Developmental Bio Flashcards
What is developmental biology?
Studying how cells acquire certain characteristics, behaviours, communications and organisations
From embryo formation to ante-, neo- and post-natal life
Describe the evolution of developmental biology from Ancient Greece to ‘cell theory’ and ‘induction theory’.
Preformation (idea that everything already formed but gets bigger over time) vs epigenesis (new structures arose progressively). Cell theory (theory that organisms are composed of one or more cells) proved epigenesis correct. Induction theory is where one cell or tissue directs development of of another cell or tissue.
What are some cellular and molecular processes underlying cell differentiation?
Cell division - can be symmetric or asymmetric (cytoplasmic determinants expressed asymmetrically causes this division)
Signal induction - e.g. growth factors causes signal transduction pathways resulting in differentiation changes
What are the types of cell-cell communication involved in generating differences?
Paracrine - protein secreted and detected by nearby cells which activates a signalling cascade
Autocrine - protein secreted and detected by the same cell
Juxtacrine - factor is not diffused, instead is attached to cell and detected by neighbouring cells
How does gene regulation support cell differentiation?
The gene content is identical in all cells but transcription and translation determine the protein content and therefore its behaviour. Can be controlled at different levels: by the production of mRNA, the processing/stability of mRNA, the production of proteins and the activity of proteins.
What are two mechanisms which control the gene transcription?
Differential gene expression - where transcription factors can promote or repress expression of certain genes
Enhancer-mediated control of gene expression - enhancers promote gene expression
What is the impact of developmental biology on biomedical science and medicine?
Stem cell therapy, cancer medicine, fertility understanding, congenital disease, degenerative disease, ageing, regenerative medicine
Why is the concept that all cells contain the same DNA but express different genes important?
Mutations will only show up in cells where the mutated gene is expressed
E.g. Shh transcribed and translated in the developing limb and a mutation in this pathway will affect hand development
What are 4 features of cell signalling?
Signal reception requires cells to be competent
Signals are instructive or permissive
Signals can act as morphogens
Require signal transduction cascade to reach nucleus
What are the 3 germ layers (and which cells are the cells not derived from one of these)?
Ectoderm (external layer)
Mesoderm (middle layer)
Endoderm (internal layer)
Germ cells are the exception
What will the ectoderm form?
Skin cells of epidermis
Neuron’s in brain
Pigment cells
What will the mesoderm form?
Cardiac muscle Skeletal muscle Tubule cells of kidney RBCs Smooth muscle in gut
What will the endoderm form?
Lung (alveolar) cells
Thyroid cells
Pancreatic cells
What processes underlie embryonic development?
Pattern formation
Morphogenesis
Cell differentiation
Growth
What is pattern formation?
The process by which cells are organised in space and time to produce a well-ordered structure within the embryo
What is morphogenesis and what processes contribute to it?
Cell and tissue movement and changes in cell behaviour that give the developing organ it’s shape in 3D
Cell adhesion
Cell migration
Cell death
Cell shape
What is cell differentiation?
The process where cells become different from each other over time and acquire specialised properties. (Governed by changes in gene expression which dictate protein synthesis)
What are the steps involved in differentiation?
Egg/stem cell —> specification —> determination —> differentiation —> maturation
What does the continuous growth process (increase in size) involve?
Cell proliferation (through mitotic divisions)
Cell enlargement
Accretion (of ECM tissues)
Also depends on age and organ
What methods do we use to study changes in cell behaviour, cell-cell communication and gene expression which underpin developmental processes?
Embryology (observational biology and experimental manipulation) Developmental biology (study of genes and proteins) Animal models and use of genetics
What makes animal models a good way to study developmental biology?
Early embryology is highly conserved therefore the foundations for all surviving organisms is very similar to that of humans
What are experimental approaches to study gene expression?
RT-PCR In situ hybridisation Northern blot Reporter lines (transgenic) High throughput analyses (microarray, RNAseq)
Describe how in situ hybridisation can be used to establish where and when a gene is expressed?
