GEN3030

C. elegans lifecycle

Egg to adult within 3 days - Really good for muticellullar organism - Go through 4 larval instars - Interesting alternate pathway: dauer phase = Due to stress or bad conditions = When they receive signals that conditions are better they come out of that phase

How can we manipulate neurons to determine if they are in the same circuit?

Electrophysiology allows you to electrically stimulate one neuron, and measure the electrical activity in a second. If stimulating the first neuron results in an electrical response in the second, they are coupled and therefore in the same circuit. If not, then these neurons are not coupled. They could still be in the same circuit, they are just not directly coupled.

Early embryogenesis in Arabidopsis

Embryogenesis occurs by a 'defined series' of cell divisions. First division ⇒ one apical and one basal cell. Apical cell ⇒ embryo Basal cell ⇒ suspensor (holds embryo in the seed coat). *cells may be connected via regulated cytoplasmic bridges (plasmodesmata) - proteins, including tranx factors potentially move between cells

Evolution of multicellularity in eukaryotic lineages

Eukaryotic lineages Evolution of complex multicellularity => Multi cell body where there is tissue division - E.g. vegetative cells vs gamete cells Division of labour Of these six groups, we are the only ones w/out a cell wall so how each of them develop is different and they're all diff from each other Important when comparing across the linages When you get close the nuc, the way things act across the lineages is pretty much the same. The way genes are regulated is very similar across all eukaryotes, but the further you get away from the nucleus, when you get to the membrane things become v different. The way cells communicate with other cells is very different. The concepts are the same but the molecules aren't and receptors/ligands aren't conserved across linages.

SHOOT MERISTEM (=SHOOT APICAL MERISTEM, SAM)

How are leaves and plant stems produced? - Shoot meristem is a saucer of stem cells at growing 5p of plant stem. - Has a 'central zone' (stem cells), surrounded by a 'peripheral zone' (organ producing cells), and above a 'rib zone' that contributes to stem development. Cells occur in three layers: L1 (epidermis), cells divide anticlinally ⇔ L2, cells divide anticlinally ⇔ (L1 and L2 expand like the skin of a balloon.) L3, cells divide in any plane (anticlinally ⇔ and periclinally ⇑⇓). (L3 cells generate ver5cal extension of plant stem.) Shoot meristems can be very long-lived - must be a balance between cells leaving the meristem (recruited to form organs from the peripheral zone) versus cell proliferation in the central zone

1. Maternal genes set up the body axes

Identified a large number (~50) of maternal genes - when mutant don't affect mother but affect development of progeny. 4 classes of maternal effect genes found, affect: anterior specification, eg. bicoid posterior specification, eg. nanos both terminal regions, eg. torso dorso-ventral specification, eg. dorsal - Maternal genes create differences and act in the ovary where mRNA is deposited. - Maternal genes create differences along axes even before egg is fertilized.

What is the predicted phenotype of ced-3 ced-4 double mutants?

If you get rid of ced 4 which is required to turn on ced 3 which will turn on apoptosis so even in you have functional ced 9 if there is a double mutant of ced 4 and 3, then there will be no apoptosis Also it's the same as single mutant, because need ced 4 to activate ced 3 and ced 3 to activate apoptosis.

Life cycle of an angiosperm (Arabidopsis thaliana)

In angiosperms, it's the opposite. The haploid male and female are dependent on the diploid part. Embryo sacs is enclosed in structure called ovule. Becomes a seed when fertilised Enclosed in fruits Both haploid and diploid stages are multicellular: this results in an alternation of generations*. Life cycle is diploid (sporophyte) dominant, with haploid (gametophytes) dependent upon diploid phase. *Germ cells are not set aside early in development as they are in metazoans.

How does induction determine cell fate?

Induction: when a group of cells directs the development of another group of cells Usually involves some kind of secreted factor or some kind of cell to cell communication The cell that is inducing has to be able to tell its neighbours, you're going to become something

Regulation of Hox gene expression

Initial domains of expression regulated by gap and pair rule genes. Then maintained in adult by polycomb and trithorax genes - chromatin regulators. *Example of chromatin modification as mechanism of gene regulation

How does genetic variation contribute to the evolution of plasticity? How can you use selection experiments to understand the evolution of plasticity?

It means that new phenotypes will evolve, and new environmental cues will trigger them. Selection experiments such as the maduca quinquemaculata allow you to see the impact of different environmental cues on phenotypes and to see the plasticity of different genes and phenotypes. Evolving a polyphenism in the lab · 28 degrees they are green · At lower temps, they're black, occurs naturally · Mutation called the black mutant, and makes it black · Heat shocked the black mutant · Found that the black ones would go green and diff individuals would go diff levels of green, starting from black. · The experiment was assuming the response was different between different individual phenotypes · And if it is different, you can perform a selection experiment · Experiment is assuming there is genetic variation and plasticity and it is being used to evolve a polyphenism in the lab · Green line is evolving different phenotype over time · Managed to evolve a polyphenism in the lab, one colour at low temps and diff at high temps. - Now get an enabling mutation, which could be covered by cryptic variation or in the case of the black caterpillars, be shown. - This mutation allows for you to occupy a different colour space, but retains the same plasticity - So will still change colours at diff temp. but instead of going from greens they go from black to greenish.

How can conserved pathways, like the melanin synthesis pathway in Drosophila, be used to understand how plasticity evolves in other insects? What types of experiments show how melanin synthesis responds to changes in temperature, and what are the genes involved?

It shows the interaction between the genes in the pathway and how they respond to environmental cues. These enzymes are changing their level activity in response to temperature Experiments in drosophila have shown that Genes are environmentally sensitive even in drosophila - Genotypes, if reared at diff temps will show diff pigmentation patterns - Bands get smaller at higher temps - Diff genotypes are diff in their degree of plasticity.

Outline how paralogous homeodomain proteins (e.g. KNOX/BELL) lead to activation of zygotic gene expression

KNOX and BELL proteins act heterodimeric transcription factors KNOX: TALE class homeobox BELL: TALE class homeobox KNOX and BELL proteins are cytoplasmic unless they can form heterodimer, which is able to move into the nucleus to control gene expression ancestral eukaryote (single cell!) had two homeodomain genes: 1. non-TALE class, i.e. Antennapedia class (e.g. Hox, body plans etc.) 2.TALE class (e.g. KNOX/BELL) ancestral KNOX function was to activate zygotic (2n, meiotic) gene expression - early targets: cell wall remodeling enzymes KNOX/BELL are supplied by the egg /sperm to initiate zygotic gene expression - MpKNOX1 is supplied by the female - BELL is provided by the male KNOX2/BELL1 are active in cell wall remodeling in the sporophyte - MpKNOX2 and MpBELL1 have retained(?) an ancestral function in regulation of cell wall remodeling to pattern later diploid sporophyte development The KNOX/BELL genetic system is conserved between Chlorophytes and land plants

What are the general differences between the fly and mammalian clocks?

Key differences to Drosophila: - the CYC equivalent is called BMAL. - the light input is not intrinsic to the clock neurons, input occurs at the retina and signal transmitted to the clock. - more elaborate regulation, more molecular players.

How does LIN-14 control developmental timing?

