PART 1 Flashcards
What are the two opposing theories of plant development?
- Preformationism= Nicolas Hartsoeker (1695)- organisms develop from miniature preformed versions of themselves, hommoculus
- Epigenesis= Aristotle- new structures slowly added to form whole body
The cell Theory- Robert crooke (1665)
- All organisms are composed of 1 or more cells
- Cell most basic unit structure
- All cells arise from pre-existing cells
What is germ plasm determinants theory?
Fredrick Weisman 1880s
Germ cells have whole collection of somatic cells which have subset of determinants.
Carry out specific characteristics determined
Wilhem Roux- 1888
Killed one half of frog egg with hot needle.
Half remains dead the other half embryo
Induction theory
Separated embryo at stage 2 and 4
both reform themselves and fully develop
NOT in contact with each other, disrupt communication and modify same outcome
How do cells aquire new properties that differentiate them from others?
Cell division- symmetric or asymmetric
Cell-cell communication
Cell adhesion- condensation or dispersal
Cell shape-epithelial or epithelial to mesenchymal
cell migration
cell death
What are the different types of cell-cell communication
Paracrine= Signal produced in one cell and binds to another
Autocrine= provides on one cell, acts on other side of same cell
WNT, SHh, TGF, BMP signalling ^both above
Juxtacrine= cell/cell contact
Cell signalling- cell-cell interaction
Cell needs to be competent- receptor for signal and machinery
signal can be instructive (initiates new programme, direct change in cell) or permissive (changing environment for another to react)
signal can act as morphogen
What does transmission of signal to the nucleus involve
Signal transduction cascade
What are the 3 germ layers?
- Ectoderm (external layer)= Skin, brain/neurons and pigment cells
- Mesoderm (middle)- Cardiac, skeletal, RBC, smooth muscle
- Endoderm( Internal layer)= Lung alveoli, thyroid and pancreatic cell
What 2 methods is tissue homeostasis maintained by?
- Negative feedback loop
2. Stem cell mediated repair
What is pattern formation in embryonic development?
Process where cells are ordered within embryo.
Need Co-ordinates to establish axis
What is morphogenesis?
Cell and tissue movement and behaviour changes in cell
Factors affecting morphogenesis of cell?
Cell adhesion
cell death
cell migration
cell shape
Step by step series of differentiation of cell in embryonic development
- egg/sperm
- Specification- early commitment, not stable
- determination- commitment to cell fate, stable , still aquisation of terminal characteristics
- Differentiation- reversible
- Maturation- irreversible
What are the processing underlying embryonic development?
Growth
Morphogenesis
cell differentiation
pattern formation
Growth of embryonic development
- continuous process
- growth rate varies depending on age
- cell proliferation, enlargement and accretion
What are the two opposing theories in early embryonic development in vertebrates?
- Funnel (Haeckal)= Few differentiate in early development X wrong
- Hourglass (van baer)= Lots of differentiation beginning and end Correct
Methods to find out where and when a gene is expressed in the embryo
- In situ Hybridisation
- Northern blott
- RT-PCR
- Microscopy
- reporter lines (transgenic)
What is a method to find out how many transcript is in an embryo?
Northern blott
RT-PCR- carry out amplifcation of gene expression
Is the protein expressed with the same timing of the gene?
Look at mRNA and protein as the protein may not be expressed where it is transcribed
look at distribution of protein- western blot (how much protein made) or immunochemistry (temporal and spatial
To find out if the gene/ protein is essential for development
Loss of function- disrupts expression
gain of function- increase amount of activity to gene product encoded by mutant gene
Forward and reverse genetics
Forward Genetics
C.elegans, drosophilla, mouse and zebrafish
Seeks to identify gene whose mutant causes a specific phenotype.
Cross mutant and WT- F3 generation gives m/m (mutant), m/+, +/+
Reverse Genetics
Target 1 gene you are interested knowing function of by two main methods:
- Straight KO
- Conditional KO
- homologous recombination(loxp)
- Es cells transfection
- ES cell selection
- Es cell injection into blastocyst
- Implant into mouse
- Selection of chimeric mouse
- breed either conventional KO or conditional KO (floxed allele with tissue specific cre mouse)
How is a gene regulated?
