Fragenkatalog

In common: Both cell-cell adhesion structures, mediated by Cadherins

Differences: Desmosomes-TM-adhesion protein-Non classical cadherins (Desmoglein, desmocollin), Intracellular Cytoskeletal attachment- Intermediate filaments

Adherens junction - TM-adhesion protein- Classical cadherins, ntracellular Cytoskeletal attachment- Actin filaments

In Common: Intracellular Cytoskeletal attachment: Intermediate filaments, both cell adhesion structures

Differences: Desmosomes- Mediate Cell-Cell adhesion, TM-adhesion proteins -Cadherine (Desmoglein, desmocollin),

Hemidesmosomes - Mediate Cell-ECM adhesion, TM-adhesion proteins-Integrin, type XVII collagen

Also different Intracellular adaptor proteins

a) Clasical cadherin (E-Cad, N-Cad) Neighbouring cells are linked through homophilic interactions between the extracellular domains of the cadherins, p120 Catenin, ß-Catenin linked to Cadherins on Cytosolic side, Anchorprotein (Vinculin) link Complex to Actinfilaments

b) In epithelia

c) Cadherin cell adhesion is Ca2+ dependent. EDTA forms a chelate complex with Ca2+ and therefore destabilizes the adhesion. Cadherins contain Ca2+ -binding sites in their extracellular domain and Ca2+ binding is required for maintaining of this domain in a conformation that allows homophilic interaction. Removal of Ca2+ induces a conformational change that prevents homophilic interaction

d) ß-Catenin is a component of the canonical Wnt pathway. Upon activation of the pathway cytoplasmatic ß-Catenin is stabilized, translocates to the nucleus and associates with Lef/Tcf Proteins to form a transcriptional activator

a) Integrin a3, Integrin a6ß4, collagen XVII, Laminin-332 make up connection of epithelia to Basal lamina in Hemidesmosomes

b) Kollagen VII mediates connection Dermis to Basallamina

Laminin: Primary organizer of basal lamina structures, joins to each other and binds to Surface Receptors (Integrins) to form a network -> Stabilization

Collagen IV: ropelike superhelix provides tensile strenght to basal lamina

Integrins: Connect Epithelial cells to ECM

Additional Components: Nidogen-Glycoprotein, Perlecan-proteoglycan -> Bring protein stability

Cell-Matrix adhesion structures that anchor epithelial cells to underlying basement membrane and provide structural stability promoting tissue integrity

a) Tight junctions-seals gap between epithelial cells

Adherens junction- connects actin filament bundle in one cell with the adjastend cell

Desmosomes- connect IF in one cell to those in adjastend cell

Gap junctions- allows passage of small water soluble molecules from cell to cell

b) Tight junction: Occludin & Claudins

Adherens junction: Cadherins (E-Cadherin)

Desmosome: Desmogleins & Desmocollins (Nonclassical cadherins)

Gap junctions: Integrins

c) Desmosomes have contact with Cytoskeleton & connect to Intermediate Filaments (Keratins)

A) Function is to inhibit Apoptose, Prevent activation of pro-apoptotic members of BCl-2 family -> inhibiting Cytochrome c release in Mitochondria

B) promote apoptose by facilitating the release of pro-apoptotic factors (Cytochrome c) - Promote Activation of downstream caspases and apoptosis initiation, Acts opposite to pro-survival BCL-2

C) Important role in Intrinsic apoptotic pathway - Mitochondria release Cytochrome c in Cytoplasm -> triggers formation of the apoptosome -> Caspase activation & Caspase cascade

D) Protease Enzymes Key executioners of Apoptose, Activated in response to apoptotic signals. Initiator Caspase (9) cleave & activate effector caspases (3,7) -> proteolytic cleavage of cellular substrates

E) Apoptose Inhibitors that inhibit caspases, regulate both intrinsic & extrinsic pathway

· Genome [numerical alteration of the complete genome or numerical alteration of a single chromosome]

· Chromosome [insertion, deletion, duplication, inversion, translocation (defects every 3 – 5 Mb)]

·  Gene [point mutation, mutations of several kilobases, mutations in regulatory sequences. 1-300bp. point mutations account for up to 70% of all types of variation]

·  SNP`s [= ”single nucleotide polymorphism”: occur ~ 1 in 300 nucleotides]

· CNV`s [= “Copy number variations”: are responsible for by far the greatest number of nucleotides that differ between two genomes,]

· Microsatellites [= “short tandem repeats” (used for DNA profiling)]

-> SNPs most common type in humans

·  SNP`s and small DNA changes are caused by DNA damage, that can occur via

- External causes: [ionizing radiation, ultraviolet radiation, environmental chemicals]

- Internal causes: [depurination, deamidation, reactive oxygen species (ROS), nonenzymatic methylation, normal DNA metabolism]

