Developmental Biology Flashcards
What cells are egg and sperm cells derived from?
Germ cells
What are the major characteristics of a sperm cell?
- Flagella
- Acrosomal vesicle
- Many mitochondria
What are the major characteristics of a egg cell?
- Large
- Contain large amounts of RNA and proteins for zygote
- Arranged asymmetrically
What happens in fertilisation?
- Sperm binds to the pellucida zone
- Acrosomal reaction - allows penetration through the bona pellucida
- Fusion of egg and sperm membranes releasing nucleus
What are the blocks to polyspermy?
- Depolarisation of membrane
- Cortical reaction
What is the cortical reaction?
- Vesicles containing cortical granules bind with plasma membrane
- Froms fertilisation membrane
- The vesicles remaining cause the hyaline layer
What happens during egg activation?
- Sperm triggers the release of Ca2+ which act on proteins that initiate cleavage
- Pronuclei fuse and cleavage is initiated
- Oscillations of Ca2+ continue for hours
What occurs during cleavage?
- Rapid and can occur in diffeerent patterns
- There is no growth - no S and M phase
- Transcription is suppressed
- Cleavage continues an forms a blastocoel - fluid filled cavity
How do cells become different?
- Cell-cell signalling
- segregation of cytoplasmic componants before division
What are the three germ layers?
Ectoderm
Mesoderm
Endoderm
What does ectoderm develop into?
- Neurones
- Glia
- Neural crest
- Epidermis
What does mesoderm develop into?
- Muscle
- Cartilage
- Bone
- Kidney
- Heart
- Blood
What does the endoderm develop into?
- Gut and associated organs
- Yolk cells
What is a blastula?
Single layer of cells surrounded by a hollow ball
What happens during gastrulation?
- Mesoderm moves inside the cavity and endoderm takes its place
- Endoderm cells buckle and move inside the cavity and fuse with the top layer of cells forming a hollow tube - this will become the gut
What is the difference between epithelium and mesenchyme?
Epithelium - have polarity - microfilm at the top and secrete at the bottom
Mesenchyme - cells exist in a matrix and are more random
What is neurulation?
Further rearrangements in cell and tissue to form the neural tube
Give an example cells the migrate after gastrulation?
Neural crest cells
Germ cells
When are body plans developed?
After gastrulation and neurulation Form anteroprosterior (A/P) and dorsoventral (D/V) axes
What are the advantages of using drosophila as a model organism?
- Small organism
- Short generation time
- Large batches of embryos
- Sequemced genome
- detailed genetic map
What are the disadvantages of using drosophila as a model organism?
- Small embryo
What are the techniques available when using drosophila as a model organism?
- Mutagenesis
- Transgenesis
- Clonal analysis
How many days after fertilisation is an adult drosophila?
9 days
What are the advantages of using worms (C.elegans) as a model organism?
- Small organism
- Invariant lineage
- Short generation
time - Sequenced
genome - Hermaphrodite
What are the techniques available when using worms (c. elegans) as a model organism?
- Mutagenesis
- Cell ablation
- RNAi
What are the disadvantages of using worms (C. elegans) as a model organism?
Small embryo
How many hours after fertilisation is a worm fully developed?
50 hours
What are the advantages of using a zebrafish as a model organism?
- Small vertebrate
- Large batches of embryos
- Transparent embryo
- Haploid development possible
What are the disadvantages of using a zebrafish as a model organism?
Large genome
What are the techniques available when using zebrafish as a model organism?
- Mutagenesis
- cell transplantations
- Injections
- Transgenesis
How many days after fertilisation is a zebrafish fully developed?
90 days (only 2 days after fertilisation hatchling)
What is the cls zebrafish mutant phenotype and what human disease does it relate to?
Caused by the sox10 gene and causes ear defects, uninflated swim bladder and enteric neurones missing
Similar to Waardenburg syndrome type IV
What are the advantages of using a mouse as a model organism?
