WHOLE MODULE Flashcards
What are the two opposing theories of early development
Epigenesis - Aristotle belief that development is a gradual process, stage by stage, whereby new functions and characteristics are added on top of others. The other theory is that everything is already present and pre-established in the fertilised egg and all is required is amplification
What is meant by preformationism
The idea that organs develop from miniature versions of themselves as a homunculus (little human)
Explain Wissman’s theory of how cells become different
Two types of cells exist, somatic and germ cells. Germ cells contain determinants (germ plasm) whereas somatic cells only contain a subset of these determinants which define its particular set of characteristics
Recall the experiments carried out by Wilhelm Roux on frog embryos
Roux killed half of the cells at the 2 and 4-cell stage and looked at this effect on the developing embryo. It was noticed that ablation of these cells lead to the production of half embryos. This lead him to believe that at that stage each cell contributes uniquely to the development of the embryo and that the cell that wasn’t killed is only capable of adopting its primary fate.
How do the experiments carried out by Hans Driesch differ to those of Roux
Hans Driesch carried out similar experiments on sea urchin embryos. He divided the embryos up at the 2 and 4 cell stage and allowed them to develop, each cell in isolation from the others. He believed that these cells would give rise to the relevant part of the body plan already decided on. However each of the separated cells gave rise to a separate sea urchin proving that these cells were totipotent and could give rise to any of the cells of the body.
How does cell shape influence behaviour
Epithelial cells are fixed and immobile whereas mesenchymal cells allow migration. Thus epithelial to mesenchymal transitions (EMTs) are essential during development particularly in gastrulation.
What are the four types of cell-cell communication
Paracrine, autocrine, Juxtacrine and endocrine
Which types of cell signalling occur in development
Paracrine, autocrine and Juxtacrine
Which type of cell-cell communication involves a factor acting on receptors on the same cell that produced it
Autocrine
Which type of cell-cell communication involves two cells communicating with each other by direct contact
Juxtacrine
Which type of cell-cell communication involves a factor acting on a receptor on a neighbouring cell
Paracrine
Give an example of a juxtacrine signalling mechanism seen in development
Delta-notch
Give example(s) of paracrine/autocrine signalling mechanisms seen in development
Shh, Wnt, TGF?, BMP and FGF signalling
What are the two types of cell division seen during development and how do they differ
Symmetric cell division - progeny divide and give rise to two identical daughter cells. Asymmetric cell division – progeny divide and give rise to two daughters with different characteristics
How is asymmetric cell division achieved
Cytoplasmic, extracellular or membranal determinants are segregated/distributed unevenly in the precursor cells so that when it divides the daughters contain different levels
Every cell has the potential to communicate with other cells, T or F
F - some cells dont
Cells need to be competent to respond to a signal by receiving and transmitting a signal to the nucleus, T or F
T
What type of signal initiates a new program
Instructive signals
What type of signal provides a favourable environment for a specific program
Permissive signals
What is meant by a morphogen
A morphogen is a chemical (usually a diffusible signalling molecule) whose concentration varies forming a concentration gradient
What are the three requirements of a molecule for it to be a morphogen
Produced from a localised source, usually sensed through cell surface receptors and cells respond differently to difference concentrations of the morphogen
What are the common features of signal transduction pathways
Ligand binding to a receptor causes its activation which leads to the transduction of the signal to the nucleus via a cascade if secondary messenger activation. This in-turn leads to the activation of a transcription factor which induces the transcription of specific target genes
Gene content is identical in most cells and transcription/translation dictates the protein content of a cell, T or F
T
At what levels does the cell operate control on a gene program
Production of mRNA, processing and stability of mRNA, production of proteins and the activity of proteins
What are the two ways in which the cell controls the genetic program at the level of gene transcription
Differential gene expression, enhancer-mediated control
What other two methods are the of controlling the genetic program
miRNA, controls at the level of chromatin
What feature of cells is essential for the normal development, growth and repair of organs and how is this achieved
Tissue homeostasis - achieved by a balance of new cell formation and knowledge of how many cells are required by a tissue and when it needs to build more
What are the effects of disruption of tissue homeostasis
Contributes to ageing and degeneration
What are the four processes underlying development
Pattern formation, morphogenesis, cell differentiation and growth
What is meant by pattern formation
The organisation of cells within the body and how they occupy particular points in the xyz axis
What is meant by the term morphogenesis
The creation of shape
Outline the process of morphogenesis
How a cell/organ/tissue adopts a particular 3D shape and how cells move and reorganise during development to generate functional organisms
What four attributes of cells determines morphogenesis
Adhesion, migration, death and shape
Define cell differentiation
The process by which cells become different from one another and acquire specialised properties
What governs differentiation
Changes in gene expression which dictate the repertoire of proteins synthesised by a cell
Differentiation is an increase in cell fate and specialisation over time that corresponds to a decrease in pluripotency, T or F
F - differentiation is a gradual restriction in cell fate over time whereby specialisation increases and pluripotency decreases
What attributes of cells in development contributes to growth or a tissue/organ
Cell proliferation, enlargement and accretion
What is meant by cell accretion
Growth of a tissue/organ that occurs by a gradual accumulation of additional layers of cells
Describe Haeckels funnel model of development
Embryos start off developing similarly but become progressively different as time goes on
How does the Von Baer Hourglass model differ to the Haeckel funnel model
Embryos start off developing differently, but then converge at gastrulation, showing substantially conserved phenotypes. After this point they then diverge again
How can the spatial and temporal expression of a gene of interest be studied in development, what can you actually measure
You can investigate where and when the mRNA of this gene is transcribed
List the 5 techniques that can be used to determine where/when a gene is expressed in the embryo
In situ hybridisation, northern blotting, reverse transcriptase PCR, micro-arrays and reporter lines
Outline the process of in situ hybridisation
A probe with complementary base pairs to the target gene is synthesised and labelled with a digoxigenin (DIG) immune-tag. This will bind to the target sequence of the gene of interest and then a secondary antibody binds to the DIG label. Binding of the secondary antibody leads to an enzymatic reaction that produces a reaction product that indirectly marks all the cells that produce that specific mRNA.
What is the major disadvantage of in situ hybridisation
You have to kill the embryo in order to carry out the technique
Explain the process of reporter line transgenics
Introduce a coding sequence for a reporter gene i.e. GFP, RFP or ?-gal into the coding sequence of the gene of interest. This will result in the reporter gene being under control of the target genes promoter sequence. Hence, wherever the gene of interest is expressed the reporter will be too. This can be easily visualised or investigated
What methods can be used to determine if a protein is actually expressed at the same time as a gene by investigating if the mRNA produced is actually transcribed
Western blotting, immunohistochemistry
Explain how immunofluorescence can be used to visualise where a protein is expressed during development
Introducing labelled tags to antibodies for various epitopes of a protein of interest allow you to visualise where these epitopes and proteins are expressed
Gain and loss of function experiments are used to investigate if target genes are essential in development, T or F
T
How can loss of function mutations be used to test for essential genes in development
By introducing a premature STOP codon into the target sequence you can investigate how essential a gene is in development. Loss of function mutations results in a disruption in the expression or function of the mutated genes
How can gain of functions mutations be used to investigate essential developmental genes
Mutations in regulatory regions of a DNA sequence can lead to increased levels of transcription. This confers a gain in the activity of the mutated protein
What is the difference between forwards and reverse genetics
Forward genetics investigates the identity of genetic mutations responsible for a specific observed phenotype. In contrast, reverse genetics seeks to characterise the phenotype produced from a particular gene mutation
How can tissue ablation be used in the study of gene regulation during development
Tissue ablation and ectopic grafting experiments can be correlated with levels of transcription factors. You can also investigate is a transcription factor soaked bead is sufficient to direct gene expression
How can the tissues/organs that are derived from cells that express a certain gene be elucidated
This is investigated in order to determine if every single tissue expressing a factor originated from the early structure expressing it. This can be investigated through fate mapping and determination experiments.