LIN-14 = transcription factor. Protein levels of LIN-14 drop as the worm goes through larval stages Lin-14 levels start high so in L1 it has high levels and go lower over each level to L4 - loss of function of lin-14 → late larval stage divisions start too early = Loss of function - No lin14 and skip L1 - gain-of-function of lin-14 → L1 divisions are maintained too long = Lin 14 stays high so repeated L1 Correlation between level of lin 14 expression and at least the L1 and L2 progression

(1) land plants have a gametophyte generation (alternation of generations) = Evolution of the alternation of generations

Land plants have an alternation of generations bc they arose from a fresh water algae, where the haploid body was a complex multicellular body that produced sperm and eggs, producing a zygote which only underwent meiosis to produce more zygotes. Then land plants evolved, the haploid body become multicellular and now has a complex diploid and haploid body - Haploid body become reduced and diploid became increased - Haploid: pollen (3 cells) and embryo sac (7 cells) - Change in body plan

Importance of syncytial cell

Large molecules such as proteins that aren't normally secreted can diffuse between nuclei (eg. transcription factors = repress/activate transcription). Single cell, many nuclei Zygotic nuclei, first nuclei of next gen Big molecules can diffuse through the space and activate diff genes in diff nuclei

How are leaves formed?

Leaves arise from primordia - outgrowths of undifferentiated cells (singular - primordium). A leaf primordium is initiated via a periclinal cell division in the L2. Primordia arise at regular intervals and spaces in the peripheral zone. Initially the leaf primordium is a dome of cells; grows laterally and differentiates into the flattened mature leaf.

Commonly used types of microscopes in cell and developmental biology

Light Microscopes Stereo/dissection microscopes designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. Compound microscopes used for viewing samples at high magnification (40 - 1000x), which is achieved by the combined effect of two sets of lenses: the ocular lens (in the eyepiece) and the objective lenses (close to the sample). Electrons Electron Microscopes electron microscope is a type of microscope that uses electrons to create an image of the target. It has much higher magnification or resolving power than a normal light microscope.

EWAS in complex diseases

Limitations/issues Reversible (unstable changes) Quantitative (small) differences Tissue heterogeneity/composition Genotype-dependent effects

What does LIN-4 do?

Lin -4 expression comes on just as lin-14 begins to die = When we keep lin4 in, we get progress from L1 Lin-4 is a miRNA which prevents the expression of lin-14 by binding to it and it makes lin-14 stop being produced So if we have lin-4 lof it has opposite effect of lin-14 - So lof of lin4 we get continued L1 while in lof lin-14 we skip L1 - And visa versa with GOF

1.2. Maternal control of D-V patterning

Main D-V morphogen is encoded by maternal gene dorsal. Embryos from dorsal mutant mothers lack ventral structures (" dorsalised"). But dorsal mRNA and protein are uniformly distributed. So what is the morphogenetic gradient? In this case it is a gradient in protein sub cellular localisation - on ventral side of embryo Dorsal protein enters nuclei, on dorsal side it is in cytoplasm. How does this occur? Localised activation of a receptor called Toll - signalling pathway activated on ventral side only. - Toll signal causes Dorsal protein to enter nuclei from cytoplasm, generates gradient of nuclear Dorsal, highest on ventral side. NB: Toll signalling pathway also very important in immunity in vertebrates and in flies.

Oogenesis

Maternal cells called follicle cells surround each oocyte. Cell-cell interactions between oocyte and follicle cells help establish body axes before fertilisation occurs. So oocyte already has some A-P and D-V organisation.

Arabidopsis male gametophyte (pollen) development

Meiosis produces 4 haploid microspores Each haploid microspore undergoes 2 mitotic divisions to produce pollen with 3 cells: 1 vegetative cell (1n) - drives growth of the pollen tube 2 sperms cells (1n) -1 fertilizes the egg cell -1 fertilizes the central cell Male: Diploid cell set aside to become germ cells, and undergo's meiosis to become 4 cells Each haploid cell will undergo 2 rounds of mitosis - Round 1: generative cell nuc, and nuc divided to produce two sperm to go to female

Early embryogenesis in Arabidopsis cont.

Meristem: self renewing group of stem cells After plant germinates the plant will produce stem and leaves, then stem and more leaves etc. Two primordia develop on the flanks of the apex (heart stage) ⇒ cotyledons (leaf-like organs). *major difference from animals: plant embryos are not mini-adults, but produce new organs throughout their lifetime. Can't run away so need to continually respond to environmental conditions by modifying their development - clones look different

PLANT MERISTEMS

Meristems: Groups of undifferentiated stem cells* present at growing points in plants. (*capable of giving rise to more stem cells and to differentiated cells) - When a plant germinates, it produces organs throughout the lifetime Made by meristems Primary meristems (many diff types of meristems): Shoot meristem ⇒ leaves, plant stem Inflorescence meristem ⇒ flower meristems Flower meristem ⇒ sepals, petals, stamens, carpels Root meristem ⇒ roots Secondary meristems: Plant stem ⇒ wood, bark

DNA methylation, detection methods

Methods - MeDIP (methylation-dependent immunoprecipitation) - Bisulfite treatment: • 5-methylcytosine resistant to deamination by sodium bisulfite treatment• Exploited for detecting DNA methylation to identify DNA sequences associated with (changes in) DNA methylation can be applied to screening the whole genome

Expression of auxin

Model proposed for how gene expression is induced by auxin *New paradigm for a signalling pathway having an F-box protein, which target other proteins for proteolysis, as a receptor

How do modularity and constraints direct how traits evolve?

Modularity imparts evolvability to a system by allowing specific features to undergo changes without substantially altering the function of other features or the functionality of the system. Can evolve in two ways - Modularity: if genes that modulate output of A are somewhat separate to B, though if you modified the genes of A you could still get the output of A through the blue genes (B). So, there is some separation in the regulation of the output A. - No modularity: get all the orange and blue talking to each other. So, if change one thing in the blue or in the orange you will change output A. System has little evolvability, bc so interconnected. - If gene network effects a lot of different systems, modularity allows you to modify part of something without messing everything up. - Keep most of the system the same while modifying one element.

Morphogen

Morphogen: concentration dependant function If you have a high concentration of a morphogen it can lead to a diff outcome compared to cell that has a low level Thresholds, diff outcomes depending on concentration Morphogenetic gradients are critical for development Start as 6 equivalent cells, source of morphogen, the cells have thresholds of response, which gives each of them one of three (in this example) different fates

Genes and mutants in flowering

Mutants isolated that flower later than normal in long days (signal disrupted). Two important genes: CONSTANS (CO) and FLOWERING LOCUS T (FT). Mutants of each (co and ft) flower late (~10 days later than wild type).Double mutants also flower ~10 days later, so likely to act in same pathway. Both genes are regulatory genes (encode transcription factors). Amount of CO protein is controlled by the circadian clock (24 hour cycle). Most CO protein is produced in the late afternoon. CO protein is degraded in the dark, so, if late afternoon is dark (short day), CO is degraded, but if late afternoon is light (long day) it is not degraded. CO activates the expression of FT. But, both CO and FT are expressed in leaves, not in shoot meristems.

Genetics in gametophyte versus sporophyte

Mutations affecting the gametophytic (haploid) versus the sporophytic (diploid) phases of the life cycle will segregate differently Consider female gametophytic versus embryonic lethal mutations in Arabidopsis Figure out if something is active during the 2n vs 1n phase Bc in 2n phase, acts like typical medallion gene, if bred with itself or heterozygote, will have 3:1 ratio with 1/4 dead etc.