- Small vertebrate
- Mammal: model for man
- ES cells
What are the disadvantages of using a mouse as a model organism?
- Poor accessibility
- Small batches of embryos
- expensive
What are the techniques available when using mouse as a model organism?
- Targeted transgenesis
- Mutagenesis
What is the mutant phenotype of a Pax6 mutation?
Wild type has normal eye
Heterozygote has smaller eye
Mutant has no eye
At what stage can cells be separated and develop into normal separate lava?
- 4 cell stage develop 4 normal slightly smaller larva
- 8 cell stage - develop abnormally e.g. missing organelle
What are the advantages of using a frog (Xenopus) as a model organism?
- External fertilisation
- Large batches of embryos
- Robust
- Large embryos and cells
What are the disadvantages of using a frog (Xenopus) as a model organism?
- Long generation time
- Yolky embryo
What are the techniques available when using Xenopus as a model organism?
- Injections
- Tissue transplantations and culture
- Transgenesis
What happens if you inject a pregnant women’s urine into a a frog?
It will lay eggs
What happens if you take two vegetal or animal cells out of the four cell stage?
Vegetal removed - only anterior structures develop
Anterior removed - only posterior structures develop
What are the advantages of using a chick as a model organism?
- Big embryo
- Good model for man
What are the disadvantage of using a chick as a model organism?
- Not accessible early
- Genetic analysis difficult
How many days after fertilisation is the Xenopus fully developed?
60 days
What are the techniques available when using a chick as a model organism?
- Tissue transplantations and culture
- Retroviral infection
- Electroporation
What is cell fate?
What cells will normally develop into
What is a fate map?
A map of structures present at a given stage back onto an embryo at an earlier stage
What are the ways to make a fate map?
Observation
- C. elegans - small cell number and transparency of embryo - trace fates using a microscope
Natural pigmentation
- e.g. early blastomeres containing pigment can be tracked
Application of a marker
- Look for progeny of labelled cell in later development
What are the qualities of an ideal marker?
- Can be applied at any region at any stage
- Readily visible
- Doesn’t change normal development
- Doesn’t leak into neighbouring cells - high mr
- Inherited in all progeny of labelled cells
Name some good markers of fate mapping?
- Vital dyes
- High molecular weight traces
- Cytological labels
What is commitment?
Commitment to s fate me reversible (specification) and irreversible (determination)
How can you test commitment?
Culturing in isolation
- Remove cells from blastula stage and culture and compare outcome to fate map - show if commitment is irreversible
Transplantation - Transplant tissue to another embryo - see whether it develops according to its new position or not
What is mosaic development?
If cells are developed early then development is said to be mosaic
What is regulative development?
if cells are not developed early
What sort of development is it if a defect in a part of the embryo doesn’t affect the rest of it?
Mosaic
What is a morphogen?
- A signalling molecule that is expressed from a localised source
- Forms a concentration gradient
- Causes different responses fro cells depending on different threshold levels
What is the French flag model?
Used to understand how a morphogen may act on an embryo depending on its concentration and threshold level
Give an example of a morphogen?
Bicoid protein
What is the role of the bicoid protein?
Regulates the expression of the hunchback gene which controls segmentation
If bicoid protein placed central in drosophila embryo then then segments form either side
Forms a concentration gradient
What is induction?
Induction is the ability of one cell or tissue to direct the development of neighboring cells or tissues
Give an example of induction?
Xenopus animal cap assay
- Cells from Xenopus that are fated to become neural , specified to become epidermis can be induced to become muscle by moving it to vegetal tissue
What is competence?
Ability to respond to a signal - e.g. having the appropriate receptors
e.g. in the Xenopus cap assay - cells are competent to be muscle
Why do cell types appear different?
Because they express different genes hat code for specific genes
What does gene expression depend on?