Chick-quail chimera labelling with dyes has been used to investigate cell fate and determination through dye labelling of progenitor cells, T or F
T
Define a morphogen
A soluble secreted molecule that acts at a distance to specify the fates of cells
What are the two main attributes of a morphogen
Morphogens act at a distance to induce different output or cell fates at different concentrations by forming a gradient in the embryo.
How is higher information encoding achieved by morphogens
More information is encoded by the higher morphogen concentrations closer to the source
What two mechanisms can account for the different cell fates achieved by morphogens
Temporal diffusion of the secreted signals from the source to where it is destroyed/sunk. Similarly, the magnitude of the morphogen concentration also causes the acquisition of different cell fates
Why are morphogen gradients usually exponential decays
Morphogens move by passive diffusion throughout the embryo leading to the exponential decay appearance of the gradient
How may it be possible to achieve a straight linear morphogen gradient
Active movement of the morphogen may achieve a linear gradient
The shape of the morphogen gradient is what encodes the information to direct cell differentiation, T or F
T
All molecule involved in patterning of the embryo are morphogens, T or F
F – permissive signals aren’t morphogens
Why aren’t permissive signals morphogens
Permissive signals only direct a cell to response to an instructive signal
Instructive signals are morphogens, T or F
T
What would be seen in a field of cells as a result of ectopic grafting of another source if the secreted factor was a morphogen
A mirror image of cell differentiation would be seen
What would be seen in a field of cells as a result of ectopic grafting of another source if the secreted factor was a permissive signal
This would have no effect on cell differentiation, all cells in the field would develop normally as the permissive signal only enables the cells to respond to a morphogen
Give an example of an experiment that has proved a gene product is a morphogen by ectopic grafting
Ectopic grafting of an shh soaked bead opposite to its normal site in the chick limb bud leads wing development that defined by a mirror image duplication of the digits etc.
What would be the effects of forcing a uniform gradient of a secreted factor across a field of cells if this factor was a permissive signal
There would be no abnormal effects on cell development. The same number of fates will be produced and each cell would adopt the correct fate
What would be the effects of forcing a uniform gradient of a secreted factor across a field of cells if this factor was an instructive signal
This would result on the field of cells only adopting one fate as all cells would receive the same ligand concentration
Explain what is meant by the bucket brigade signalling mechanism and why this is therefore not used by morphogens
The bucket brigade mechanisms uses sequential signalling of the cells that acts immediately on the adjacent cell. Expression of one signal induces a target cells to produce another different signal. This signal acts on the next cell in the sequence to induce production/secretion of another different factor and so on. As this signalling occurs between adjacent cells and not over distance, this isn’t a type of morphogen signalling
How can you experimentally modify a potential morphogenic molecule to make it juxtacrine
Using genetic engineering to add a transmembrane domain region to the protein sequence to make it a membrane bound signal
What would be the effects of making a morphogen juxtacrine and how would this differ if the signal was part of a bucket brigade signalling mechanism
If the ligand was a morphogen, making it juxtacrine would lead to only the first cell being induced to adopt its fate as the ligand will be unable to act at a distance and direct differentiation by diffusion. This would be due to it becoming membrane bound and only able to act on adjacent cells. If the factor was part of a bucket brigade mechanism, then the signalling would be unaffected as adjacent cells would still receive their ligand and thus go onto produce the next sequential secreted factor.
How would creation of a genetic mosaic lacking a morphogen receptor influence differentiation
The cell lacking the receptor would adopt the same fate as the terminal cell in the field would normally. This would be due to the fact that the terminal cell usually receives the lowest/no morphogen signal. Cells after the one lacking the morphogen receptor would however development normally
How would the bucket brigade signalling mechanism be affected by a lacking receptor
Cell fate wouldn’t be effected if the receptor knocked out from a cell was one for a ligand that acts upstream of the cell in the sequential signalling pathway. This is because at this point in the cell field, this ligand is no longer acting and instead a different ligand is acting on the target cell produce by the proximal adjacent cells. However, if the receptor knocked out was the first receptor in the sequential sequence then none of the cells would adopt their fate and would remain undifferentiatiated
Shallowing morphogen gradients would encode far less information than steeper ones, T or F
T
How can a higher effective concentration of a morphogen be achieved
Preventing the morphogen from diffusing in axes that aren’t desirable
How can non-beneficial morphogen diffusion be prevented
Morphogen binding to molecules in the extracellular matrix such as heparan sulphate proteoglycans allows sequestration and facilitation of morphogen diffusion, effectively increasing the size/steepness of the gradient in desirable planes. This acts to increase the amount of information encoded by the morphogen gradient
Heparan sulphate proteoglycans are sometimes referred to as co-receptors and bind many different ligands, T or F
T
How can rapid degradation of the morphogen at the sink increase information encoding
Increases the steepness of the morphogen gradient
Decreasing the range of the morphogen gradient can be achieved by localisation of the morphogen in the desired range, T or F
F – this acts to extend the range
How can morphogens move through cells in a tissue and give an example of a molecule for which this is the cane
Planar transcytosis - Repeated cycles of endocytosis and re-secretion. Dpp is transcytosed and antibody staining has revealed its presence in vesicles
How is the timing of morphogen gradient establishment critical to cell differentiation
The cells are prevented from responding at an inappropriate time where the morphogen concentration won’t have reached that required by the particular cell to direct its correct fate. This is likely achieved by the cell waiting for a steady state of receptor activation to be achieved but the molecular mechanism by which this occurs isn’t understood
Explain how a cell interprets a morphogen gradient before responding and determining its fate
Morphogen concentration is directly correlated to the activation of transcription factors inside the cells. Higher concentration of morphogen often results in a higher concentration of an activated transcription factor. In this model, receptor activation causes transcription factors to enter the nucleus and direct transcription. Levels of activated transcription factor determine the fate of the cell
Which morphogen is the only known one which itself is a transcription factor and how does it act
Bicoid is a morphogen and a transcription factor. Bicoid mRNA is localised at the anterior of the egg and is translated into protein during early embryogenesis. Bicoid protein then diffuses through the cytoplasm and accumulates in nuclei of the syncytial blastoderm generating a concentration gradient