C. elegans biology

Not quite rotting, with little bacteria, is dauer habitat, but when more rotten with more bacteria it will come out of the dauer phase and continue with lifecycle Small worm about 1 mm long, grows on decaying matter Can be grown on a petri dish of bacteria (e.g. Escherichia coli) Can be frozen (at some stages) and resuscitated - storage Sexes are male and hermaphrodite (no females) - hermaphrodites first produce sperm and then make eggs and can self-fertilise - hermaphrodite is XX, male is XO

Nurse cells and follicle cells

Nurse cells and follicle cells deposit mRNAs and proteins into the oocyte to control early development - maternal gene products Follicle cells: Positional information - Important for embryo to know what is up and down

DNA methylation and imprinting

One allele transcriptionally inactive (silenced), depending on the parent it was inherited from Imprinted alleles usually heavily methylated (or have chromatin or histone modifications, ie epigenetic changes) Selective gene silencing impacts phenotypic expression Silencing takes place early in development and is transmitted across generations (via reprogramming)

C. elegans advantages

One of the simplest organisms with a nervous system - 302 neurons, with connections that have been completely mapped. = Not a lot of behaviour besides moving but can image all of the neuronal activity and behaviour and only animal this can be done in Can see all the nuclear divisions and map put all the cell divisions in c.elegan development = transparent

shoot meristemless mutants fail to form a SAM

One screen, seedlings that germinate but don't produce leaves Lof This gene needs to be activated in those cells during meriogeneisis otherwise you wont get a meristem

What dictates the patterns of modification? CONSTRAINTS

Opposite of modularity. - System is set up so there are only a few ways you can do something. - Constraints to number of phenotypes we can produce. · Constraints by things like DNA code, has very specific relationship with types of amino acids. If DNA can't code for it, then you can't get it. · Only a limited number of ways to create structures e.g. eye · Constraint by how a skeleton, or body is set up. - Absent phenotypes: Centipedes, always have an odd number of legs - Parallel evolution: Even though in separate areas, they seem to form the same phenotypes

How do these changes in growth rate and duration affect the size of individual organs? How does this work?

Organs respond differently to nutrition. Organs show different sensitivities to insulin signalling. - Wing size is substantially reduced when we down-regulate insulin signalling. - Palp size is also reduced when insulin signalling is down-regulated. - However, reducing insulin signalling does not significantly affect genital arch size. How does this work? Reducing insulin signalling (via starvation) extends the duration of the growth period, but also severely reduces wing growth rates. This results in much smaller wing sizes when compared to fully fed animals (with high levels of insulin signalling).

What is significance of the three classes of homeotic mutant?

Overlapping domains of functions suggest combinatorial activities specify floral organ identity, class A, B and C Hypothesis: • A function alone controls sepal identity • A + B function control petal identity • B + C function control stamen identity • C function alone controls carpel identity. Additional components of hypothesis: • A function represses C function in outer 2 whorls • C function represses A function in inner 2 whorls

What tools do we need to activate neuron A and examine the response in neuron B?

Pair the GAL4/UAS and LexA/LexA Op systems with a transgene for quantifying neuronal activity. GCaMP is a transgenic protein that binds to calcium. In the absence of calcium, like when a neuron is inactive, it does not fluoresce. When a neuron becomes activated, calcium floods into the cell. This calcium binds to GCaMP, changing its conformation and causing it to fluoresce. You can measure the intensity of the fluorescence of a neuron, using techniques like two-photon microscopy, to quantify its activity.

What tools do we need to activate neuron A while inhibiting neuron B?

Pair the GAL4/UAS and LexA/LexA Op systems with transgene for manipulating neuronal activity.

What dictates the patterns of modification? MODULARITY

Parcellation: differential elimination of pleiotropic effects between members of different complexes - Everything is connected Integration: creation of pleiotropic effects among primarily independent characters - One gene, one trait. Evolve to something where one gene effecting two traits.

Phylogeny of eukaryotes

Plants are the descendents of the primary endosymbiotic event that gave rise to the chloroplasts Plants represent one of the 6-8 major lineages of eukaryotes Secondary endosymbiotic events have disseminated the chloroplast to multiple other eukaryotic lineages Secondary endosymbionts: eukaryotic cell, non photosynthetic, that engulfed an algae that was photosynthetic. So genes from algae can be transferred into eukaryote. Genes: gene conservation increases towards the nucleus (e.g. transcriptional regulators), and decreases towards the plasma membrane (e.g. signaling molecules).

Phylogeny of plants

Plants consist of 4 major lineages: - glaucophytes - rhodophytes (red algae) - chlorophytes - charophytes + land plants Multicellularity evolved multiple independent Genes Land plants evolved from a multicellular fresh water green algal ancestor whose life cycle was haploid dominant Only in seed plants have plants completely lost their reliance on water

Double fertilization and the formation of the embryo in angiosperms

Pollen tube delivers 2 sperm cells - One fuses with egg cell to form embryo - One fuses with diploid cell to form endosperm = 2 maternal copies and one paternal = triploid embryo (2n) - the next generation of life endosperm (3n) - nutritive for the embryo, either before or a\er seed germination; - e.g. in grasses, popcorn, rice

Early embryogenesis in Arabidopsis cont.

Produce outside and inside cell - Future epidermal cell, exposed to environment - Future inside cell Every time a cell divides a cell wall is set between the cells - When a cell in a plant is born, it is stuck in position Apical cell divides three times ⇒ octant stage (8 cells) Then divides to give outer and inner cells ⇒ globular stage (16 cells) Suspensor divides forming a linear chain of 8 cells. Upmost cell* becomes root meristem Outer cells ⇒ epidermis (L1) Inner cells ⇒ L2 and cortex (L3) *major difference from animals: plant cells are surrounded by a cell wall - and thus cannot move; need to differentiate appropriate to their position

Tools for models

RNAi, mutants, natural variation, selection experiments, experimental evolution Molecular biology: various forms of PCR, Northern/Southern/Western blot, in situ hybridization, immunocytochemistry, etc Transgenics: transcriptional/translational reporters, single transgene, GAL4/UAS, Cre/LoxP, homologous recombination, CRISPR Genomics: genome sequencing, transcriptomics, proteomics, chromatin immunoprecipitation, microarray Population genetics: QTL mapping, genome wide association (GWAS) mapping

How does the flowering signal reach the target shoot meristem?

Recent evidence shows that the FT protein (not RNA) moves from the leaf to the shoot meristem through the phloem (sugar transmitting veins). FT is a "florigen" (flower inducing signal)

Epigenome-wide association studies (EWAS)

Recruit cases and controls into the study, aiming for whole (epi)genome analyses Select tissue of interest Extract, quantify, QC DNA Sodium bisulfite treatment (5meCpG vs CpG) a) hybridize to microarrays (targeting known CpGs from reference genome) b) direct next generation sequencing (whole genome, every CpG covered and compared to reference genome)

Magnification is no good without resolution

Resolution the shortest distance between two points on a specimen that can still be distinguished by the observer

Protein gradients in early embryo

Result is four protein gradients in early embryo: - All are TFs, activate or repress zygotic genes. - At different positions along A-P axis different combinations and levels of these 4 genes expressed, positional information.

FLORAL ORGAN IDENTITY

Screen for mutants in which floral organ identity is altered; e.g. recessive homeotic mutants were found where the identity of an organ is inappropriate for its position (like bithorax in Drosophila). wild-type flower 4 sepals - whorl 1 4 petals -whorl 26 stamens -whorl 3 2 carpels -whorl 4 agamous mutant flower 4 sepals 4 petals 6 petals 4 sepals (another flower) Mutations at two loci, APETALA3 and PISTILLATA, affect the identity of floral organs in whorls 2 and 3 pistillata mutant flower and apetala3 mutant flower 4 sepals 4 sepals 2-6 carpels (fused with whorl 4) 2 carpels Mutations at two loci, APETALA2 (and APETALA1) affect floral organ identity in whorls 1 and 2. apetala2 mutant flower 2-4 carpels 0-4 stamens 0-6 stamens 2 carpels(some organs absent)

Building the apoptotic pathway

Screen for mutants of C. elegans that have fewer cell corpses, or more cell corpses (easy due to transparency) ced-3 and ced-4 loss of function mutants - the 131 cells survive ∴ activators of apoptosis = Mutation of these genes cause the cells NOT to die so ACTIVATOR ced-9 loss of function mutant - many more cells die ∴ inhibitor of apoptosis In a normal cell, that wont undergo apoptosis has high levels of ced 9 and that represses ced 4 and ced 3 Ced 9 expression is HIGH = NO apoptosis Represses ced 3 and ced 4 Ced 9 expression is TRUNED OFF/LOW = APOPTOSIS = Ced 3 and 4 become active and trigger apoptosis

1.3 The terminal patterning system

Set of terminal genes specifies the structures at the extreme ends of the A-P axis. A key gene is torso, embryos laid by mothers homozygous mutant for torso lack terminal structures. Torso protein is a receptor tyrosine kinase, ubiquitously expressed but only activated at the termini. Activated Torso is at top of intracellular signal transduction pathway (highly conserved Ras/MAPK pathway), result is activation of transcription factors that specify terminal structures. Thus mechanism of specification is localised activation of a receptor. Involves localised production of ligand for Torso - mechanism unclear. Involves two other genes - trunk and torso-like.

