 cell died
the other survived and gave rise to smaller blastula and larva
Can do this on 4 cell staged blatomere but not on 8 cell stage - 21Amphibian organiser (Spemann & Mangold, 1924)Dorsal lip of blastopore grafted to blastocoel roof
Secondary embryo is induced -> can induce development pathway in the cells of the 'host' embryo - 22Chicken developmentInternal fertilisation - cannot see early stages of development
Gastrulation:
1) One layer of cells forms on top of yolk - forms primitive streak
2) Cells move to make more layers
3) Cells roll up into tube, forms the body plan - 23FateWhat cells will normally become in development
- 24Fate mapA map of structures present in a given stage back onto an embryo at an earlier stage
Can be done by looking under a microscope at (fluorescently labelled) cells - 25Orthotopic transplantationTransplantation of cells to the same place in the embryo
- 26Ideal marker for a fate map Characteristics?Applied at any region, at any time
Readily visible at later stages
Doesn't perturb normal development
Doesn't leaking into neighbouring cells
All progeny of labelled cells inherit it
Not diluted on cell division - 27Useful markers for fate mappingVital dyes
High molecular weight tracers
GFP
Cytological labels - 28Clonal analysisShows what fate a cell can give rise to
Show lineage of final cell types - 29CommitmentCommitment to a fate can be:
Reversible - specification
Irreversible - determination - 30Test for commitmentCulture in isolation - does it have the same fate?
Heterotopic transplantation (different place)
Cells can pass through stages of commitment before acquiring their final fates
Commitment experiments can show when cells become committed to a fate - 31Mosaic developmentIf cells are determined early on, no further signalling should be necessary
Isolated parts should develop as normal
Cells develop in autonomous fashion - aren't affected by signals from other cells
Very few cells show mosiac development - 32Roux, 1888Killed one cell of a two cell stage amphibian embryo using a hot needle
Half of the normal embryo developed - 33Regulative developmentCells signal to each other
Driesch experiment - splitting the 2 cell embryo, 2 normally patterned sea urchins developed
Rather than self-differentiating into its future embryonic part, each isolated blastomere regulates its development to produce a complete organisms - 34Mechanisms of cell developmentAutonomous mechanisms:
Intrinsic factors making cells different
Non-autonomous mechanisms:
Extrinsic factors making cells different - 35MorphogensA chemical whose concentration varies, forming a concentration gradient
Expressed from localised area
Cells respond differently to different threshold concentrations of the morphogen - 36Bicoid proteineg. of morphogen
Transcription factor
Bicoid mRNA localised to anterior end of egg - 37Bicoid cytoplasmic transplantFrom wild-type egg to bicoid mutant egg causes some anterior structures to develop
- 38Bicoid regulation of hunchbackLocalised expression of hb is an interpretation of positional information provided by the bicoid protein gradient.
hb gene switched on when bicoid is present over a certain threshold concentration - this is only attained in the anterior half of the embryo.