How do different levels of transcription factors dictate the different fates of cells under the influence of a morphogen
Binding of a transcription factor to the promoter seuequence of genes that confer a particular cell fate is an equilibrium reaction with an on and off rate. The strength of the equilibrium that’s moving gene expression towards its on state will control how strongly the gene is expressed. Increasing transcription concentration factor would increase on state by shifting the equilibrium to the right to counteract this increase in transcription factor levels. The concentration of activated transcription factor determines if it binds to high or low affinity sites. Each of these sites dictate a different cell fate and differential gene expression
What happens at low-medium transcription factor levels and how does this confer a particular cell fate
At low-medium concentration of the transcription factor only the high affinity sites will elicit binding which will direct a particular cell fate and specific subset of gene expression
What happens at high transcription factor levels and how does this confer a particular cell fate
At high concentrations of the transcription factor the low affinity sites will now allow binding. Binding of the transcription factor to these sites will result in differential gene expression and a different cell fate. More ligand will increase activation of low affinity sites
How do the low affinity sites allow differential gene expression if the high affinity transcription factor binding site are also being activated
One of the genes switched on by transcription factor binding to the low affinity sites will turn off by transcriptional repression) the high affinity genes in a process called crosstalk
Explain how strict thresholds of cell differentiation are achieved even if the morphogen gradient isn’t steep
Positive feedback helps a cell commit to its specific fate. If one of the genes switched on by high affinity site binding encodes a transcription factor – it can, amongst other things, activate its own expression
Drosophila melanogaster is a segmented animal, T or F
T
The genome of Drosophila has been sequenced, T or F
T
Drosophila can regulate their own body temperature, T or F
F – they cannot
What two temperatures are stocks of Drosophila kept
18 and 25°C
Which temperature stocks tend to have a lifecycle of around 10 days
25°C stocks
Which temperature stocks tend to have a longer life cycle of upwards of 21 days
18°C stocks
How much time elapses from laying of a fertilised Drosophila egg to hatching
24 hours
Briefly outline Thomas Hunt-Morgan’s contribution to Drosophila research
Hunt-Morgan noticed a white-eyed fly in his lab one day. He bred this fly with wild type flies to investigate the phenotypes produced
What was Sturtevants contribution to Drosophila research
Constructed the first genetic map and arranged it in a linear order
Bridges showed that chromosomes must contains genes, T or F
T
Muller showed that radiowaves caused mutations and chromosomal rearrangements, T or F
F – he showed that X-rays caused mutations
Outline Nusslein-Volhard and Wischuas contribution to Drosophila research
Carried out a saturation mutagenesis to identify genes in Drosophila involved in development and patterning. This was an absolutely massive screen and lead to the identification of 139 complementation groups
Describe the general arrangement of the Drosophila genome
4 chromosomes consisting of 3 autosomes and 1 sex chromosome. Roughly 17,000-18,000 genes, 14,000 of which code for proteins
What was seen as a result of alignment of homologous sequences of genes between Drosophila and other organisms
The protein encoding exons were almost perfectly conserved. The non-coding introns were seen to only be randomly conserved and showed differences between organisms most likely due to not being under any selection pressures. Regulatory sequences in the genes also showed high levels of conservation between species
Drosophila can produce 200 progeny in less than 2 weeks, T or F
T
Put these Drosophila courtship processes in order, orientation, licking, attempted copulation, tapping, wing vibration
Orientation, tapping, wing vibration, licking, attempted copulation
Female Drosophila mate several times in their life cycle while males mate only once, T or F
F – vice versa
The strongest Drosophila reproducible behaviour is genetically encoded, T or F
T
Outline spermatogenesis in Drosophila
The testes located within the abdomen on the male contain hub cells. These cells secrete factors such as unpaired which maintains stem cell fates in the cells adjacent to the hub. Unpaired is a JAK/STAT ligand and its signalling decreases with distance from the hub. Cells further away from the hub differentiate due to decreased levels of unpaired signalling. These cells divide and give rise to 64 sperm cells
What is remarkable about sperm produced by male Drosophila
Spermatids are 25x the length of the adult Drosophila body
Explain the interplay between the sexual evolution of male and female Drosophila
The sperm produced by male Drosophila is coated in a sex peptide. This binds to receptors in the brain of female Drosophila and prevents them from being able to respond to subsequent courtship dances by other males. This increases the likelihood of the males passing on their genetic information. However, females evolved the ability to repress the effects of the sex peptide. This caused the males to evolve and produce larger and larger sperm coated in more and more sex peptide to try and increase the females inability to respond to other courtship dances.
Outline the process of oogenesis in Drosophila
Stem cells are maintained at one end of the bundled linear structures that make up the ovaries. Cells in the ovaries undergo 4 incomplete cytoblast mitotic divisions. Most cells become nurse cells which undergo endoreduplication making multiple copies of the genome and expressing a huge amount mRNA that supplies the oocyte with the genetic information to synthesise proteins etc. The cells that go on to form oocytes do so by undergoing meiosis to produce cells containing mostly cytoplasm.
What is meant by cytoplasmic dumping and how does this occur
The nurse cells that supply the developing oocyte contain massive amounts of genetic information and are transcribing a vast amount of mRNA. This mRNA is then deposited through ring canals into the cytoplasm of the oocyte so that it can synthesise useful products.
Explain how polytene chromosomes are produced and what they represent
Polytene chromosomes are chromosomes stained for polymerase activity. Bands represent regions of the genome where there is little activity. Interband regions contain puffs which represent extremely active and transcribed genes.
Explain how some polarity is established in the oocyte even before fertilisation
Some of the factors deposited by the nurse cells through cytoplasmic dumping become localised (i.e. through gravity and other mechanisms). This localisation of factors accounts for the inherent polarity of Drosophila oocytes
Describe some of the features of the Drosophila egg
Chorion acts as the eggshell surrounding the embryo. The vitelline membrane lies beneath the chorion and provides waterproofing. The micropyle located at one end of the egg is the opening through which sperm enter.
Describe what is meant by a syncytial blastoderm and how this is achieved in the Drosophila embryo
Syncytium is a multinucleate cell. This is achieved in Drosophila through nuclei duplication in the centre and then migration to the periphery. After 14 rounds of nuclei duplication and after they have relocated to the edges of the embryo the cell membrane moves in and encapsulated the nuclei creating a cellular blastoderm.