Meristems

Shoot meristems (indeterminate) → leavesInflorescence meristems (indeterminate) → flower meristems Flower meristems (determinate) → sepals, petals, stamens, carpels Identity of flower meristems is controlled by the APETALA1 gene

Cilia and ciliopathies

Single cilia (ie kidney and most other cells) Multiple cilia (ie olfactory, epithelial cells) Motile (ie embryonic node, sperm, airways) Primary or sensory (ie photoreceptor cells, olfactory neurons). Motile cilia move extracellular fluids (ie clearing mucus from the lungs) or propel cells (sperm) Primary cilia function as "sensors" and transduce signals (ie kidney cells) Ciliopathies: Complex syndromes caused by genetic mutations that result in defective or dysfunctional cilia Can affect any gene/protein involved in cilia development and function, as well as many organs in the human body

Genetic control of developmental timing

Some cells keep dividing in the larval stages - Really stereotyped set of divisions - In each stage L1, L2 etc , each has a different pattern of cell divisions - Each pattern is recognisable bc they're so different from each other in each stage Lin 14 GOF will be stuck in L1 and never progress to L2,3 and 4 division factors

Epigenetics

Something that is not directly related to the DNA sequence but is on top of or additional to the nucleotide sequence "Heritable" changes in gene expression (phenotype) that do not involve changes in the DNA sequence (genotype) (reversible changes)

What is auxin?

Synthesised locally, and can move around the plant but usually don't as much Receptor for auxin is in the nuc Synthesised in shoot meristems, primordia of leaves/floral organs and roots. Transported directionally down the plant (apical to basal) - "polar" transport. Auxin controls a range of processes: • phototropism (see below) • initiation of new meristems and organs (see below) • development of vasculature (not discussed). • cell elongation (discussed in text) Cell elongation is important in growth of plants - auxin is involved in loosening the cell wall (extracellular matrix of cellulose etc.). Auxin provides the basis for positive phototropism of plant stems. Auxin concentration is reduced on the light side, cells expand more on the dark side, and so the stem bends towards the light. Also the basis of negative geotropism, stem bends upwards if placed on side. Herbicides: Activity of one class is based on the growth promoting action of auxins. Auxin is made at the 5p and moves down the stem and promotes cell expansion.

2.4. The homeotic selector genes cont.

Expressed in overlapping domains and act combinatorially. Expression is controlled by gap and pair-rule genes. Encode homeobox transcription factors. Bind DNA and control activity of other genes. Target genes are those needed to determine particular structures in segments. First found in Drosophila, but also control segment identity in vertebrates.

Evidence bicoid encodes the anterior morphogen

Expression pattern bic mRNA in anterior region. Not translated until after fertilization. Protein forms a gradient with high point at anterior end, site of synthesis. Diffuses through embryo and breaks down rapidly, establishes concentration gradient (morphogen). Functional evidence 1. Female flies lacking bicoid produce embryos lacking anterior regions. 2. Overexpression of bic - more of embryo devoted to anterior structures - If you have more than one copy of bic then you have larger and larger heads Thus gradient system at play

Induction of cell fate - vulval development

External genitalia of the adult hermaphrodite. Forms from an initial 4 cells - one inducing cell and three responding cells. Vulva = egg depositing cells in the organism P cells are either going to give rise to vulva or skin Vulva, where the eggs come out (opening) To differentiate these cells there is an inductive event from other cell type, the anchor cell Provides signal to 3P cells to become vulva And other 3 P cells will become epidermis

Flowering of Plants

External triggers to flower may include day length (photoperiod): • longer days - plants that flower in spring/summer, or • shorter days - plants that flower in autumn, or • not responsive - plants that flower year round (e.g. tropical species). exposure to cold temperature (vernalization) plants that grow in one year and flower in the following spring Internal triggers include age.Will not flower until plant reaches certain age, i.e. it must be competent. Arabidopsis is a long day plant, spring flowering is induced by longer days. A flowering signal (florigen) is produced in the leaves. Moves to, and acts on, the shoot meristem (shown by grafting experiments). Leaf from induced plant triggers un-induced meristem on a different plant to produce flowers (providing it is competent). Flowering is controlled by genetic and environmental factors.In long days, CO and FT genes switch shoot meristem to inflorescence m.

Arabidopsis female gametophyte development

Female: - Diploid cell set aside and undergo meiosis (a) to produce 4 haploid cells, but 3 die like in humans (3 of 4haploid cells undergo programmed 2n cell death) (b) - Surviving cell will undergoes 3 successive mitotic divisions (c-e) to produce a syncitial embryo sac with eight nuclei nuclear migration followed by cell wall formation results in a 7 celled embryo sac: - 2 synergid cells (1n) - abracts the pollen tubes - egg cell (1n) - will give rise to the embryo following fertilization - central cell (2n) - will give rise to the endosperm following fertilization - 3 antipodal cells (1n)

2.2. Pair-rule genes define segments

First sign of segmentation - transient grooves on embryo surface define 14 segments along the A-P axis. Segments initially similar but will eventually each acquire a unique identity. Segments defined by action of pair-rule genes, each expressed in every second segment, therefore a series of seven transverse stripes across embryo. Therefore, mutations affect alternate segments. Each stripe of expression is specified independently, in each stripe pair-rule genes activated by different concentrations and combinations of gap genes and maternal genes.

Screening for mutants altering meristem activity

First thing we do is a mutagenesis - Lets look for mutants in gene that affect meristem function = Meristem forms but less function = No meristem = Meristem functions too much, GOF Complications: if genes are used in early mutagenesis, then may not come up in screen mutations in genes required for meristem forma5on could lead to seedlings lacking a meristem mutations in genes required for central zone activity could lead to: - meristems in which the central zone is lost- meristems in which the central zone is too large depends upon the timing of gene activation in the meristem:screen mutagenized population for seedlings either lacking a SAM, or in which the SAM fails to be maintained or grows larger than normal

2.1. Gap genes divide the A-P axis into broad regions

First zygotic genes to be expressed along the A-P axis. All encode TFs. Mutant phenotypes - large regions of the A-P pattern are missing. Expressed in fairly broad stripes. Expression initiated when embryo is a syncytium. So protein products can diffuse, but very short-lived and mostly act in region of expression.

Angiosperm organs

Floral meristems are determinate Floral organs often arise in whorls Four types of floral organ (outer to inner): • sepals (green protective organs) • petals (showy organs to attract pollinators) • stamens (male reproductive organs ⇒ pollen) • carpels (female reproductive organs ⇒ ovules)

Segmentation Genes

Function in a regulatory hierarchy, action of one set essential for next - Divide A-P axis into broad regions - Define 14 parasegments Pattern the parasegments, divide into compartments Specify segment identity

CCR5

G protein-coupled receptor C-C chemokine receptor Binds CCL3, CCL4, CCL5 (RANTES) Likely involved in inflammatory responses to infection (and cancer) Expressed on immune cells - T cells - macrophages - dendritic cells - eosinophils

Control of gap gene expression

Gap gene promoters are differentially sensitive to different concentrations of one of more morphogens (the 4 maternal genes). Leads to stripes of gap gene expression at different positions along A-P axis. Expression of gap genes regulated by a balance of transcriptional activation and repression. - both maternal genes and gap genes themselves play a role in controlling gap gene expression. eg. hunchback also transcribed zygotically, controls some other gap genes. Example: Regulation of KruppelFor Kruppel to be expressed needs activation by bicoid, but also low levels of its repressors hunchback and knirps. Therefore get precise band of Kruppel expression in middle of embryo.