The target mRNA in a fixed embryo is recognised by a DIG-labelled probe with an antisense strand complementary to the mRNA sequence.
The DIG is recognised by an anti-DIG-AlkPhos antibody which is attached to alkaline phosphorylase that can cause a reaction that can be detected.
Describe how a reporter line can be used to establish where and when a gene is expressed?
A reporter gene (e.g. betaGal or GFP) is added to the genome near the regulatory sequence of gene of interest.
The transgenic gene is then introduced into the animal model where it is expressed.
The reporter gene will give indication of when and where the gene of interest is expressed.
Describe how high throughput analysis (microarray) can be used to establish where and when a gene is expressed?
The RNA is isolated and cDNA is generated.
The probe is labelled with fluorescent tags and hybridised to array.
When imaged, the colours will show if the particular cDNA is attached.
Describe single-cell RNA Sequencing (scRNA-Seq)?
Individual cells from a tissue are separated and suspended in oil droplets with barcodes so that each transcript or can be sequenced.
Look at the individual genes in each cell and compare them.
Why is it important to study whether the protein is expressed with the same timing and place to the gene? What techniques could you use to investigate this?
Location gives an idea of function.
Want to find out spatial and temporal expression pattern.
Western blot
Immunohistochemistry
Describe how immuno-detection methods (immuno-histochemistry, immuno-fluorescence) can be used to investigate the distribution of proteins?
Each section of a sample will be incubated with a primary then secondary antibody.
The secondary anti-IgG is coupled to a fluorescent tag.
Describe how fusion protein construct can be used to investigate the distribution of proteins?
GFP is fused to the gene so when the protein is expressed, hybrid mRNA and a hybrid protein is produced.
How can you study whether a gene/protein is essential for development?
Genetics: gain- or loss-of-function
What is forward genetics and how do you study it?
Seeks to identify a gene whose mutation caused a particular phenotype.
Expose animal model to a mutagen and breed with WT for 3 generations.
Identify the gene which has mutated and caused the malfunction in this mutant.
(Phenotype —> genotype)
What is reverse genetics and how do you study it?
Seeks to characterise the phenotype of particular mutated gene.
Homologous recombination knock-in of target gene.
ES cell is transferred, selected and injected into blastocysts.
Chimeric mouse selected and bred to study phenotype.
(genotype —> phenotype)
How would you study how genes are regulated?
Embryology tissue manipulation (graft, ablation)
Manipulating signalling pathways with drugs, transferring, electroporation or genetics
Describe how tissue graft can be used to demonstrate inductive function?
Inserting presumptive epidermis into a blastocoel of zebrafish on the opposite side to the primary invagination.
This will result in a phenotype e.g. a second head and will tell you the function of this tissue.
Describe how bead/cell implantation (e.g. signalling molecules or drugs) can be used to demonstrate inductive function?
They mimic signalling molecular instead of using an ectopic piece of tissue.
E.g. transplanting ZPA in ectopic location results in mirror image
What techniques can be used for studying fate mapping (i.e. tissues and organs derived from cells that express gene)?
Embryology (chick/quail chimera, labelling with dye)
Genetics (labelling with retrovirus or GFP, brainbow)
How can cell/tissue transplantation be used to study fate maps?
Transplanting part of embryo from quail into chick.
The quail tissue can be detected by antibodies and traced over time.
How can cell/tissue labelling genetically be used to study fate maps?
Different cells can be marked with different colours and traced over time.
What is a morphogen?
A soluble secreted molecule that acts at a distance to specify the fates of other cells.
It may specify more than one cell type by forming a concentration gradient.
How do morphogenesis gradients cause different fates?
Cell have certain thresholds of concentration. If it reaches the threshold for a certain fate then the cell will follow that fate.