What happens to the cells that are retained in the centre of the embryo before cellularisation
These become pole cells and give rise to the gametes and gonads of the adult organisms
What are the names given to the two regions of the embryo lower order animals (not humans) that give rise to the three germ layers and extraembryonic structures
Epiblast – gives rise to the germ layers, hypoblast – forms vegetal pole/hemisphere
In which regions of the morula would you find the structures that give rise to the embryo and to the extraembryonic structures
Top of the morula gives rise to the epiblast, bottoms gives rise to the hypoblast
What changes govern the formation of these two layers in the morula
Governed by morphogens and transcription factors that lead to changes in gene transcription
What causes the hollowing of the morula during development alongside squashing
Changes in osmolarity
Which region of the morula ultimately ends up undergoing apoptosis
Hypoblast
At which stage does the primitive streak in chick embryos form at the posterior of the embryo
Stage 3
At stage 4 of chick development, the primitive streak has elongated and Hensen’s node has formed anteriorly, what does this structure then begin to express
Expresses and secretes BMP antagonists such as chordin, noggin and follastatin
At what stage in chick development does the notochord begin to form
Stage 5-6
What structure then extends forward from the node and is involved in anterior-posterior axis formation
The notochord
Why are the events from morulation to gastrulation less studied in mammalian embryos and why is this
Mammalian embryos have been less extensively studied because they are difficult to maintain in culture after the blastula stage due to this being the stage at which they normally implant into the uterus
At five days post-fertilisation what structure forms in the mammalian embryo that is similar to the hypoblast
Trophoblast
What day corresponds to chick development stage 2 and corresponds to the embryo implantation into the uterus lining
12 days post-fertilisation
At what stage in mammalian embryonic development is the embryo referred to as a blastula
12 day stage
The processes occurring in chick embryos from stage 2-6 occur in 2-4 week old human embryos, T or F
T
At which region of the developing embryo does gastrulation and germ layer formation begin at
Posterior of the embryo
What is the first structure to become visible in the initial stages of gastrulation
Formation of the primitive streak due to cells moving into the midline forming a line that elongates anteriorly
What forms at the most anterior point of the primitive streak
(Hensen’s – in chicks) node
Which regions of the embryo does the primitive streak denote
Posterior/caudal regions
The epiblast gives rise to mesoderm, endoderm and ectoderm, T or F
T
Explain how signals from the hypoblast layer of the developing embryo accounts for the formation of three germ layers by the overlying epiblast
Signals from the hypoblast (trophoblast) induce some epiblast cells to become mesoderm and definitive endoderm. These cells involute and ingress along the anterior-posterior axis by losing contact with one another and migrating into the embryo. Superficial migrated cells become the mesoderm and the first cells to have ingressed, those now lying deeper within the epiblast will become endoderm.
How does the migration of cells into the embryo at the primitive streak account for the formation of both mesoderm and endoderm
Cells that migrated first from the epiblast layer will lie deeper within the embryo and will become the endoderm. Those that migrated later will be lying for superficially and will become mesoderm.
Once primitive streak reaches maximum extension and forms Hensen’s node, a similar event takes place whereby cells ingress but begin to move directly anteriorly/rostrally, T or F
T
What is different about the gastrulation of Xenopus embryos compared with mammal/human early embryonic development
The Xenopus embryo doesn’t compact to form a blastodisc as mammalian embryos do. It remains morula-like with an animal cap and a vegetal hemisphere
What structures in the Xenopus embryo correspond to the epiblast and hypoblast seen in mammalian embryos
Animal cap – epiblast, vegetal hemisphere – hypo/trophoblast
Explain how oocytes are already polarised before fertilisation
Particular determinants inside the egg have become localised in the vegetal hemisphere of the cell. This is due to gravity in Xenopus and an interaction with the placenta in mammals
What is the result of the inherent polarisation of the egg prior to fertilisation
Different cytoplasmic determinants have sunk to one part of the egg. This leads to the cells that come from this region being different and having specific factors localised/activated
In an early cleavage event, the vegetal and animal hemispheres of the now fertilised egg are separated, what is the effect of this on cytoplasmic determinant localisation
Cytoplasmic factors restricted to the vegetal part of the oocyte are now restricted to vegetal hemisphere cells
What is the effect of the factors that become localised to the vegetal hemisphere cells of the embryo as a result of early cleavages in the embryo that separate the animal and vegetal cells
These factors localised in the vegetal part of the embryo bind to promoters for particular transcription factors and upregulation. In-turn, these particular transcription factors become solely expressed in vegetal hemisphere cells
What is the name of the transcription factor that is specifically expressed and localised to vegetal hemisphere cells
VgT
Describe what happens as a result of the localisation of a specific transcription factor in the nuclei of cells in the vegetal hemisphere of the early embryo
Localised VgT in the nuclei of cells in the vegetal hemisphere leads to its binding to the promoter of a gene called Nodal and upregulate its expression. The Nodal morphogen then leaves the cells of the vegetal hemisphere and binds to receptors on cells in the animal hemisphere
What is the name of the morphogen expressed in the vegetal hemisphere cells that is upregulated as a result of VgT binding to its promoter
Nodal
What is the effect of the morphogen produced by cells of the vegetal hemisphere
Nodal binds to receptor on cells in the animal hemisphere and dictates the mesoderm and endoderm formation via cell proliferation, migration and differentiation.
What happens to cells at the top of the animal hemisphere as a result of VgT activity on its promoter
The Nodal morphogen produced as a result of this leaves the cells and binds to receptors in the animal hemisphere. It forms a concentration gradient throughout the animal hemisphere. Cells at the top of the animal hemisphere don’t receive nodal signalling and so remain as ectoderm. This is thought to be due to either not expressing the nodal receptors or by not receiving a threshold concentration of the nodal ligand
Nodal is expressed throughout the vegetal hemisphere/hypoblast in the developing embryo. What aspects/axes of early embryonic development does this signalling mechanism not account for
Can’t account for anterior-posterior axis formation and the appearance of the primitive streak at the distal region of the embryo
Which signalling pathway is therefore responsible for anterior-posterior patterning and formation of the primitive streak
Wnt signalling
Wnt signalling mediates a secondary symmetry breaking event but unlike nodal, it is activated on one side of the embryo. Explain how this is achieved and where wnt activity is seen in the early embryo
The Wnt signalling pathway is activated on one side of the embryo, opposite to the point of sperm entry. This region is referred to as the dorsal embryo but also represents the site where gastrulation movements will begin, i.e. the posterior embryo.
Nodal is activated throughout the animal cap of the embryo, T or F
F – it is activated throughout the vegetal hemisphere
Which gene product accumulates in the nuclei of cells in the posterior/dorsal embryo as a result of increase Wtn signalling and promotes the expression of Wnt target genes
?-catenin
What is the Nieuwkoop centre
The region of the embryo where ?-catenin has accumulated in the nuclei and there is activation of Nodal signalling
How does ?-catenin and nodal interact
?-catenin and nodal interact directly and result in enhanced activation of Wnt signalling in regions of the embryo where ?-catenin is also expressed. The causes the conversion of what was a uniform gradient of nodal in stage 8 to a clear concentration gradient from anterior to posterior in stage 9 embryos.
Where is the highest nodal activity seen as a result of its interaction with ?-catenin
Highest nodal in the dorsal/posterior embryo
What is the role of the Nieuwkoop centre
Region of high nodal and ?-catenin signalling that induces the formation of the organiser
What happens to regions with low nodal activity
Induction of ventral mesoderm
What are the two crucially different types of mesoderm induced in the early embryo
Organiser mesoderm, ventral/lateral mesoderm
The mechanism of ?-catenin and nodal interactions is poorly understood, T or F
T
What two factors are required for goosecoid expression
Nodal downstream effector known as Smad2/4 binding to its distal region of the promoter and a Wnt/?-catenin downstream effector called Xtwn binding to the proximal element of the promoter.