Body plan development in Drosophila

General body plan of Drosophila is the same in embryo, larva and adult: - From anterior to posterior have head end, tail end, and a number of repeating segmental units in between. - From dorsal to ventral have four regions - Each segment in adult has its own identity, eg. T2 has wings and legs, T3 has legs and halteres.

Plant Tumours

Genes from bacteria to go into plant and creates cells that won't stop growing. And produces aa that plant can't use, and so producing food only bacterium can use

Molecular characterization of ABC genes

Genes have been cloned. All of the B and C class genes and one of the A class genes (APETALA1) encode similar genes - MADS box transcription factors - compare with metazoan Hox genes. Expression (mRNA) mapped by in situ hybridization, or by using reporter genes (see Practical Topic 1). Occurs in concentric overlapping fields within the new flower primordium just as predicted. Most ABC genes encode MADS transcription factors, bind to CArG boxes. ABC genes act combinatorially to regulate expression of target genes.

Plants compared with animals

(1) land plants have a gametophyte generation (alternation of generations) (2) land plant embryos are not mini-adults (3) land plant meristems are present throughout life (4) plants have no germ line (5) plant cells are often totipotent (6) land plant cells do not move (7) plant cells have walls (8) land plant cells are interconnected by plasmodesmata Major differences: 1. Plants have a gametophyte generation (alternation of generations). Also, double fertilization in angiosperms.(both common and distinct gene expression during the two genera4ons) 2. Embryogenesis in plants does not result in development of all the organs of the mature body. Plants have vegetative meristems, undifferentiated stem cells, from which organs (roots, leaves, stems) arise throughout life.(Postembryonic development is much more important than in most animals.) 3. Plants do not have a germ line set aside early in development. (Flowers (⇒ gametes) arise as fl. meristems from shoot meristems that may be hundreds of years old, or that have undergone thousands of cell divisions.) 4. Many plant cells are totipotent. (Now know the same is true for some mammalian cells, but a wide range of plant cells can regenerate a whole plant relatively easily e.g. new carrots from phloem cells of root (see also Wolpert 3, Fig 6.8).) Conclude that many plant cells are not determined (i.e. not irrevocably committed to a specific fate.) 5. Plant cells do not migrate. Plant cells are constrained by a cell wall. (Much movement in gastrula4on and organogenesis in animal embryos, none in plants.)

Embryonic origin of shoot and root meristems

- Auxin produced in basal cell, moves up to 2, 4, 8 cell embryo, promotes its growth - Auxin produced in upper region, moves down to root meristem precursor, promotes its growth - Top develops: shoot meristem, bottom forms: root meristem - Auxin produced in cotyledons, moves down to shoot meristem precursor, promotes its growth

Are biological clocks operating in all cells?

- Clock mechanism operates at the level of single cell. But not all cells have one, only a group of specialised ones. - In mammals the "clock" is a group of neurons called the suprachiasmatic nucleus (SCN), in hypothalamus. Sometimes called the circadian pacemaker. - SCN receives information about light from photoresponsive retinal ganglion cells, contain a photo pigment called melanopsin. These cells signal to the SCN. o SCN receives signal from the eyes (light), then tells part of the brain what time of day it is. - The SCN cells generate signals that are communicated to relevant parts of brain.

What is phenotypic plasticity? What are the different types of plasticity?

- Definition: the ability of an organism to react to an environmental input with a change in form, state, or behaviour = phenotype change - Many different structures are plastic o e.g. plants, leaves can change due to light o structure change due to the environment - Many different inducers and many degrees of plasticity (reaction norms) o Way of characterising how a phenotype might change across an environmental gradient. o Linear: § e.g. body size § blueline, more food, bigger body § ENVIRONMENT: a stimulus e.g. food o Non-linear: § blueline, first section: one set of responses § second section: another set of responses § switch like reaction

What are the major types of genetic modifications that can change morphology?

- New Genes - New Tricks: Gene Duplication & Evolution - Teaching Old Genes New Tricks: Gene Co-Option & Evolution · Take a gene in an existing network and have it be expressed elsewhere in the organism. · Genes are switches that control transcription so you can tap into a pathway and control what is and isn't expressed. · Three ways: 1. Change expression domain: Change timing of expression of gene 2. Change target gene 3. Change protein function

How does this localisation of bic and nos mRNAs occur?

- system of microtubules running from anterior to posterior of oocyte - bic and nos mRNAs tethered to opposite ends of cytoskeletal microtubules via their 3' UTRs * Example of mRNA localisation as mechanism of gene regulation and of setting up morphogenetic gradient.

Describe the molecular genetic control of the clock in Drosophila. What is the key gene that entrains the clock?

Genes: timeless (tim), clock (clk), cycle (cyc) - also encode TFs cryptochrome (cry) - encodes blue light photoreceptor double time (dbt) - encodes a protein kinase (regulates other proteins via phosphorylation). Day: - CLK and CYC TFs combine to activate transcription of per and tim. - PER and TIM proteins in cytoplasm form heterodimer. TIM protects PER from degradation by the DBT kinase. Night: - PER/TIM heterodimers reach a critical level at which point they enter the nucleus. - Here they interact with CLK and CYC and prevent them activating transcription at the per and tim promoters. - Thus per and tim transcription stops during the night. Dawn: - Light activates CRY, causing it to degrade TIM. - PER is no longer protected by TIM and is degraded by DBT. - CLK and CYC are free to once again activate transcription of per and tim, and the cycle starts again

What are genetic accommodation and genetic assimilation? How are these related?

Genetic Assimilation is a subcategory of Genetic accommodation Genetic accommodation: Trait changes in sensitivity to environmental changes (becomes more or less sensitive) Genetic assimilation: Trait becomes insensitive to environmental changes (becomes LESS sensitive) - The fixation of an environmentally-dependent phenotype by a genetic change in the regulation of the genes involved. - E.g. ostriches are born with calluses o calluses are an environmentally induced phenotype, but ostriches are born with them so calluses for them have been genetically fixed, thus don't need envo cue.

Next generation sequencing (NGS)

Genome sequencing Metagenomics/microbiota studies Expression profiling RNA-seq (diagnostic) • Mutation/SNVdetection - whole genome sequencing (WGS) - whole exome sequencing (WES) - targeted sequencing - gene panel (ie cancer)

Early development of the embryo

- zygotic nucleus undergoes 13 rapid rounds of nuclear division without cell division, results in a syncytium - single cell with many nuclei. - After 13 mitoses nuclei move to periphery, membranes form and enclose nuclei to form cells, cellular blastoderm. 13 nuclear divisions, now cell division but the nuclei End up with thousand of nuclei in the end of 90 mins Then they move to the outside edge of the egg Then membranes come in from outside and split the nuclei into separate cells

Why study cell biology & development in Drosophila?

1. Ease of study: - Rapid life cycle - Low chromosome number (4) - Sophisticated genetic & molecular genetic techniques - Genome sequenced - Drosophila community - Embryos develop externally to mother - History and resources. flybase.org 2. Conservation of developmental genes and mechanisms

What are the key properties of a circadian rhythm?