Morphogens decay exponentially. What would happen if a morphogen is:
- Increased
- Reduced
- Decays slower (‘long range’)
- Ectopic (e.g. morphogen produced in two places)
- Mutated
- Uniformly expressed
- More cells with highest response, same at lower concentrations
- Loss of highest response
- Less information as will not reach lower concentrations
- Mirror image
- No information so all one cell type
- All one cell type
What is the difference between an instructive signal and a permissive signal?
Instructive provides information whereas permissive signals provide a switch (already had signal but waiting for timing cue) and is not a morphogen
How would you test whether a signal is instructive or permissive (2 methods)?
Adding an ectopic source with the the signal. An instructive signal will produce a mirror image whereas permissive will have no effect.
Introducing a signal at a uniform concentration will induce one cell fate if instructive and have no effect if permissive
Why is a “bucket brigade” mechanism not used by morphogens?
Morphogens act directly on cells. Bucket brigade involves induced cells producing their own signals.
How would you test whether a ligand is a morphogen or using the bucket brigade mechanism?
Using genetic engineering to make proposed morphogen juxtacrine so that it is on the membrane of the producing cell and only affects neighbours. If morphogen then other cells will not see signal and will not respond whereas bucket brigade will.
Make a cell lose receptor for original ligand. Bucket brigade will not be affected as does not respond to this whereas if morphogen cell without receptor will not respond.
What helps establish a morphogen gradient?
Restricted diffusion generates a steep gradient by having high concentrations of binding molecules (e.g. HSPGs) and receptors which bind molecules to the extracellular matrix
Rapid degradation of the signal can also help generate the gradient as well as planar transcytosis across cells
How is timing important in establishing morphogen gradients?
There must be a mechanism which blocks premature specification as the first cell would see all fates and if it continued to be produced then all cells would adopt the same fate. A ‘check point’ where the steady state of receptor activation is reached must be involved for cells to determine their fate.
What is the transcriptional read-out model?
Higher concentration of morphogen often results in a higher concentration of an activated transcription factor
How does enhancer affinity and genetic crosstalk result in reading the gradient?
Genes in a cell could have low or high affinity to the TF. If the concentration is lower then the high affinity gene is likely to be expressed. If the concentration is higher the both affinity enhancers will be activated. The low affinity enhancer will produce a repressor to inhibit expression of the high affinity enhancer (crosstalk).
How are strict thresholds achieved when the gradient is not steep?
Positive feedback - a transcriptional activator which binds to its own enhancer
What is hedgehog and what is its role in the drosophila embryo?
A segment polarity gene which acts in segmental patterning via a feedback loop with Wg/Wnt
How is the Hh ligand formed and released from membrane?
The protein is targeted and undergoes autoproteolyis and cholesterol modification. It then undergoes palmitoylation and as it is hydrophobic can move through membrane but is not easily diffused. The dispatched receptor and its ligand Scube are important in the diffusion of Hh where it interacts with (lipoprotein particles?, cytonemes? and) HSPG.
How is the Wnt ligand formed and released from the membrane?
The protein undergoes palmitoylation and palmitoleic acid modification. Wntless receptor helps membrane targeting, presentation and release as Wnt is very hydrophobic. Once released it interacts with (cytonemes?, lipoprotein particles? and) HSPG
What are cytonemes and how do they aid Wnt signalling?
Cellular protrusions which allow cells to reach out and contract other cells. Wnt becomes exposed to the tip of the cytoneme and as it grows out via anterograde transport it will reach a Wnt-receiving cell and change its fate.
What is the “handover” mechanism of WNT diffusion?
Dlp protein binds to Wnt. Dlp is coupled to the membrane via GP1 link so can be motile within the cell and laterally diffuse over membranes. Could also be possible that they can hand over Wnt molecules to other Dlp molecules on another cell.
Palmitate can block the binding pocket shielding the hydrophobic nature of Wnt
How is the Hh signal signal received?
When Hh binds to receptor Ptch, it no longer inhibits Smo. Smo relocates, accumulates and phosphorylates and increases the level of cholesterol on the inner leaflet.
What are the Hh downstream events?