Brachyury is induced in response to high levels of Nodal in the dorsal/posterior embryo, T or F
F – brachyury expression is induced in response to low Nodal signalling in the ventral/anterior embryo
What levels of nodal signalling are required for expression of T, another transcription factor involved in anterior-posterior patterning
T is expressed at low Nodal signalling levels
Chordin, Xnot and Xlim1 are other genes expressed alongside goosecoid that act as inducers of various mesodermal fates, what is required for their induction
High levels of nodal signalling and wnt signalling activation and subsequent ?-catenin presence in the nucleus
Different types of mesoderm have different abilities in terms of proliferation, migration and differentiation etc, T or F
T
The combinatorial expression of T, Gsc, Chordin and Tbx6 accounts for the differences seen in the mesodermal tissue, T or F
T
What rod-like structure does the organiser/node differentiate into
Axial mesoderm
Explain how goosecoid and siamois act in a cell-autonomous way to direct the differentiation of the node/organiser
Siamois and goosecoid expressed in the organiser/node in turn act on the cells that express them in a cell-autonomous, or intrinsic manner to alter their fate. They begin induce the cells of the node/organiser to differentiate into axial mesoderm
What is one key property of axial mesoderm and accounts for rolling up of the neural plate into the neural tube during neurulation
Axial mesoderm is able to undergo convergent extension
Siamois and goosecoid are expressed at uniform levels but only transiently in different cells. This accounts for the formation of the 3 different types of mesoderm derived from the axial mesoderm, T or F
F – whilst gsc and siamois expression is only transient, they are also expressed at marginally different levels in the cells too
What are the three different types of mesoderm derived from the axial mesoderm induced by the transient expression of goosecoid and siamois
Prechordal mesendoderm, prechordal endoderm and notochord
Which of the three mesodermal tissues derived from the axial mesoderm are most anterior and most posterior
Prechordal mesendoderm are most anterior and the notochord lies posteriorly
Explain the two hypotheses by which Hensen’s node is thought to express chordin and goosecoid
Either cells in Hensen’s node co-express both gsc and chordin or there are individual cells that express each transcription factor
Explain how the expression of Gsc and chordin differs between notochord and prechordal mesoderm
Gsc is only expressed in the prechordal mesoderm (and not in the notochord) whereas chordin is only expressed in the more posterior, notochord and not at all in prechordal mesoderm in the anterior embryo
Explain how anterior-posterior axis extension is mediated and refined in the posterior embryo
Signals that were initially close together become spatially separated through the anterior-posterior axis. High FGF signalling and the presence of RA in the posterior embryo keep cells in a proliferative state and confers them to a posterior identity. The proliferation of posterior cells causes the addition to the axis as it elongates posteriorly
What happens in the anterior embryo
BMP and Wnt antagonises maintain the anterior identities
Anterior-posterior identity is through to be realised through the Hox code, T or F
T
Neurulation of ectoderm and ventralisation of the mesoderm are both mediated by BMP antagonists, T or F
F – neurulation of ectoderm and dorsalisation are mediated by BMP antagonists
BMP antagonists are secreted by which structure
The organiser/node
Other than chordin, name four other BMP antagonists
Follistatin, frizbee, cerberus and noggin
Where do the BMP antagonists end up and prevent the BMP signal in
Top layer of epiblast
How do BMP antagonists act
Block the interaction of BMPs with their receptors
What happens to the cells where the BMP signal is inhibited
They will become neural plate
Explain how BMP antagonists lead to the refinement of cell fates in the mesoderm
Initially, low levels of Nodal give ventral mesodermal fate. Subsequent inhibition of BMPs in the adjacent mesoderm to the neural tube induces the dorsalisation of what was ventral mesoderm initially. By this process the most proximal mesoderm to the organiser become the somites
What feature of the mesodermal tissue leads to the rolling up of the neural plate during neurulation and how does this occur
Convergent extension of the axial mesoderm creates a force in the anterior-posterior direction that is translated to one which drives curling up of the neural plate with somites residing either side.
Where does wnt derive its name from
Amalgam of wingless Drosophila gene and Int vertebrate proto-oncogene
Int-1 is a proto-oncogene, what causes its activation
Integration of the mouse mammary gland tumour virus
Wingless (wg) mutants initially discovered produced wingless but viable flies, T or F
T
Explain how wg and hh maintain each other’s expression in an auto-regulatory loop
Wg maintains hh by controlling the expression of engrailed (en), a transcription factor that regulates hh expression. Hh then in-turn maintains and directly upregulates wg
What is significant about hh and wg knockout mutants
They exhibit the same phenotype – larvae with a lawn of denticles
Unlike similar developmental signalling pathways, wnt expression is highly conserved throughout Kingdom Animalia, T or F
T – even found in sponges
Why is it that vertebrates have more wnt genes
Due to genome duplication throughout evolution
The wnt protein is produce by a cleavage event that separates its signalling sequence from the initially translated protein, T or F
T
What is the role of porcupine in the early modification of wnt
Porcupine is an acyl transferase that adds palmitoleic acid modifications to a serine residue at point 209 in the wnt3a structure
What is the hypothesised role of wntless in wnt signalling and what is its basic structure
Wntless is a 7 transmembrane domain protein potentially required for the transport of wnt to the plasma membrane and its subsequent release/presentation to target cells
What is the effect of palmitoylation and palmitoleic acid modification of the wnt protein
Addition of these hydrophobic groups makes wnt insoluble in water
What components of the extracellular matrix are involved in mediating the diffusion of wnts away from the sending cell
Heparan sulphate proteoglycans (HSPGs)
What is significant about the fact that most of the effects of Wnt signalling can be elicited by a membrane bound form of the protein in Drosophila
It suggests that wnts act as juxtacrine signalling molecules or that they don’t diffuse far and act on adjacent cells in Drosophila
Recall the two main receptors involved in reception and transmission of wnt signalling in Drosophila
Frizzled and Arrow
What is the name of the nuclear factor in Drosophila that is induced as a result of wnt signalling and its corresponding vertebrate homologue
Armadillo (vertebrate homolog – ?-catenin)
What are the names of the arrow receptor homologues found in humans
LRP5 and 6
Both the frizzled genes and arrow/LRP5&6 act in combination as receptors for wnt signalling molecules, T or F
T
Describe the structure of the frizzled receptor and how it interacts with wnts
7 transmembrane domain protein. Wnt binds to the cysteine-rich domain (CRD) in the N-terminus of the Fz protein
Describe the structure of the LRP5&6/Arrow receptor for wnt
Single pass transmembrane protein
What happens when wnt binds to LRP5/6/Arrow and the Fz receptors
These two receptors come together to form an active wnt signalling complex
Which important extracellular wnt inhibitor is overexpressed in order to downregulate wnt signalling in experiments
Dickkopf1 (Dkk)
How does Dkk act to downregulate wnt signalling
Dkk is coupled to Kremen. Activation of Dkk by wnt binding promotes the internalisation of the LRP receptors
Describe the composition of the degradation complex involved in wnt signalling
Consists of the scaffold protein axin bound to APC, GSK3?, CK1? and slimb
Describe what happens in the absence of wnt signalling
Also bound to the degradation complex via an interaction with APC is ?-catenin. In the absence of wnt signalling ?-catenin is phosphorylated by CK1? and then by GSK3?. This poly-phosphorylated ?-catenin is then recognised by the slimb protein which ubiquitinates the ?-catenin marking it for degradation by the proteasome system. With low levels/absence of ?-catenin T cell factor (TCF) transcription factors are bound to the promoter regions of wnt target genes. Also bound to these TCFs is a transcriptional repressor known as groucho. Groucho inhibits the transcription of wnt target genes
Describe what happens in the presence of wnt signalling
Wnt binds to its Fz and arrow/LRP 5&6 receptors in the membrane. These receptors come together and form an active complex which recruits the dishevelled protein to the complex. Dishevelled is then phosphorylated and as a result may bind to axin in the intracellular destruction complex. Arrow/LRP is then also phosphorylated this time by GSK3? and the receptor recruits axin also. Binding of the destruction complex to the receptor complex displaces the slimb protein. With slimb lost the destruction complex is inactivated. ?-catenin then accumulates inside the cell due to it not being ubiquitinated and marked for degradation by slimb. It then translocates to the nucleus of the receiving cell and displaces groucho from the TCF DNA binding proteins. In combination with additional downstream transcriptional activators this leads to the transcription and expression of wnt target genes.