1. Have an ~24-hour periodicity. 2. Entrained by environmental signal: · Some signal coming in that helps drive the rhythm · Interaction between internal clock signals and external environmental signals (zeitgebers) produces a functional circadian rhythm. - Rhythm is synchronised (entrained) or reset by environmental cues - Primary one is daylight, most rhythms synchronised to the day-light cycle. 3. Endogenously generated: · Clock maintains itself when the signal is taken away · Circadian rhythms persist when cyclic environmental cues are absent (e.g. in continuous darkness). · In this case called "free-running".

Advantages as a model system

1. Transparent adults and embryos = See what is going on, and good for reporter genes (e.g. GFP), so able to see it 2. Small number of cells (959 cells in adult) with invariant cell lineage =Always have the same number 3. Whole genome mutant screens in 2 months = Can screen all the genes bc of fast Lifecyle, often done with RNAi 4. RNAi very easy and effective - first organism for large scale RNAi genetic screens in vivo 5. Easy to make transgenic constructs 6. Can freeze, and thaw out years later 7. Hermaphrodites can self fertilise

Give an example of a circadian rhythm.

Good example is sleep-wake cycle. - more commonly called the rest- activity cycle (as rest is not always sleep).

Segmentation along A-P axis

14 segments: - three for head - three thoracic (T1-3) - eight abdominal (A1-8) Basic segmented body plan will be maintained in the adult.

Layer mosaic of shoot meristem

4n cells larger than 2n cells- allows one to see clonal periclinal sector Can follow lineage of L1, L2 and L3 cells if genetically marked. All descendant cells stay in the same layer - periclinal mosaic, or layer mosaic. Conclude: cells seldom migrate vertically between layers.

GWAS (genome wide association study)

A genome-wide association study (GWAS) is a hypothesis-free method used to identify regions of the human genome that are associated with a disease or trait of interest, through the analysis of allele frequencies at hundreds of thousands of SNP markers, at once in large populations and samples or two groups of cases and controls A GWAS produces millions of tests associated with the same hypothesis (ie SNPàdisease), performed on the same study sample A consensus Bonferroni-corrected level of significance is usually considered, with set p < 5x 10-8 (1 million tests) Associations that withstand this correction are regarded as (genome-wide) significant, usually corresponding to several regions (and several markers) from the genome in well-powered (large sample-size) GWAS analyses GWAS risk loci contain multiple variants (at SNPs) from multiple genes associated with disease risk effects, eventually affecting various pathways How to infer "causation", ie what alleles and what genes are mechanistically conferring risk and are therefore responsible for the observed associations? Opposite to rare diseases (coding mutations), risk of complex diseases is often mediated by multiple SNPs/variants affecting gene (mRNA, and/or protein) expression

What is a microscope? How do they work?

A microscope is an instrument used to see objects that are too small to be seen by the naked eye How do they work? Lenses to magnify and resolve

what it means to change the slope (α) of the allometric equation

A proportional increase between wing and thorax size is called ISOMETRY, where α = 1 Increase α: hyperallometry α > 1 If α > 1, the relationship is hyperallometric. This means that the wing becomes disproportionately larger with increases in thorax size. Decrease α: hypoallometry α < 1 = If α < 1, the relationship is hypoallometric. This means that the wing would not change much in size with increases in thorax size.

Identity of floral organs controlled by ABC genes

A → sepals A + B → petals B + C → stamens C → carpels A inhibits C C inhibits A A - AP1, AP2 B - AP3, PI C - AG

DNA methylation

Addition of a methyl (-CH3) group to position C5 of a cytosine residue Due to the action of DNA methyl transferases (DNMTs) Almost exclusively taking place at the level of cytosines followed by a guanine base (CpG dinucleotides) Most (70%) of CpGs sites appear to be methylated in humans Addition of a methyl (-CH3) group to position C5 of a cytosine residue Due to the action of DNA methyl transferases (DNMTs) Almost exclusively taking place at the level of cytosines followed by a guanine base (CpG dinucleotides) Most (70%) of CpGs sites appear to be methylated in humans CpG reach regions ("islands") are found upstream of many human promoters and are generally hypomethylated Methylation status at CpG islands correlates with gene expression

Reduce insulin signalling to alter growth rate and developmental timing

Although we reduced the growth rate of the larvae, we also extended developmental time. This particular combination results in slightly larger pupae. By further reducing the growth rate, we find that the extension in developmental time can no longer generate pupae of the same size. When insulin signalling is very low, growth rates are severely impacted. Even with a greatly extended developmental time, the animal ends up pupating at <50% the size of a fully fed animal.

Fluorescence microscopy - king of contrast!

An optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances.

Why don't all 6 p cells become vulva cells?

Anchor cell is secreting peptide signals (EGF) and the P cells all have the receptor (let23 lin 3 receptor) only 3 of these cells will become vulva cells But why if all of them have the receptor will only 3 become vulva not all 6? Lin 3 doesn't diffuse far enough = So if lin 3 is over expressed there will be more vulva cells so that's k=how we know expression levels influence how many cells become vulva and how many become epidermis

HLA and autoimmune disease

HLA genes in complex diseases Because of their high degree of polymorphism, and their known role in antigen presentation, HLA molecules have been among the first factors studied in human complex (immune- mediated) conditions Most of the initial associations with human genetic conditions have been detected for genes within the HLA region

Hormones

Aspects of plant development are controlled by low molecular weight chemicals -hormones. Formal definition Hormones are molecules that: • act at very low concentrations (<10-6M) • are synthesised by the organism • are transported within the organism • have specific effects on physiology, growth or development in vivo • are recognised by specific receptor(s).

Auxin and phyllotaxis and leaf initiation

Auxin is very important for leaf initiation - NPA inhibits the activity of PIN proteins - Addition of auxin (in red blob) to NPA treated apices induces leaf formation PIN-FORMED1 is a member of the PIN gene family. PIN proteins act as auxin efflux carriers - i.e. they export auxin out of the cell Can detect movement of auxin by the expression of special auxin transport proteins, PIN proteins (transmembrane channels). Can follow direction of transport of auxin by observing the polar distribution of PIN proteins. Highest auxin concentration is furthest away from where the formed leaves are

Chromatin structure/status

Heterochromatin: Condensed, not accessible - Histone 3 and 4 are more often targeted, but all do get targeted Euchromatin: Accessible to dna binding proteins, due to no. of enzymes that modify histones

Cell lineages in Arabidopsis

Bc cells are born in particular positions, in epidermis almost all cell divisions occur like this (perpendicular to the surface) - Rare cases you get a periclinal division - Inside cell that's mother was an epidermis cell, so not good if inside cell is still epidermis cell, so what happens is this cell will dedifferentiate and re-differentiate as an internal cell - Gene regulation wise, plant cells are not locked down to a fate, they can change - More flexible Since cells do not move, cell division paBerns determine relative positions of cells. Cells displaced into new position differentiate according with position, not lineage. Suggests an ability to dedifferentiate and re-differentiate. *Major difference from animals - most plant cells are not irreversibly determined.Can regenerate an en?re plant from most plant cells by additional of a couple hormones (cytokinin and auxin), can use this attribute to clone plants and create transgenic plants. *positional information can be supplied maternally, but is not absolutely required

Embryogenesis in Arabidopsis

Because angiosperms are placental, the embryo gets bigger and bigger over developmental time

what it means to change the intercept (b) of the allometric equation

Butterflies have proportionally larger wings than flies, so they will have a larger value for b. Plant hoppers have proportionally smaller wings than flies, so they will have a smaller value for b.

Insulin signalling affects growth rates and developmental timing

By affecting the timing of ecdysone synthesis in the prothoracic gland, insulin signalling affects developmental time - thus the duration of growth. By affecting the growth of tissues throughout the body, insulin signalling affects the size of organs.