When Hh binds to Ptch, Smo is no longer inhibited. Smo causes the Cos2-Fused-Ci complex and the SuFu-Ci complex to modify and become CiAct (activated TF). This is due to the CKI-PKA-GSK3 complex (which normally phosphorylates Cos2 complex) and Slimb (which usually is involved in ubiquitination of phosphorylates Cos2).
How does Hh signalling act on its own pathway?
Negatively by limiting activation levels of Ptch
Positively by inducing Gli1 (acts similar to Ci) which cannot be proteolysed into a repressor
How is Hh involved in Drosophila wing patterning?
It’s expressed on the posterior side and diffuses across to induce a BMP like signalling molecule which acts to pattern the anterior-posterior plane
How is Hh involved in neural development?
It is produced in the floor plate and notochord. Cells in the neural tube differentiate into different types of Neuron’s depending on how long and how much Shh they receive
How is Shh involved in limb development?
It creates a ZPA which forms the anterior-posterior pattern.
It also has inhibition pathways and a signalling loop to produce outgrowth.
What are two diseases caused by both a loss of Hh signalling or too much Hh signalling?
Loss: holoprosencephaly, cyclops
Gain: polydactyly, syndactyly
What could cause a gain of Hh signalling resulting in cancers such as basal cell carcinoma, medulloblastomas and rhabdomyosarcomas and Gorlin Syndrome come about?
Inactivation of Ptch1 or Sufu (tumour suppressor genes)
Activating mutations of Smo (proto oncogene)
Gorlin syndrome is due heterozygosity for Ptch1 therefore damage to other copy of Ptch1 e.g. by sunburn will result in activation of Hh signalling
How is Wnt received?
Ligand binds to both Arrow/LRP5&6 receptor and Frizzled receptor. This brings them together forming a receptor complex and it is likely this is what causes downstream activation of pathway
What are the downstream effects of Wnt binding?
Dsh phosphorylated and binds to Frizzled. Arrow is phosphorylated and binds to APC-Axin-CK1-GSK3 complex. This is phosphorylated and Slimb (ubiquitination complex) is lost. Beta-catenin can be released and displace the Grouch transcriptional repressor, activating expression of target gene.
What are the roles of Wnt in Drosophila and C elegans?
Segmentation and expressed in D/V boundaries in Drosophila
Regulation of neuronal fate and migration in C elegans
What are some diseases related to loss or gain of Wnt signalling?
Loss of Wnt can cause a loss of stem cells in the gut
Gain of Wnt signalling results in colon cancers, breast, ovarian, uterine cancers, melanomas, prostate cancer, bone diseases, tooth agenesis
What is an example of non-canonical Hh signalling?
In myocytes and adipocytes Shh activates Smo which activates Ca2+ and Ampk signalling. This results in metabolic reprogramming towards aerobic glycolysis producing lactate and ATP.
Inhibitors of canonical Hh signalling activate the non-canonical, and could lead to too much Ca2+ and issues with muscles.
What is an example of non-canonical Wnt signalling?
Planar cell polarity/convergent extension pathway. Mediated by Wnt11, Wnt5, Frizzled and Dishevelled. These act to polarise cells within a sheet so affecting this pathway affects polarity (hairs on wings Drosophila and axis elongation zebrafish)
What are advantages and disadvantages of Drosophila as an animal model?
Advantages: accessible embryology and adult development stages, very low cost, fast, excellent genetics, no ethical concerns
Disadvantages: not a vertebrate, kept as live stocks
What are the main features of a Drosophila larvae?
3 thoracic segments and 8 abdominal segments. Mouth apparatus on thoracic end and posterior spiracles (for breathing) and anal pads on posterior end. Dentricles (lines of hair) on underside of larvae along abdominal segments.
What are techniques that use transgenic animals to either visualise, misexpress or reduce gene expression?
P-element transformation - transgenics Enhancer trap - promoter trapping Gal4/UAS - gene misexpression FLP/FRT - ‘clonal’ mutant analysis RNAi - both ex vivo and in vivo ‘Omic’ technologies - genome, transcriptome, proteome etc
Describe spermatogenesis in the Drosophila testes.