Explain how ?-catenin degradation is achieved by the destruction complex
?-catenin is phosphorylated by CK1? first, which primes it phosphorylation by GSK3?. Phosphorylation by both kinases is required for ?-catenin recognition by an E3 Ubiquitin ligase complex (which contains b-TrCP/Slimb) and subsequent degradation by the proteasome. The serine/threonine phosphates and surrounding amino acid sequence in ?-catenin as a result of phosphorylation forms an optimal binding site for b-TrCP/Slimb. ?-TrCP/Slimb binds only after GSK3 phosphorylates the 3rd and 4th phosphorylation sites
Where does CK1? phosphorylation occur within the ?-catenin/armadillo structure
Sites within the N-terminal tail
The S/TXXXS/T(P) is the ideal site for GSK?, what does this mean
A serine or threonine residue followed by 3 residues of any identity and then another serine or threonine that has been phosphorylated by CK1?
What is the name of the vertebrate homologue of slimb
?-TrCP
Describe the structure of the SCF E3 ubiquitin ligase complex involved in ?-catenin/armadillo degradation
The Skp1-Cullin-F-box E3 ubiquitin ligase complex consists of the ring finger protein Roc1 which binds to an E2 ligase, the scaffold protein cul1 and skp1
Explain the role of SCF in ?-catenin/armadillo degradation
The F-box protein interacts with Skp1 via its F-box. The F-box of also interacts with the substrate via the WD40 domain that interacts specifically with phosphorylated targets
How does the absence of Wnt signalling lead to no expression of wnt target genes
Without ?-catenin binding to TCF, groucho remains bound. The transcriptional repression by groucho is mediate by its recruitment of histone deacetylases thought to make DNA refractive to transcriptional activation
How therefore does wnt signalling lead to expression of downstream target genes
In the nucleus increases levels of ?-catenin displace groucho from the TCF complex. Displacement of groucho leads to the recruitment of histone acetylase CBP/p300 and another transcriptional activator called BRG-1. These lead to transcription of wnt target genes
Explain how interactions between TCF/?-catenin and chromatin could also be mediated by legless (Bcl9) and pygopus genes
Mutations in these genes result in wingless-like phenotypes in Drosophila. Both genes also promote wnt signalling in mammalian cell cultures.
Wnt signalling components are also involved in directing planar cell polarity and convergent extension, T or F
T
Explain the negative feedback mechanism of wnt signalling
Dickkopf1 (Dkk) activation by wnt binding promotes the internalisation of the LRP receptors. This decreases further wnt signalling activation and has important homeostatic roles.
Give examples of planar cell polarity events caused by wnt signalling
Wnt signalling aligns all the hairs in the skin in a certain direction
Explain how defective wnt signalling can cause cancer, particularly in the gut
Patients who are heterozygotes for APC loss of function mutations suffer from familial adenomatous polyposis. This is where sporadic loss of the other functional wild type APC allele in the gut results in activation of the wnt signalling in such cells. This causes hyperproliferation and culminates in the formation of polyps which may accumulate further mutations and cause colon cancer.
The APC gene is a proto-oncogene, T or F
F – it’s a tumour suppressor gene (loss of function results in tumorigenesis)
Give an example of another disease phenotype caused by mutation(s) in wnt signalling
Tetra-amelia is a disease where the infant is born without limbs. This is caused by a mutation in wnt3
What are the two main problems faced during segmentation
Organism needs to increase from one cells to many cells and makes these cells different from each other
Segmentation is highly conserved in all organisms, T or F
T
Where is segmentation easily visible in the Drosophila embryo
IN the anterior abdomen
When does segmentation occur in Drosophila embryos
Around 24 hours after fertilisation
The Drosophila embryo must set up all of the body axes in order to undergo segmentation, T or F
F – the anterior-posterior and dorsoventral axes have already been partially established in the oocyte by the adult fly. The embryo just needs to define and redefine this pattern
What was the contribution of Christiane Nüsslein-Volhard and Eric Wischaus to the study of Drosophila
They undertook a saturation genetic screen to identify all the genes involved in development and patterning of the larval cuticle. This lead to the identification of 4332 embryonic lethal mutations and 139 complementation groups involved in patterning
Most genes in development require both alleles to be functional to be expressed properly and provide normal function (haploinsufficient), T or F
F – most genes involved in development are haplosufficient
Describe how complementation testing can be implemented to determine if mutations lie in the same gene or different ones
If you cross two parent individuals that both have the same mutant phenotype you can determine if these mutations lie in the same gene or different genes. If the progeny produced by breeding these two heterozygotes do not show the mutant phenotype, then the mutations are said to complement each other. If 25% of the progeny do show the mutant phenotype, then the mutations of the parents must lie in the same gene and thus fail to complement each other
Recall the hierarchy of genes that dictate patterning in the Drosophila embryo
Maternal genes–> Gap genes–> Paired-rule genes–> Segment polarity genes
What type of gene is gooseberry
Segment polarity gene
What type of gene is bicoid
Maternal gene
What type of gene are knirps, giant, Krupel and tailess
Gap genes
Paired and fushi tarazu are hox genes, T or F
F – they are paired-rule genes
Where is paired expressed in the Drosophila embryo
In alternating parasegments
Gooseberry is expressed in alternating parasegments, T or F
F – it is expressed in all parasegments
Genes higher up the patterning hierarchy have a greater influence over segmentation, T or F
T
Give an example of another maternal gene, other than bicoid and explain where its expressed and its role in development
Nanos is another maternal gene. It is expressed in posterior embryo and is responsible for patterning the posterior larvae
What is seen in bicoid mutants and why is this
Bicoid mutants develop without head structures. This is because bicoid is at its highest concentration at the anterior end and dictates formation of the head structures
What is unique about bicoid and its role in development
Bicoid is a morphogen but is also in itself a transcription factor, unlike other maternal and patterning genes that are usually just transcription factors.