hb codes for another TF that causes a gene cascade to establish segmentation in the embryo. - 39InductionSignalling from one cell/tissue type to another, with a change in specification of the responding cell or tissue
eg. Spemann's amphibian organiser,
Anchor cell signalling to vulval precursors in c. elegans embryo - 40CompetenceAbility to respond to a signal
May involve expression of appropriate receptor molecule
More cells are usually competent to respond than actually do
Competence to signals change over time in embryonic development - 41Xenopus animal cap assayInducing tissue = vegetal cells
Animal cap cells are competent to respond to this induction
Different areas of the embryo produce different inducing signals
Can be used to identify the molecules (signals involved) - by testing proteins on the animal cap - 42Transcriptional activity of a gene depends on:External signals received by the cell
TFs present in the cell
Set of binding sites present in the regulatory regions of the gene
Accessibility of chromatin to TFs and RNA Polymerase - 43SomitesVertebrate embryos
Segmented blocks of tissue that contain undifferentiated mesodermal progenitor cells
Skeletal muscles develops from the somites (myotome)
Also dermis (dermatone)
vertebrae and ribs (sclerotome) - 44Stages of skeletal muscle development1) undifferentiated cells - mesodermal progenitors
2) determination cells receive external signals and upregulate MyoD expression - myoblasts
3) cells are committed to adopt a muscle fate, but don't express muscle specific proteins
4) differentiation - cells stop dividing, align, fuse and start to express muscle-specific proteins - multinucleate myotube
5) maturation - arrangement of proteins into muscle fibres - 45MyoD geneTF
Expressed only in muscle precursors and muscle cells
Controls expression of genes for muscles differentiation
Sufficient to direct muscle cell fate
Not required - mice without MyoD function still develop normal skeletal muscles because of Myf5 which acts redundantly with MyoD
However a double MyoD/Myf5 knocknout lacks all skeletal muscle - 46Stem cellsRemain in undifferentiated state
Self-renewing
Continue to give rise to differentiated cells throughout their lifetime - 47Apoptosis - Programmed cell death ExamplesVertebrate limb development - cells die in limb bud to form the fingers
Vertebrate CNS - neurone refinement
C. elegans embryo
(Hard to discover because macrophages are efficient at savaging the dead cells) - 48Forward geneticsMutant phenotype is starting point
Assumed that the inactivation of a gene required for a particular process will have a specific and reproducible effect on that process
No need for any prior knowledge about the function of the gene product
Clone gene - 49Reverse geneticsStarts with an identified gene
Sequence of gene must be known
Different approaches are used to interfere with its function
eg. targeted transgenesis to knock out genes in mice - 50White mutant phenotype in DrosophilaMutation affecting eye colour
White gene required for the production of red eye colour
Recessive mutation (-/-) gives white eyes
One wt allele is sufficient for normal function -> gene is haplosufficient - 51BrachyuryMutation affecting tail length in mice
wt (+/+) = long tail
heterozygous mutant (+/-) = short tail
homozygous mutant (-/-) = embryonic lethal
Brachyury is required for development of mesoderm in embryo - is a TF
Semi-dominant mutation because an intermediate phenotype is visible in heterozygote - 52Dominant mutationsOne mutant copy has phenotypic effect
One wt copy is not sufficient for normal function -> haploinsufficient
Mutant copy product interferes with wt function (dominant negative, antimorphic form) - 53AmorphsComplete loss of function
Protein nulls
Recessive or dominant
eg. deletion of a gene
premature truncation
receptor unable to bind ligand - 54HypomorphsPartial loss of wt allele
'Weak allele'
Recessive or dominant
eg. missense mutation,
temperature sensitive alleles - 55HypermorphsOver-expression of transcription unit
Over-activity of the gene product
Dominant
eg. ligand-independent activity of a receptor - 56AntimorphsDominant negative
Competitive inhibitors - lack of activity but retain co-factor interation
Semi-dominant - receptor sequestering ligand but unable to signal - 57Mutagenesis Ionising RadiationX-rays / gamma rays
Breaks phosphodiester bond in DNA
If repair incorrectly can lead to larger lesions in DNA - eg. inversions, deletions, translocations - 58Mutagenesis Chemical MutagensAlkylating agents
Donate alkyl groups to nucleotides
Changes their base pairing properties
Point mutations occur when the DNA is replicated - 59Complementation testing for recessive mutationsM1/M2 - mutant phenotype?