From candidate gene to GWAS approach

Candidate gene requires disease knowledge in order to formulate a hypothesis in relation to specific gene function(s) GWAS does not require disease knowledge, nor insight into gene function(s), hypothesis- free since all genes are implicitly tested

Heterochrony

Heterochrony - developmental change in the timing or rate of events Somethings considered heterochronic would be skipping a stage, such as going from am embryo straight to an adult and skipping all the juvenile stages

What types of approaches can we use to understand how morphology evolves?

Candidate gene: a gene or variation on a gene that may relate to the construct of interest, given the role of the gene in a distinct biological pathway or findings from previous studies - E.g. pigmentation, look at pigmentation genes and see if there is variation Genetic mapping: Two species, interbreeding them and tracing that traits - Mapping quantitative trait loci: finding the loci contributing to variation - Can't infer how many loci o So, keep backcrossing to see which regions across genes are associated with particular phenotypes such as light blue tip is associated with little trichomes. o But now cross F1's to themselves and look at F2's to see how many genes are involved in the trait. o Look to see if a certain locus is associated with a particular marker (QTL mapping)

Genetic mechanism of apoptosis is conserved out to vertebrates

Caspases are universal regulators of apoptotic cell death = Same pathway being activated when neurons are fated to die as we found in c.elegans bc they're conserved across organisms

EWAS in cancer

Cell Reports 2018;25:1066-1080 6010 tumor samples from 23 different cancer types (TCGA) identified aberrant DNA methylation and associated changes in RNA expression chromatin remodeling and Wnt signaling pathways key to DNA methylation instability most silenced and enhanced genes involved in apoptosis, DNA repair, cell metabolism pan-cancer map of aberrant DNA methylation to inform therapeutic studies

What is the role of cell death in development?

Cell death is important for many organisms E.g. with digits, cell death in between the digits in organism Important for organisms that go through metamorphosis, so organisms that need a structure during a certain life stage but as it moves on it doesn't need it. E.g. frogs. Tadpoles need tails but adults don't so apoptosis to remove it

C. elegans cell lineage

Cell lineage is invariant The cell divisions and cell fates are 100% the same in every C. elegans! Adult worm has exactly 959 cells. Complete connectome map pf all neurons And fate maps This is because it's invariant so all cell fates are the same for every organism All the cells that will form in a c. elegan = Most divisions happen during gastrulation but there is still some cell division in each successive malt

Susceptibility to infection

Cell signals to neighbouring cells that something is going on, it has been invaded Being able to adhere and enter the cell

Apoptosis - programmed cell death

Cells can die from necrosis (die and release contents - inflammatory response) OR apoptosis - controlled cell death (no inflammatory response) Important for maintaining new cells, Old cells die of and are replenished = Apoptotic signal is produced by neighbouring cell and cell programmed to die will begin to go through processes for death e.g. blebbing of membrane = Also signals for cells to come and get the organelles to recycle them

What has changed in the Ubx gene to prevent the development of limbs on the abdominal segments of insects? What effect does this have on the Dll gene?

Change in protein structure of ubx that changes the function of ubx and how it works with dll.

Variation in body size - growth rate and growth duration.

Changing growth rates, while maintaining constant duration of the growth period, will alter final body size. - Reducing growth rate without affecting developmental time results in smaller pupae. - Reducing developmental time without affecting growth rate also results in smaller pupae.

How do the concepts of homology versus novelty relate to how traits can be modified?

Homology: Structure or gene with a common decent. - If we want to modify a human hand to look like a whale limb, we can modify that existing structure to become something different, which is homology. Novelty: structures that do not have homologous structures in the ancestral lineage nor are they serially homologous to other structures in the same organism - Make something new, to create diversity - E.g. flowers, feathers, scales on insects, horns on beetles. - Ancestors don't have these structures.

What is cryptic genetic variation? When would you expect to see/expect not to see phenotypes associated with cryptic genetic variation?

Cryptic genetic variation: standing genetic variation that does not contribute to the normal range of phenotypes observed in a population, but that is available to modify a phenotype that arises after environmental change or the introduction of novel alleles Why can't we see it? - developmental processes are buffered (buffer against slight variation) - candidate for this buffering mechanism: heat shock proteins - BUFFERING: most of the time you won't see genetic variation because the buffer system is good at keeping everything normal, however, if overwhelmed... - if you produce environmental stress, the buffer will generally take care of it, however, if envo stress is strong and overwhelms the buffer system, you then get a new phenotype. - chaperone proteins generally fix these developmental mistakes by helping proteins to fold correctly.

Histone Mods cont.

DHS: Mapping where the dna was accessible, indentify the footprint of TF Tells where dna is accessible and what was bound there ChIP: Can be used to identify TFs bound to specific DNA sequence motifs Can be used to characterize DNA sequence motifs recognized by TFs Can be used to study the effect of DNA polymorphisms (SNPs)

How cells become different?

Development is the process of cells becoming different Positional information: one cell inherits something from the original cell and one cell doesn't Then you can get INDUCTION, with enough cells, where one cell tells the neighbouring cells to do something or INHIBITION where one cell tells the neighbouring cells not to do anything.

What is developmental buffering and how does it normally relate to cryptic genetic variation?

Developmental buffering is a process that accounts for the slight genetic variation, meaning you generally don't see genetic variation as much due to this. However, if the system is overwhelmed, the buffering process can be disrupted, and a new phenotype can be formed. This relates to cryptic genetic variation, because it buffers against this variation, thus we don't normally see it.

Histone modifications

Different residues and mono/di/tri methylation has different effects in same histone Activity that is taking places in a region of euchromatin Lots going on, a lot of transcription activity Methods DHS (DNase I hypersensitive site) assay ChIP (chromatin immunoprecipitation) to identify DNA sequences associated with histone and other modifications, resulting from increased DNA accessibility (usually because actively transcribed) can be applied to screening the whole genome

Genetic screen to identify auxin resistant mutants

Do mutagenesis's to understand action of auxin Screen for plants that cant respond to light in proper way Screen for mutants that are resistant to high levels of a hormone or hormone analogs => Often identify genes encoding components of perception, transport, or response.

Apoptosis was first discovered in C. elegans

Easy to do a screen for apoptosis in c.elegans bc they leave cell corpses and they're easy to count. Easy to screen for embryos with too many or too little corpses = Did a screen and found 3 genes that were very important

The "Hygiene hypothesis"

The hygiene hypothesis proposes that overcrowding and unhygienic contacts (microbial exposure, infections etc) early in life may protect from (auto)immune diseases

Two homeotic gene complexes in Drosophila

The order of the genes is the same as the order of expression along the A-P axis and of the segments each gene controls. Example: The bithorax complex specifies posterior segments If delete whole complex, every parasegment from 5-13 is transformed into parasegment 4 (14 is OK). Therefore essential to diversify 5-12. Default state is 4. To examine role of each gene, make single, double or triple mutants. Shows that identity of each parasegment specified by combinatorial action of the 3 genes.

What gene is altered in sticklebacks to control the presence or absence of pelvic spines, and how is it altered?

The study found that there are most likely multiple regulatory elements controlling Pitx1 gene expression in different tissues. The research has identified a major chromosome region that controls the loss of pelvic structures in a natural population of sticklebacks. Their study suggests that regulatory mutations in key developmental control genes may also be responsible for major morphological changes in limb/fin structures during the evolution of these two separate fish populations. - The freshwater stickleback lacks an enhancer for expression in the hindlimbs= Modularity of enhancers

How can gene duplication generate genetic variation and how does this allow for traits to evolve?

Three possibilities: a. Subfucntionalisation: 2nd copy confers a benefit o eg. the expression increase is beneficial, so both copies retained as functional genes o same function but in a new way (benefit) b. Neofunctionalisation: One copy acquires mutations that confer a new function that is advantageous, so both copies retained (2nd gene has a completely new function) c. Pseudogenisation: 2nd copy confers no benefit and one copy rapidly accumulates deleterious mutations as not under any selective pressure. o This happens to the majority of duplicated genes, acquire mutations that inactivate them and become pseudogenes.