What is the role of bicoid in establishing the anterior-posterior axis prior to fertilisation
Bicoid mRNA is deposited in the anterior oocyte by the adult female fly in a process called maternal loading. This leads to the localisation of bicoid protein at the anterior region of the embryo
Describe the bicoid gradient in the early Drosophila embryo
Bicoid has its highest concentration at the anterior region of the embryo and drops away towards the middle
What unique feature of the Drosophila embryo allows the bicoid gradient to be established easily and accounts for the simple early patterning
At this stage in development the Drosophila embryo is a syncytial blastoderm whereby many nuclei are contained in the same cytoplasm. This allows the bicoid protein to diffuse easily through the embryo and establish a gradient easily
Explain the transplantation experiments carried out on the Drosophila embryo and what they showed
Transplantation of wild type Drosophila embryo cytoplasm into a bicoid mutant was sufficient to rescue some of the head structures. Transplantation of wild type cytoplasm to an ectopic site in the middle of the embryo lead to a duplication of the embryo with head structures developing in the middle flanked by thoracic segments either side. This shows that bicoid is both necessary and sufficient to dictate head structure formation
What happens if both copies of the bicoid gene are mutated, why is this
If both copies of bicoid are mutated then the embryo will try to pattern normally using other maternal genes that are functional such as nanos. However the resultant patterning would still be abnormal
What is the effects of forced overexpression of bicoid
Shunting of the segments towards the posterior end
With morphogens, the ligand doesn’t act as the signal to dictate differentiation as such but it is in fact the gradient of the morphogen that encodes information on cell fate, T or F
T
How can bicoid expression be visualise in situ
Fusing reporter genes for chaperones such as Hsp70 or genes easily stained for such as LacZ to the promoter sequence for bicoid allows easy visualisation. Wherever bicoid is expressed the reporter gene will be to as its under the control of the same promoter sequence. These reporter genes can then be easily visualised
What attribute of morphogens accounts for there ability to be used to carry out multiple different functions
The there are different affinity sites for transcription factor morphogens such as bicoid. Some sites in the DNA have low affinity for the transcription factor ligand and so will only elicit binding at really high concentrations. However, other sites will have high affinity and will always elicit binding of the morphogen regardless of concentration
What is the role of gap genes
Subdivide the embryo into different parts once the basic pattern is established
What is significant about gap gene interactions
The gap genes actually also interact and repress each other as well as their targets
Hunchback is a gap gene that regulates points in the embryo where other gap genes are switched on, what is significant about its expression with regards to other maternal gene(s)
Hunchback expression directly mirrors bicoid
How are the paired-rule genes expressed in the Drosophila embryo
Expressed in alternating parasegments whereby their expression is controlled stripe by stripe
What is paired-rule gene expression dependant on
Interactions of positively and negatively acting transcriptional regulators, many of which are gap genes
Even-skipped is a paired-rule gene only expressed in parasegments 3. What conditions are required for even-skipped expression
Very low concentrations of giant and kruppel, high concentrations of hunchback and a little bit of bicoid
Describe the expression pattern of segment polarity genes
Segment polarity genes are expressed in all 14 parasegments
What has happened in the Drosophila embryo by the time that the segment polarity genes are expressed, and what are the implications of this
Cellularisation has occurred meaning that signalling pathways dictating patterning and development now need to get more complicated. Factors used for signalling now need to be secreted
Engrailed is a segment polarity gene that is always expressed in the very anterior part of the segment, T or F
F – this was initially thought to be the case but in fact, engrailed is aways expressed most posteriorly in the segment
What common phenotype is seen in wingless and hedgehog mutants in Drosophila and why is this
Both mutants are said to have a lawn of denticles in the larvae. This is because both genes maintain each other, hh maintains wg which supresses denticle development
What is the function of wingless and hedgehog together
They work together to inhibit the formation of the denticles
The segment polarity gene engrailed is switched on by wingless, what is the role of engrailed
Engrailed then switches on hedgehog
Explain how wingless and hedgehog interact
Hedgehog signals in a paracrine manner to the adjacent cell to switch on wingless expression. Wingless then acts on its own receptors in an autocrine manner to upregulate is expression but also acts via engrailed to upregulate hedgehog too
Hh and wg feedback onto each other to maintain their expression and refine segment borders, T or F
T
The hedgehog-wingless signalling loop is present in in the posterior parts of the segments, T or F
T
Wg is expressed uniformly throughout the Drosophila embryo, T or F
F – its expressed as a gradient
What is the role of the Hox genes (homeobox domain containing genes)
Hox genes provide identity once the segments have already formed
Hox genes are transcription factors, T or F
T
From which genes do the hox genes receive input from
Pair rule and gap genes
What is unique about the expression of the hox genes
They are expressed along the anterior-posterior axis in the same order in which they lie in the genome
What happens due to a of loss of function in a hox gene
The segment will adopt a different identity, this is known as a homeotic transformation
What is meant when Drosophila are referred to as long germ band organisms
All 14 segments are defined at once. All tissues are present they just need defining during development. This process is relatively quick (24hours in Drosophila)
How do short germ band organisms develop differently to long germ bands
Similar process to short band organisms in the anterior head and thoracic segments. However, the abdominal segments are added sequentially by a region known as the posterior proliferative disc budding off segments as it gets smaller
Short germ band segmentation is more complex and slower than that of long band organisms, T or F
T
What signalling pathway mediates the segmentation of short band organisms
Delta-Notch
Explain how the segmentation clock of delta, notch and Hes1 accounts for segmentation in short germ band organisms
Notch and delta signal to each other in a juxtacrine manner. Activation of the Notch receptor leads to the expression of Her/Hes1 which inhibits delta expression in the nucleus of that cell. At high concentrations Her also inhibits itself. This mechanisms acts as a molecular oscillator or clock whereby notch activity rises and then triggers the expression of Her. Her then switches off delta and then itself. This causes notch to rise again and the process repeats. This pattern the segments at exactly the right time as the organism grows based on the time taken to produce Her mRNA and then translate it and switch delta off and then itself
How is the segmentation clock relevant to vertebrates
Humans are short band organisms. The primitive streak in vertebrates also use the notch pathway and its oscillations to produce and pattern body segments
Which axis is the first to be laid down during embryonic development
Anterior-posterior axis
Which germ layer forms at the primitive streak during gastrulation
Mesoderm
What experimental method lead to the identification of the origin of mesodermal tissue development
Lineage tracing
What determines the adoption of different mesodermal fates in the developing embryo
Where the cells ingress along the anterior-posterior axis into the primitive streak
Put these different mesodermal tissue types in order from most anterior to most posterior. Intermediate mesoderm, axial mesoderm, lateral mesoderm, paraxial mesoderm
Axial mesoderm, paraxial mesoderm, intermediate mesoderm, lateral mesoderm
The axial mesoderm is one of the mesodermal tissues formed by the ingression of cell into the primitive streak, what two subtypes of mesoderm does it give rise to and where
Prechordal mesoderm (anteriorly) and the notochord (posteriorly)
Which mesodermal tissue gives rise to the somites
Paraxial mesoderm
Cells that ingress posteriorly to the paraxial mesoderm give rise to which tissue