NO:
M1 and M2 complement each other and are usually on different genes
(M1/+; M2/+)
YES:
If 25% of progeny show mutant phenotype
M1 and M2 fail to complement each other so are usually alleles of the same gene
(M1/M2) - 60Kit and Steel Complementation testingCT showed they were two different genes
But kit and steel mutants show similar defects:
Coat pigment abnormalities (white spotting)
Anaemia
Sterility - 61Kit and Steel Molecular analysisThe proteins work together in the same pathway
Steel codes for a ligand - steel factor
Kit codes for receptor for steel factor
No signal for melanoblast to develop - 62PiebaldismMutations in KIT and SNA12 genes
Signalling required for the correct development of:
Melanoblasts (pigment abnormalities)
Haemotoietic stem cells (anaemia)
Germ cells (sterility) - 63Epistasis experimentsShow order of gene action in a pathway:
1) make a cross between animals heterozygous for both mutations (+/A; +/B)
2) examine phenotypes
3) If there is no double mutant phenotype (looks like B) - B phenotype is overriding A phenotype
Recessive epistasis - B is epistatic (masks) A - 64Drosophila hedgehog pathway Epistasis experimentsRequired for segmentation patterning
Hedgehog like class (hh, smo, wg):
lawn of denticles, loss of naked cuticle
Hh -| Ptc -| Smo -> Ci -> wg expression
Patched like (Ptc):
excess naked cuticle, loss of denticles - 65Drosophila hedgehog pathway MechanismHedgehog (secreted signalling molecule) binds to patched (transmembrane protein)
Patched lifts inhibition on smoothened (tranmembrane protein transducer)
Smoothed activates Ci (a transcription factor) which allows wingless (wg) expression (another secreted signalling molecule) - 66Maternal effect mutations affecting DV patterning in DrosophilaDorsalised class:
Dorsal, spätzle, toll
Required for development of ventral fates in embryo
Ventralised class:
Cactus
Required for development of dorsal fates in the embryo - 67Toll Signalling PathwaySpätzle -> Toll -| Cactus -| Dorsal -> Twist
Binding of spätzle (ligand) to toll (transmembrane receptor)
Releases cactus kinase which phosphorylates cactus causing it to be degraded, activating dorsal
Dorsal (a TF) enters nucleus and activates other genes - 68Gradient of nuclear dorsalDorsal protein uniformly distributed in egg
Only enters nucleus on ventral side of embryo where toll signalling is active
Created concentration gradient of nuclear dorsal protein - 69Why is the Toll pathway only active on the ventral side?Because the active spätzle fragment only present on ventral side
Spätzle is a maternally derived ligand (processed in the perivitelline space after fertilisation)
Reflects DV patterning of the egg chamber set up by signalling between the oocyte and the follicle cells - 70Dorsal as a transcription factorActivates the transcription of zygotic genes required in developing mesoderm on ventral side of embryo (eg. twist and snail)
Represses transcription of zygotic genes so they are only activated in dorsal regions (eg. dpp)
Twist, snail, rhomboid and dpp genes contain binding sites for dorsal in their regulatory regions - 71Dorsal as a morphogentwi, sna, rhomboid and dpp respond to different thresholds of dorsal protein concentrations
Dorsal acts as a morphogen
Zygotic gene expression along DV axis:
Dorsal ectoderm - dpp
Neuroectoderm - rhomboid
Mesoderm - snail, twist - 72Analysis of maternal effect mutations affecting DV patterning 1) ventralised mutantIn ventralised mutant (eg. cactus), dorsal protein enters nuclei across entire DV axis
Mesoderm genes (twi, sna) are activated throughout embryo
dpp expression is repressed throughout
All cells adopt a ventral fate - 73Analysis of maternal effect mutations affecting DV patterning 2) TwistTwist is expressed in all cells
Above the threshold that dpp is repressed
No three zones - 74Analysis of maternal effect mutations affecting DV patterning 3) dorsalised mutantIn a dorsalised mutant (Toll, spätzle, dorsal) dorsal proteins are excluded from the nuclei
Mesodermal genes (twi, sna) not expressed at all
Dpp expressed throughout embryo
All cells adopt a dorsal fate - 75Drosophila Segmentation: Ultrabithorax geneRecessive mutation:
3rd thoracic segment transformed into 2nd -
2 pairs of wings instead of one pair of wings and halteres
Homeotic transformation - one stucture replaced by another, no alteration of number of segments
Ubx acts as a control (/selector/master) gene to specify haltere (balance organs) formation on 3rd thoracic segment - 76Drosophila Segmentation: Selector genes - The Bithorax Complex (BX-C)Ubx - abd-A - abd-B