Hub cells in apical top of testes secrete factors e.g. Unpaired (JAK/STAT pathway ligand). The germline stem cells are located adjacent to the hub and cells further away will differentiate forming spermatogonial cells. Eventually will divide mitotically and meiotically into mature sperm.
Describe oognesis in the Drosophila ovarioles.
Stem cells at tip of ovariole maintained by JAK/STAT pathway. They divide until 32 cells (4 cytoblast mitotic divisions) but remain attached by bridges. One of the cells which has 4 attachments will become the egg, and undergo ‘meitotic’ recombination, and surrounding cells will become nurse cells and undergo endo-reduplication.
As it moves through the ovariole, follicle cells and nurse cells provide the oocyte with yolk, fats, lipids and maternally supplied proteins so it can grow and become fertilised.
How do nurse cells provide food and proteins etc to the developing oocyte?
Cytoplasmic dumping through ring canals
How do bicoid (anterior), oskar mRNA (posterior) and other mRNAs (dorsal) become localised in the oocyte?
Microtubule transport
Minus and plus ended motors
Glue anchors them in position
What happens in the first 14 divisions of a fertilised oocyte?
The nuclei of sperm and oocyte fuse and divide. By the 8th division most of the blastoderm nuclei are around the outside of the egg and this continues. At the 14th division there are vitellophages in the middle and pole cells at posterior as well. After the 14th division cellularisation occurs.
What are some common intracellular signals used Drosophila?
Hh Wnt Delta and Serrate TGF-alpha and -beta FGF
What is the role of the gap genes in Drosophila, what is an example and what would a mutation in this gene look like?
Gap genes are involved in development of a section of an organism. A mutation results in loss of a body segment e.g. knirps mutation sees loss of middle of the body
What is the role of pair rule genes in Drosophila, what is an example and what would a mutation in this gene look like?
Pair rule genes help define alternating segments. A mutation will result in loss of every other segment e.g. paired mutation will see every second segment pair lost therefore half the size.
What is the role of segment polarity genes in Drosophila, what is an example and what would a mutation in this gene look like?
Segment polarity genes help define the anterior and posterior polarities within each embryonic parasegment. A mutation will result in loss of naked cuticles e.g. gooseberry mutation will see all dentricle belts fused together.
What is the role of Bicoid and what does a Bicoid mutant look like? Can it be rescued?
Bicoid is a DNA binding transcriptional activator maternally loaded into a developing oocyte.
Mutant loses its anterior structures.
Yes can be partially rescued by transferring Bicoid WT cytoplasm to anterior part of body. Transferring to middle of body results in ectopic head structures and mirror image thoracic segments.
How does different Bicoid concentrations affect the segmentation pattern?
No Bicoid pushes everything anteriorly and top two segments missing.
Increased Bicoid pushes the pattern backwards.
How do enhancers on DNA help Bicoid form the French Flag Model?
Strong (high affinity) AND weak (low affinity) enhancers on a gene will be expressed at very low concentrations.
Just strong enhancers will express a gene at low concentrations.
Just weak enhancers will require higher concentrations to be expressed.
How do segmental genes form expression patterns?
From maternal genes gradients e.g. bicoid. Read these gradients to define blocks or domains of gene expression
Where is engrailed segment gene expressed in parasegment boundaries (and how does this compare to segment boundaries)?
On the posterior.
Segment boundaries are not molecularly determined but instead define each segment so engrailed is expressed anteriorly.
Why do both Hh and Wg mutants result in loss of naked cuticle and so show lawn of dentricles?
They feedback on each other to maintain each other’s continued expression and refine segment borders. As Wg is needed to suppress hair growth and Hh maintains this, loss of either will result in loss of naked cuticle.
True or False?
Expression of homeostatic genes along the D/V body axis occurs in the same order as the genes are within the genome.
False. They are the same as the A/P body axis.