Intermediate mesoderm
The more posterior a cell ingresses into the primitive streak, the more posterior the mesoderm it will give rise to will be, T or F
F – more posterior ingress, the more lateral the mesoderm
Alike the axial mesoderm, the posterior paraxial mesoderm is also subdivided, what are these subdivisions
Unsegmented posterior paraxial mesoderm and the segmented posterior paraxial mesoderm that give rise to somites
What structures do the intermediate mesoderm give rise to
Kidneys and gonads
How many components is the lateral mesoderm divided into and what do these divisions give rise to
The lateral mesoderm divides into 3 components, two of which give rise to the circulatory system and the other which contributes to extraembryonic structures and limb bones
List the structures that the paraxial mesoderm give rise to
Axial skeleton, heart, somites, cartilage and tendons
The formation of somites occurs in a sequential manner on both sides with the size of somites being preserved throughout, T or F
T
The pre-somitic mesoderm is unsegmented, T or F
T
Describe the rough structure of the somites viewed as a horizontal cross section
The somites show a clear metameric structure with a clear repeating pattern and defined anterior and posterior boundaries
Somites are the earliest evidence of segmentation in vertebrates, T or F
T
How do the number of somites relate to segmentation in vertebrates such as humans
Somite number dictates the number of vertebrae
How many somites are present in humans and how does this relate to their segmentation
The human embryo has between 38 and 44 somites, this correlates to the 33 vertebrae which we are born with
The number of somites is fixed for any given species and the timing of somite formation remains constant, T or F
F – whilst the number of somites does differ between species, so too does the timing of somite generation
What five factors must cells in the paraxial mesoderm be able to respond to
Positional information, mechanisms that coordinate left and right, anterior and posterior boundary formation and the formation of the cleft
What structure separates the left and right somites
Spinal cord
What model describes the periodicity of somite formation and how is this achieved
Clock and wavefront model. The clock explains the temporal component whilst the wavefront provides spatial information to drive somite formation. Where cells hit the travelling wavefront an abrupt change of property leads to the decision to form somites
Explain how a molecular oscillator drives mesodermal segmentation in chick embryos
In the embryo levels of the helix-loop-helix transcription factor C-hairy was found to fluctuate at different embryonic stages. Later genes were discovered that regulate the timing of this clock oscillation and are members of the notch, wnt or FGF signalling pathways
What is meant by the determination front
Wavefront that travels from the anterior part of the presomitic mesoderm towards the posterior embryo
How does the determination front dictate somitigenesis
When the cells of the paraxial mesoderm encounter oscillations from the molecular clock an abrupt change determines their formation of the next somite pair
What aspect of the paraxial mesoderm determines somite boundary formation
Position of the somite minus II (S-II)
Explain the results of ectopic grafting of boundary cells
Somite boundary cells isolated from one embryo transplanted into another embryo is sufficient to induce the formation of a new boundary. Where you’d expect to see one somite you would now get two. This shows that boundary cells instruct cells that are anterior to it to form a boundary
What family of genes were discovered to be expressed at the somite boundaries
Notch family genes. They are selectively expressed in the anterior or posterior part of the somite
Explain the results of forced lunatic fringe expression in the paraxial mesoderm and the significance of this
Lunatic fringe is a gene that blocks notch activity and thus forced expression results in an inhibition of notch signalling. This results in the formation of a new boundary and hence an additional somite
Give an example of a human disease that proves a role of notch signalling in mesodermal segmentation
Jarcho Lewin syndrome causes spondylocostal dysplasia due to problems with the segmentation of the axial skeleton. This occurs due to a mutation in the delta 3 ligand that alters notch signalling and problems with somite segmentation
Explain how the determination front is positioned
The determination front is determined at the interface of two opposing gradients. Retinoid acid which is high anteriorly and fibroblast growth factor 8 which is high posteriorly. When these gradients are equal the determination front forms.
Where is retinoic acid produced to dictate determination front positioning
Somites
RA and FGF8 agonise eachother, T or F
F – they antagonise each other
Explain how RA and FGF8 interact to dictate determination front positioning
High levels of FGF8 result in high levels of Cyp26 which inhibits RA synthesis. High levels of FGF8 also inhibit the production of the Rhald2 enzyme that is normally required for RA synthesis
What transcription factor do RA and FGF8 regulate the expression of
Mesp2
How do RA and FGF8 interact to regulate expression of transcription factor(s) involved in somite boundary formation
RA activates Mesp2 expression whilst FGF8 inhibits it. Mesp2 expression in turn blocks local Notch signalling. Lower Notch signalling on one side of the border results in high notch signalling in adjacent cells on the other side of the boundary.
What are the downstream effects of opposing notch signalling in cells either side of the boundary
High notch acitivity in one side and low activity in the cells opposite leads to formation of the somite boundary. The boundary itself forms from physical formation of cleft within the mesenchymal tissue. Downstream extensive changes in cell morphology and adhesion leads to creation of this cleft and is mediated by ephs and ephrins
Whose saturation mutagenesis experiments lead to the discovery of the hedgehog signalling pathway
Nusslein-Volhard and Wieschaus
What was seen in hh mutant Drosophila embryos
Defects in segmentation. The segments contained a lawn of denticles and no naked cuticles
What type of gene is hedgehog
Segment polarity gene
Explain how hh and wg interact
Hh directly upregulates wg. Wg then controls the expression of the transcription factor, engrailed (en) which in turn then regulates hh expression
Hh and wg are said to be dependent on each other, what does this mean
These genes require each other to be expressed. Loss of either gene will lead to the loss of the others expression. Thus mutations in either gene will give rise to similar phenotypes
Discuss the genetic conservation of the hh signalling pathway
Hh signalling is relatively conserved amongst metazoans and kingdom Animalia however not to the extent that wnt signalling is. For example C.elegans lacks hh signalling mechanisms
Recall some of the vertebrate homologues of hedgehog
Sonic hedgehog, Indian hedgehog and desert hedgehog
What accounts for the increase in hedgehog genes seen in vertebrates compared to invertebrates
Vertebrates contain more homologues of the hedgehog family genes due to genome duplication throughout evolution to account for the importance of the pathway in patterning
What is the significance of the N-terminal signal sequence of the hedgehog ligand when first synthesised
It targets the protein to the secretory pathway
What happens to the hedgehog ligand once it reaches the membrane
The N-terminal signal sequence is cleaved off by an autoproteolytic cleavage catalysed by the C-terminus of the protein
What enzyme carries out the autoproteolytic cleavage of the N-terminus of the hedgehog protein
Hedgehog acetyltransferase in vertebrates and skinny hedgehog in Drosophila
Cleavage of the N-terminal part of the hedgehog protein is coupled to a cholesterol molecule addition. This occurs as well as palmitoylation. What are the combined effects of these modifications and the effects on the signalling pathway
Addition of these groups makes the hedgehog protein very hydrophobic. This renders the molecule insoluble in water and acts to target its localisation to the membrane. In addition, it makes hedgehog unable to leave the membrane and hence restricts it to signalling only to neighbouring cells
What proteins are required for hedgehog release from the signalling cell and its long-range diffusion
Dispatched, Scube and other HSPGs
Recall some of the hedgehog receptors in vertebrates
Patched 1 and 2, Hedgehog interacting protein, Smoothened, CDO, Brother of CDO (BOC)
How many transmembrane domains does patched have
12