Gene complex with three transcription units
Arrangement of the complex reflects the order in which they function along the AP axis of the embryo
Each gene contains a highly conserved 180 nucleotide motif - the homeobox
Codes for the homeodomain - a sequence specific DNA binding motif that acts to regulate other genes - 77Spatial expression of BX-C genesDifferent anterial domains but not defined/cut off after
Can build up different domains that give rise to different characteristics:
No gene expression - all T2 segements
Just Ubx expression - all A1 segments
Segment number not affected - BX-C not required for segmentation - 78Antennapedia complex (ANT-C)Comprised of 5 homeobox genes
Controls development of head and thoracic segments
Expressed in and controls T2 identity
Dominant mutations cause a transformation of the antenna (head) to legs (thoracic segment) - 79Hox genesSpecify segment identity
Highly conserved through evolution
Order in chromosome reflects order in which they are expressed
Mammals have 4 copies of hox genes
In vertebrates, hox genes are expressed in spatially restricted patterns in the developing limbs - 80Mutations of Hox genesMutations of Hox genes can lead to homeotic transformation in vertebrates
eg. loss-of-function homozygous mutants of Hoxc8
The first lumbar vertebra is transformed into a rib bearing 'thoracic' vertbra - 81What determines the sites of limb bud formation?Hox genes
eg. homozygous Hoxb5 knockout mouse - forelimbs develop at a more anterior level
Hox genes may restrict expression of FGFs (fibroblast growth factors) to limb-forming regions
FGFs are REQUIRED and SUFFICIENT for limb bud formation in the correct place - 82FGF signalling is required for limb bud formationEvidence from mouse:
Homozygous knockout of FGF10 or FGFR genes result in embryos lacking limb buds
Tests requirement of gene - 83FGF signalling is sufficient for limb bud formationEvidence from chick:
Local application of FGF4 protein in the chick embryo results in the ectopic position of limb buds - 84Apical Ectodermal Ridge (AER)Ridge of thickened epithelium around the tip of the limb bud
Rigid - keeps the limb bud flattened
Maintains progress zone by signalling activity
AER signalling is essential for normal vertebrate limb development - 85AER grafting experimentRemoval of AER from developing limb bud leads to truncation of limb
Limb develops in a proximo-distal sequence
The later the AER is removed, the more complete the resulting limb - 86FGFs can substitute for the AER1) remove AER
2) replace with beads soaked in FGF4 protein
Formation of digits (slightly abnormal) - 87Progress ZoneDistal region of limb bud mesenchyme (mesodermal)
Undifferentiated, rapidly dividing cells
Length of time in PZ determines cell fate - according to position on PD axis
Cells that leave the zone early form proximal structures, where as cells that leave it last from the tips of the digits - 88Progress Zone: Grafting experiment - testing the role of PZYoung tip and old stump -
gives rise to extra radius and ulna
Old tip and young stump -
old tip already undergone lots of differentiation so radius and ulna don't form correctly - 89Zone of Polarising ActivityRegion of posterior limb bud mesenchyme
When transported underneath apical AER, induces symmetrical duplications of normal limb bud elements about the AP axis
Sonic hedgehog is expressed in the ZPA -
a Shh-soaked bead can mimic the action of the ZPA - 90Hox gene expression in the limb budHox a - distal/proximal
Hox d - anterior/posterior
May specify the ZPA
Deletion of the these two clusters results in the absence of Shh expression and the truncation of the limb
ZPA graft results in new expression of Hox genes - new expression of Hoxd13 around graft
(feedback loops) - 91Formation of digitsTwo ways:
1) programmed cell death (mammals, birds)
2) differential growth
Transplant experiments demostrate that the mesoderm of the limb bud determines the pattern of cell death
Patterns of cell death are regulated by Hox genes - 92Evolution of limb patterningInvolves looking at morphological differences in adults and working backwards
Loss of limbs in snakes:
Hox gene expression expands thoracic identity and represses limb formation - 93Proneural clusters in DrosophilaSmall groups of cells set aside in embryo at regular intervals in the neuroectoderm
The cells in each cluster are in an equivalence group - each has the potential to become a neurone.