- External signals received by the cell
- Intrinsic factors e.g.. proteins present in the cell
- Set of binding sites available on the gene
- Availability of RNA polymerase and transcription factors
How can gene expression be regulated?
Feedback loops:
- Positive - A gene transcripts a protein which activates another gene by binding to its binding site. Protein produced up regulates the original gene
- Negative - Gene transcripts protein which activates another gene. That protein will inhibit the original gene
What are somites?
Somites are segmented blocks of tissue that contain undifferentiated mesoderm cells that differentiate into muscle, cartilage and bone
What is the role of MyoD?
It is a transcription factor that controls the expression of genes for muscle differentiation
What is the difference between sufficient and required?
Sufficient mean the gene is enough to for the desired production in its own
Required means that without the gene then the desired product would not be produced
Is MyoD sufficient or required?
- It is sufficient as when transfected into fibroblasts, they develop into muscle
- It is not required as Myf5 acts redundantly with MyoD as mice lacking functional MyoD develop with normal skeletal muscle
What are satellite cells?
Muscle stem cells that lie between the basal lamina and cell membrane of muscle cells
What is apoptosis?
Programmed cell death
e.g. inner digit cells
What are the two types of genetic analysis?
Forward genetic analysis
- mutant phenotype as its starting point
Reverse genetic
- Uses an identified gene as its starting point - sequence must be known
What is the white gene responsible for and what is the mutant phenotype?
Responsible for production of red eyes in drosophila
Homozygous ressive mutant (-/-) have white eyes
What is the difference between haplosuffiicent and haploinsufficent?
- Haplosufficient is when one copy of the allele is enough for normal function of the gene - dominant
- Haploinsufficient - is when one copy of an allele is not enough for normal formation
What is the brachyury gene?
- Wild type has long tail
- Heterozygous +/- has short tail
- Homozygous -/- embryonic lethal as its required for mesoderm development in the embryo
What is mutagenesis?
Rate of spontaneous mutation is very low
Mutations can be induced by treating with mutagens
Name some chemical mutagens?
Alkylating agents - EMS, ENV - Adds alkyl groups to amino acid - mutation - Leeds to point mutations Ionising radiation - Breaks phoshodiester bonds in DNA - Repaired incorrectly - mutation
What is complementation testing?
Sorts mutations into complementation groups
After interesting mutation picked, cloned and analysed
- It shows that some mutants with similar phenotype result from mutations in different genes suggesting gene work together in a pathway
What are the characteristics of Kit and Steel?
They compliment each other so not same gene but share some abnormalities so suggest they are involved in the same processes
Kit is the receptor and steel the ligand and are required for development of melanoblasts, blood stem cells and germ cells
What is the human condition relating to abnormal Kit and Steel?
Piebaldism
What is epistasis?
The suppression of one gene by another (one gene is epistatic to another)
What is the hedgehog pathway responsible for?
Responsible for segment polarity in mutant drosophila
What is the hedgehog pathway?
Hh—| Ptc –| Smo –> Ci –> Wg expression
When is the difference between dorsal and ventral sides of the embryo decided?
Blastoderm stage
What is required for ventral cell fates?
- Dorsal
- Spatzle
- Toll
What is required for dorsal cell fates?
Cactus
What is the pathway for making an axis (repressing dorsal)?
Spätzle –> Toll –| Cactus –| Dorsal
How is dorsal protein distributed in an embryo?
It is uniformly distributed across cytoplasm but only enters nucleus on the ventral side
Spätzle present on ventral side meaning dorsal protein is repressed
How does the dorsal protein act as a transcription factor?
on the ventral side it activates the transcription of twist and snail
It also represses the transcription of dpp so its only active in dorsal regions
What happens in a ventralised mutant?
Dorsal protein enters nuclei across entire DV axis
Twist and snail are activated and dpp is repressed
All cells adopt a dorsal fate
What happens in a dorsalised mutant?
Dorsal protein does not enter the nucleus
Twist and snail are not expressed
Dpp expressed everywhere
All cells adopt a dorsal fate
What is the roll of the toll pathway in the immune system?