What is the Target? Identifying the target(s) for your experiment

Tissue, cell type, organelle, protein/mRNA/other Identifying target: Is it obvious to you or somebody that can show you? --> NO - then you need a marker - Protein tag (fusion or reporter constructs) - Stain (pH, nucleic acids, lipids, etc) **Some work on live tissue, some only work on fixed (dead/preserved) tissue --> YES Is identifying the target sufficient to perform your experiment? YES - if no, the need a marker

What tools do we need to tell if neuron A makes a synapse with neuron B?

To be able to differentiate between two neurons, it's useful to have different coloured fluorescent proteins. RFP, or red fluorescent protein, can be used to distinguish between cells expressing GFP.

Toll signalling pathway

Toll uniformly distributed in plasma membrane. Ligand (spatzle) also uniformly distributed, but only activated on ventral side (controlled by signal from follicle cells). Dorsal is a transcription factor, usually prevented from entering nuclei by being bound to Cactus protein. Toll activation leads to Cactus degradation, Dorsal can enter nucleus.

Describe differences in body shape using scaling relationships between organs

Traits tend to show exponential growth. We can turn the exponential function into a line. This is called the allometric equation. log (y)= log (b)+ a log (x)

Transgenic Tools

Transgenic tools can be used to either stimulate (turn on) or inhibit (turn off) neurons. The following transgenes are commonly used in D. melanogaster to manipulate neural activity.

CFTR

Transmembrane protein (5 domains) ATP-binding cassette transporter Chloride channel Controls production of mucus Expressed on epithelial cells in - lungs - digestive tract - liver - pancreas - reproductive tract Delta F508 mutation no expression on membrane most common mutation (70% of CF) high frequency of carriers (1:25)

2.3. Segment polarity genes

Turned on around time of cellularisation, and mediate interactions between cells. Roles of segment polarity genes: 1. Fix the segment boundaries, and then establish segment boundaries in the larva. 2. Establish cell fates across each segment Segment polarity genes encode components of the wingless and hedgehog signal transduction pathways. Pathways highly conserved in vertebrates. Expressed in 14 transverse stripes, one stripe per segment. Each gene activated by different combinations of pair rule and gap gene expression. Embryos mutant for these genes have defects in A-P polarity of segments. Deletion of part of each segment and replacement with mirror image of next segment. eg. in larval segments anterior portion bears denticles, posterior region naked.In wingless mutant posterior region now mirror image of anterior, posterior region lost.

Sector mosaic of shoot

Two types of sector can be observed: 1) a mericlinal mosaic - a strip of about 1/3 of the circumference 2) a periclinal chimera, where the L2 layer has a mutation resulting in a loss of chorophyll. Also, can follow sequential production of leaves along plant stem, if sector of meristem is genetically marked. Sector stays in a strip up the plant - mericlinal mosaic, or sector mosaic. Conclude: cells do not migrate sideways around the meristem.

-omics

Very complex/demanding/expensive, not the first line method What omics type, tissue source, analysis etc ("how/where to look...") Usually used in integrative analyses ie multi-omics Mostly applied to complex diseases for patients stratification Individual omics may help selecting candidate causative mutations (example metabolomics in rare disease lactic acidosis and epilepsy)

When do you need to develop a new model organism? What types of features help to make a species a good candidate for a new model?

What makes a good model? These features help, but are not all necessary: - Short life cycle - Rear in the lab - Genetic tools - Closely related to an - existing model - Genome sequence (or - access to material to - sequence) - Economic importance - (honeybees)

2.4. The homeotic selector genes

What makes the segments different from each other? - specification of segment identity involves master regulatory genes called homeotic selector genes (Hox genes). Mutations in Hox genes change identity of segments, not number - result in transformation of whole segments or structures into another one = homeosis Many mutants in homeotic genes are recessive lethal, but can see phenotype in embryo and early larvae before they die. Some mutations which affect regulatory regions, not coding sequence, are adult viable. These mutants led to identification of the genes. eg. ultrabithorax mutation, antennapedia mutation

Commonly used types of compound microscopes

Widefield fluorescence Widefield fluorescence microscopy is an imaging technique where the whole sample is illuminated with light of a specific wavelength, exciting fluorescent molecules within it. Emitted light is visualised through eye pieces or captured by a camera. Confocal fluorescence Is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. --> Advantages over conventional widefield optical microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens.

1.1. Four maternal mRNAs establish the A-P axis

bicoid and hunchback, critical for head and thorax formation. - If lost these genes then no head and thorax, so realised they're crucial for these body parts nanos and caudal, critical for forming the abdominal segments. Before fertilisation all four mRNAs deposited in egg by maternal cells - bicoid mRNA located in anterior of egg and nanos mRNA in posterior - hunchback and caudal mRNAs uniformly expressed Upon fertilisation all four are translated, encode TFs, but can diffuse because embryo is a syncytium. Protein gradients then established: - Bicoid in protein gradient highest at anterior. - Nanos forms gradient highest at posterior. - Caudal protein gradient formed because Bicoid inhibits translation of caudal at anterior end. - Hunchback protein gradient forms because Nanos inhibits translation of hunchback at posterior end.

Pair-rule genes example

eg. eve stripe 2 - bicoid and hunchback activate eve transcription. - boundaries set by giant and Kruppel, which repress eve transcription at a certain threshold level.

Establishment of polarity in Fucus zygotes

initial signal: mum provides asymmetrically distributed product environmental stimulus What was the original asymmetry, and how do you get it when all cells start as the same cells? E.g. drosophila, mum puts nanos and bic at either ends so when dad fertilises it, the ends already know what they're going to be E.g. In plants, initial difference arises from environmental signals - Where light is coming from - And where gravity is pulling you Polarity due to shaded side having a calcium flux the other side doesn't have. And if these cells divide, you could get two different cells

Genes involved in vulval induction

lin-3 (lineage-3) - all 6 P cells develop into epidermis - encodes a ligand of the EGF family (secreted protein) - LIN-3 is expressed in the anchor cell let-23 (lethal-23) - same phenotype as lin-3 (all cells epidermal) - encodes an EGF (paracrine) receptor - LET-23 is expressed in all 6 P cells

miRNA/siRNA

miRNA found in animals and plants from endogenous genes single strand, stem-loop structure partial match with target genes (3'-UTR) often multiple targets inhibition of translation siRNA found in lower animals and plants exogenous (ie viruses) + endo-siRNAs double strand perfect match with target genes usually one target (same gene) mRNA cleavage therapeutic potential: Manipulating siRNA/miRNA expression may result in gene-specific effects of therapeutic potential

Genetic defects in the development of the immune system

nude mouse: no thymus Foxn1 mutations - When something does really wrong= No thymus IPEX Many organs and tissues are effected Gene usually controls inflammation When mutated, no regulation, so lots of immune cells and antibodies are proliferated Severe condition One transcription factor can have effects in many organs

Phyllotaxy

origin of leaves and their location along the stem

'ABC' model of floral organ identity specification

sepals: A function petals: A + B function stamens: B + C function carpels: C function In mutants, individual functions lost ⇒ organ identity changes.

wuschel and clavata mutants

wuschel and clavata mutants have opposite effects on meristem maintenance wuschel mutants fail to maintain the SAMclavata mutants (3 complementation groups) have overgrown SAM Wuschel mutant: - Meristem used up over time - Produces leaves at the start and then leaves used up - Then bc plant cells can differentiate, they become a new meristem, then used up etc. - Essential for maintenance of meristem Double mutant of wuschel and clavata 3 Just get wuschel