Tissue manipulation- drugs transfection and electroporation
Method:
- Tissue ablation, graft and transplantation. (surgical)
- Bead/ cell implantation (signalling molecules, drugs)
Observational biology to see use of gene, experiment in chick and quail
Inject quail into chick Carry out early in devel when cells aren't committed to fate Use antibody to recognise quail cells Precisely trace all 300 Basic genetic construct now possible
What is a morphogen?
Soluble secreted molecule that acts from a distance to specify the fate of cells.
Not all molecules that are involved in patterning are morphogens
Need to make and break otherwise lose info
What must a morphogen do?
- Induce different outcomes at different concs
Instructive-provide info to cell next to it
permissive- Have knowledge about what they are/gonna be PERMISSIVE NOT MORPHOGEN - Act directly at a distance and NOT be diffusible through cells (bucket brigade)
Tests to show that permissive are not morphogens for
- Provide a second source of signal in ectopic expression- opposite side
- instructive would get mirror image
- permissive no effect - provide red signal at a uniform conc
- instructive all see same gradient of signal so all same
- permissive no effect
Test for morphogen acting directly at a distance
- Use genetic engineering to make the proposed morphogen a juxtacrine (eg add a trans membrane domain)
Stop peptide from diffusing
Morphogen - adjacent cell to signal will see it but none of the others - Make genetic mosaic that lacks signal
NO COLOUR
Binding of molecules in extracellular matrix and high conc of receptor can generate a steep gradient,
What are HSPGs?
Heparan sulfate proteoglycons found in extracellular matrix and bind to ligands Also known as co-receptors Regulate morphogen diffusion by: - slowing diffusion (BMP) -facilitating diffusion (Hedgehog)
How does planar transcytosis play an important role in establishing morphogen gradient?
Repeated endocytosis and restriction of morphogens allow it to travel through the cells
Evidence= antibody staining shows dpp found in vesicles and mutations that block vesicle formation cause dpp to act in a juxtacrine manner
How do cells read or interpret a gradient to make a cell fate decision?
Transcriptional read out model
- Higher conc= higher conc of activated TF
- More TF enter nucleus and direct transcription
Bicoid= morphogen and TF
-accumulates in nuclei of synaptical blastoderm generating conc gradient
How is TF concentration interpreted at the DNA level?
Enhancer have different affinities *medium TF concentration low affinity=OFF High affinity= ON Even though both recieve same conc only high affinity enhancers can bind enough to TF to cause gene expression *Low TF conc Low affinity=ON High affinty =ON
How are strict thresholds achieved when concentration is not steep?
Positive feedback
The lifecycle of drosophilla melanogaster
- fertilised egg
- cleavage
- gastrulation
- hatching
- larva- pupa
History of drosophila
1910- morgan discovers a while eye fly (genetic map formed
1913- Sturtevant constructs first genetic map
1914/16- show chromosomes must contain genes
1927- muller shows x-rays cause mutations
1979/80- saturation mutagenesis to identify genes involved in development
1980-00s- p-element, enhancer trap, gal 4/UA5 missexpression, FLP/FRT clonal mutant anaylsis
2000- Genome mapped!!
Drosophilla courtships- what are the genetically encoded behaviours?
- Orientation
- Tapping
- Wing vibration
- Licking
- Attempting copulation
what is the timeline of development of drosophilla egg?
- Stem cell maintenance= divide and move away fro source, 4 incomplete cytoblast mitotic divisions
- 2 nurse cells and 2 have full connections around (1 becomes egg)
- Selection of future oocyte:
- nurse cell= endo-replication (DNA replication without cytokinesis, many copies of genome
- Oocyte= diploid nucleus or meiotic recombination
what is the subcellular localisation of maternal factors?
A/P Localised by microtubule transport minus and plus ended motors glue anchors them in position waterproof and sealed
What is chorion?
Egg shell secreted by follicle cell
What is the vitelline membrane?
Hydrophobic, protects from drying out
How do you transform a single cell into an embryo?
- Set aside ‘top’ cells versues ‘bottom’ cells by differentiation
- ‘Bottom’ cells signal to ‘top’ cells so that the latter start to differentiate further forming 3 germ layers
What is cell differentiation?
Process by which cells become different from each other and acquire specialised proteins. Governed by changes in gene expression which dictate protein synthesis
How is gene expression governed in a cell?
Intrinsic and extrinsic factors
What are the top and bottom layer in xenopus and chick/humans?
Xenopus
Top= animal
Botttom= vegetal
Chick/ Humans
Top=epiblast
Bottom= Hypoblast `