Toll pathway is stimulated by bacterial and fungal infection
- Toll like receptors are present on macrophages
- Homologue of dorsal regulates gene expression of B cells in immune system
What is the phenotype of the a mutation in ultrabithorax (ubx) gene in the drosophila?
Causes a transformation of the third thoracic segment leads to two pairs of legs and wings
What is the difference between autonomous and non-autonomous function?
Autonomous
Mutation that affects the cell in which it is present
Non-autonomous
Mutation affects other cells
In the drosophila, what is the halter?
The balancing organ that are behind the wings
What happens to cells in the halter if they are lacking the ubx function?
They are automatically transformed into a wing
What is the bithorax complex?
Ubx—abdA—-AbdB
They cause anterior transformation of segments
In the bithorax complex what does the homeobox DNA do?
Codes for the homeodomaine
What is a hox gene?
A gene that codes for a body plan
What is ANT-C?
Controls the development of head and thoracic segments - contain homeoboxes
Dominant mutation a gene that causes a transformation of the antenna to leg
What would happen in a homozygous HoxBF knockout mouse?
Fore limbs develop at a more anterior level
What is the role of Hox genes in limb bud formation?
They may restrict the expression of fibroblast growth factors (Fgfs) to limb forming regions
What evidence is available to show that Fgf signalling is REQUIRED for limb bud formation?
Homozygous knockout of fgf10 or fgfR results in embryos lacking limb buds
Only occurs in the homozygous knockout - recessive
What evidence is available to show that Fgf signalling is SUFFICIENT for limb bud formation?
Local application of FGF4 gene into the middle of a chick embryo results in an extra limb bud forming in the middle
Limb buds form on an axis
What is the apical ectodermal ridge?
A ridge if thickened epithelium around the tip of the limb bud - rigid
What happened if the apical ectodermal ridge is removed?
If removed than only the most proximal part of the limb bud is formed
The later you remove it, the more distal elements present in the wing
Can anything substitute for the removal of the apical ectodermal ridge?
Yes - Fgfs can substitute for the AER - if you remove the AER and replace with beads soaked in FGF4 then normal limb develops
What is the progress zone?
The distal region of the limb bud
Mesodermal cells that are rapidly dividing
The length of time in the progress zone determines cell fate
What is the evidence for the role of the progress zone?
If you put a new tip (progress zone) on an old limb bud stump then you get a much longer limb (distal axis)
If you put an old tip on a new stop then the limb doesn’t develop properly
What is the zone of polarising activity and where is it present?
A region of posterior limb bud mesenchyme - both, of the limb bud more proximal to the progress zone
What happens when the zone of polarising activity is transplanted into the anterior part of the limb bud?
Two zone of polarising activities (ZPA) leads to duplication of digits - symmetrical
What is the sonic hedgehog gene (Shh)?
The morphogen present in the ZPA in the limb bud
If beads socked in Shh are implanted in the anterior part of the limb bud then it can cause duplication of the digits - mimics the role of ZPA
What genes specify the ZPA in a limb bud?
Hoxa and Hoxd gene
If absent then no shh present and trunction of the limb occurs
How are digits formed in mammals and amphibians?
Mammals - apoptosis (programmed cell death) controlled by the mesoderm of the limb bud - regulated by Hox genes
Amphibians - Differential cell growth
Controlled by evolutionary factors
What is a proneural cluster?
Groups of cells in the neurectoderm
One cell in the cluster will become a neuroblast and the others will become epidermis
What is lateral inhibition?
When the central cell in the pro neural cluster inhibits the surrounding cells and stops from becoming neuroblast
Are all cells in a proneural cluster capable of becoming a neuroblast?
Yes the proneural glosser is an equivalence group
Notch -/- or Delta -/- causes all cells to become neuroblast - must be competent
What are delta and notch?