One cell becomes a neuroblast (primary fate)
The other cells become epidermis (secondary fate)
This process is called lateral inhibition - 94Lateral inhibition - Mutation phenotypes Notch and DeltaNotch (-/-) and Delta (-/-):
all cells in proneural cluster become neuroblasts
Delta and Notch are (membrane bound) ligand and receptor pair
When binding occurs the notch intracellular domain is cleaved
Enters the nucleus changing repressor of complex -> target genes are expressed
Notch downregulates level of delta -> levels of delta decrease -> level of notch received decreases - 95Drosophila - neuroblast cell behaviour: delamination and asymmetric divisionSegregation of cytoplasmic factors to one daughter cell
Reorientation of the spindle
Neuroblast remains as a stem cell
Progeny of ganglion mother cells (GMC) differentiate
Neuroblasts -> GMCs -> Neurones - 96Neuronal differentiation: Axon growth and pathfindingGrowth cone controls path taken by a growing neurone
Specialised for movement and sensing its environment for guidance clues
Specialised part of cytoplasm of axon
Many filopodia are continously extended and retracted to explore surrounding environment
Diffusible molecules act as long range cues
Cell surface molecules acts as short range cues
both can be attractive or repulsive - 97Synapse formation and pruningMore than one connection between neurones
Not all these excess connections are needed
Some of the connections are removed to make more refined synaptic connections
In the developing vertebrate CNS neurones are created in excess, neurotrophic factors are required for neuronal survival
Ensures making number of neurones to number of targets - 98Potency of cellsCells gradually become committed to their fates and show a gradual restriction in potency
Hypotheses to explain this restriction in potency:
1) Gene loss
2) Differential gene activity - 99Lens regeneration in the newt1) Lens removed
2) Proliferation of dedifferentiated cells from the dorsal iris
3) Synthesis of crystallin proteins
In embryo lens tissues forms from the eye placode
This shows the newt has evolved to cope with injury
Cells must still have the crystallin gene - 100Genes are not lostGenomic sequencing has shown that genes coding for cell-specific proteins can be found in the DNA of other cell types
Conclusion - Gene expression in nuclei of differentiated cells can be changed (under special circumstances )
Gene expression can be controlled by cytoplasmic factors
Genes are not lost when cells differentiate, they are simply switched off
Whilst cells lose their potency, nuclei retain it - 101Hedgehog pathway and human diseaseShh in vertebrate embryo:
Midline (notochord and floorplate)
ZPA of limb bud
Mutations can cause defects in any place where Shh is expressed - 102Hh signalling: HoloprosencephalyHoloprosencephaly is a range of severe midline defects
Cephalic disorder in which the forebrain fails to develop into two hemispheres
Defects in the development of the face, and in brain structure and function
Caused by a loss of Shh signalling - 103Hh signalling: CyclopiaCaused by cyclopamine, a steroidal alkaloid from the Californian corn lily
Tetratogen -> causes malformation of an embryo
Inhibits Hh signalling pathway by inhibiting the action of smoothed
Gives rise to similar defects as loss of function mutations in Shh - 104Hh signalling: CancerInappropraite activation can cause a range of aggressive cancers
Basal cell carcinoma and Gorlin's syndrome
Mutations that cause BCC:
Ptc - loss of function, hypomorphic, tumour-supressor
Smo - gain of function, hypermorphic, oncogene - 105Tumour cells share many characteristics with embryonic stem cells:Rapidly dividing
Undifferentiated
Undergo epithelial to mesenchymal transition (EMT) - 106FGF PathwaysLarge family of signalling molecules
Work through stimulation of transmembrane receptors
Binding of ligand -> dimerisation of receptor and activation of intracellular tyrosine kinase domains
Many of the mutations affecting FGFR genes act in a domniant fashion - 107CraniosynostosisPremature ossification of the cranial structures
Abnormal head shape and facial features
Other skeletal abnormalities eg. fingers and toes fused together
Mutations map to FGF2 - 108AchondroplasiaMost frequent form of short limb dwarfism
Limbs patterned normally, but long bones fail to grow
FGFR3 - dominant mutations in transmembrane domain - 109FGF3 Mice ModelsHomozygous FGF3 knockout:
Phenotype -> bone overgrowth
Conclusion -> FGF3 normally restricts done growth
Dominant mutation/Hypermorph
FGFR3 replacement: G380R/+
Model for achondroplasia
Mouse is smaller
Shorter bones than normal due to less bone growth
Some facial/cranial abnormalities
Información
- Descripción
- BSc Biomedical Science Year 1 Fichas sobre Dev Bio Flashcards, creado por rachel-chads el 01/06/2014.
- Creado por
- rachel-chads · jun 2014
- Actualizado
- 3 feb 2018
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