Notch is a transmembrane receptors and delta is a ligand
High delta leads to the cell being a neuroblast
High notch leads to the cell becoming epidermis
How does the neurectoderm become a neuroblast and epidermis cells?
- Cells express both Notch and Delta equally
- One cell starts to express more delta
- Other cells increase in notch in order to receive the amount of delta
- The increase in notch causes the inhibition of delta in those cells
- The difference in notch and delta increases leading to a neuroblast (with high delta) and epidermis (with high notch)
=> Lateral inhibition
How do the neuroblasts divide to form neurones?
Asymmetric divison - Numb protein in one side of the cell
Divides into a neuroblast (stem cell) and a ganglion mother cell (divides into neurones)
What cells are present in drosophilla PNS mechanosensory bristles?
Hair cell
Socket
Sensory neurone
Sheath cell
How are cells in the drosophila PNS formed?
- Lateral inhibition of delta notch signalling
- Singles out sensory organ precursors (SOPs)
- Assymetric decision to form each cell
What happens in the drosophila PNS when notch is over and under-expressed?
Under-expressed - All cells from SOP become neurones
Over-expressed - All cells from SOP become socket cells
What are the roles of growth cones?
Axon guidance - responds to diffusible molecules and may be either attracted or repulsed by them
When axon reacts its target it synapses with them
How do identical chromosomes direct differences between cytoplasmic contents of different cells?
Differential gene activity
Not gene loss
Name some evidence for differential gene activity as the method for different cytoplasmic contents between cells?
Lens regeneration in the newt
- Remove lens from the newt and replace with dedifferentiated cells from the dorsal iris - lens not originally developed from the iris suggesting its potency
Liver cells
- Grow liver and muscle together
- Liver nucleus moves into larger muscle cytoplasm
- Liver genes switched off and muscle genes on
Kidney cells
- Nucleus of kidney implanted in oocyte - kidney genes off oocyte on
How does nuclear transfer in the Rana suggest that the nuclei of differentiated cells do not remain totipotent?
- If you removed nucleus from an egg cell and replace with a nucleus of a blastula
- Then tadpoles develop normally but dot survive to adulthood
- Suggest pluripotent but not totipotent
- As the age of donor nucleus increases than the success rate decreases
How does testing in the Xenopus suggest that the nuclei of differentiated cells remain totipotent?
- Nucleus from an albino gut cell implanted in pigmented egg - offspring expected to be albino
- Only had cleaved
- Took nucleus from successful half and planted in another egg
- Normal albino tadpoles develop
What are some of the criticisms of testing in the xenopus to show the uncle remains totipotent?
- Low success rate
- Germ cells are sometimes present in the gut which could have been the reason for the success
- Gut epithelium not fully developed - may still have some potency
How does testing in the adult Xenopus (second time) suggest that the nuclei of differentiated cells remain totipotent?
- Skin cells taken from adult xenopus and tested fro the presence of keratin to show that they are fully developed
- Cleavage occurs on one side
- Nucleus from healthy side and put in another egg
- Normal tadpoles develop but didn’t reach adulthood
How does the cloning of sheep suggest that the nuclei of differentiated cells remain totipotent?
- Breast cell taken from an adult white faced sheep
- Fusion to put nucleus inside black faced sheep egg
- Embryo cultured and placed in a surrogate
- Normal white face sheep developed - dolly the sheep
What is the exception to the differential gene activity theory?
Parascaris
- Genes are lost when differentiate
- Somatic cells lose genes
- Germ cells do not
Why do we need animal models of human disease?
- Understand disease mechanisms
- Development of diagnostic tools
- Development of therapeutics
- Understand protein function
Give an example of where developmental animal models has helped in he understanding of a disease?
Shh mutations
- Cause midline defect and limb defects
- Now known to underlie many cancers
- Cyclopamine - a steroidal alkaloid - inhibits hedgehog pathway and had been developed as a treatment for cancers