· CNV`s (and microsatellites and SNP`s)  are generated by errors in recombination and errors in replication

· Microsattelites: Errors in replication (backward spillage)

· All DNA Variations are caused by failure in repairing DNA

·  Derived from a SNP

· Exchange of the encoding amino acid leads to premature stop codon ( ->termination signal)

· Possible consequence truncated protein, protein degradation, NMD (nonsense-mediated mRNA decay)

· Copy number variation = sections of the genome are repeated

· Surprisingly frequent in human genome (~5%)

· Possible consequence: parts of a chromosome are deleted or duplicated

a) Autosomal dominant

b) 50%

c) Dominant negative effect (Osteogenesis imperfecta)

Haploinsufficiency (Aniridia)

a) X-linked recessive. Only male individuals are affected females are only carriers

b) Red-green color blindness, Haemophilia A & B, Duchenne muscular dystrophy, Fra (X) syndrome

Autosomal recessive - both parents are carriers but only homzygous mutation leads to phenotype

Both alleles carry the same mutation - individual homozygous for that mutation

Presence of 2 different mutant alleles at the same locus

· A loss-of-function mutation is a genetic mutation that leads to a reduction or complete loss of the normal function of a gene -> no protein, non-functional protein, disrupted functional domain

· Examples: Cystic fibrosis (CF), Hemophilia, Tay-Sachs disease

usually (autosomal) recessive pattern of inheritance

·  Autosomal recessive - One affected allele is tolerated, typically unaffected carrier parents, affected individuals homozygous or compound heterozygous E.g. Cystic fibrosis

·  Autosomal dominant - One affected allele is not tolerated, affected individuals usually 1 affected parent, 50% chance of passing mutant allele to offspring, E.g. Huntington disease

·   X-Chromosomal - Mutations on X chromosome either Dominant or Recessive (in Males single copy sufficient, females homozygous, E.g. Hemophilia)

(Y-chromosomal - Only males affected from father to all sons)

· A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of a species’ set of genes. They are often assembled from the sequencing of DNA from a number of donors.

·  Reference genomes do not accurately represent every individual among a population.

· SNPs (inducing small insertions, deletion of a few base pairs)

the main principle of NGS involves parallel sequencing of millions of DNA fragments, generating massive amounts of sequence data simultaneously.

· Fragmentation of DNA & adaptor ligation (library preparation)

· Immobilization on a glass slide surface (flow cell) & amplification of DNA fragments (clonal amplification by bridge PCR, cluster generation)

· Cyclic sequencing of clusters with fluorescently labelled nucleotides

· Imaging of flow cell after each cycle (emission from each cluster is recorded, emission wavelength are used to identify the base)

· Cycling is repeated ‘n’ times to create read length of ‘n’ bases

· Millions of short reads are aligned to a reference sequence with bioinformatics software

1. in Promotor (e.g. TATA Box) - affects expression levels

2. in splice site (Enhancer, Silencer…) - affects splicing, exons skipping, intron retention

3. in 5` UTR (cap) - affects mRNA stability

4. in 3’ UTR (Poly-A) - affects stability & export mRNA

Silent Mutation - same amino acid (rarely pathogenic), different tRNA

Missense Mutation - different amino acid by single nucleotide substitution, conservative change (AA similar properties), non conservative change ( AA different properties -> Alters structure, function of protein) E.g. Sickle Cell Disease

Nonsense Mutation - nucleotide substitution leads to premature stop-codon (termination) -> truncated protein, protein degradation, NMD-nonsense mediated mRNA decay

Readthrough Mutation - Nucleotide substitution -> Stop codon is lost -> Extended Protein

Frameshift mutations:

• Insertion (addition of nucleotides)

• Deletion ( Loss of nucleotides)

  -> can lead to stop codon, degradation by NMD

A-P

· mRNAs provided by the nurse cells use the microtubule scaffold to differentially localize to the two poles of the oocyte. Location of the mRNAs define the anterior (bicoid) and posterior (nanos trapped by Oscar) pole

• Anterior: Bicoid transported by Dynein to minus pole of microtubule

• Posterior: Oscar transported by Kinesin to plus pole of microtubule where it traps nanos

·  Oocyte nucleus associates with the microtubuli network and is transported to the minus end. The location of the nucleus defines the dorsal pole. Nucleus travels to anterior dorsal side -> Gruken recieved by Torpedo in dorsal follicle cells -> Inhibition of pipe dorsally

Ventral secretion of pipe - cleavage of spätzle binds toll & activates dorsl

Maternal gradients (Bicoid,nanos…)

Gap genes: regulated by maternal gradients, expression controlled by concentration of bicoid & hunchback at different points along A-P axis, Regulation of Gap genes by themselves ( Krüppel, Hunchback…)

Pair rule genes: formation of striped pattern that’s a precursor for segmentation. Each stripe requires a specific gap protein activity. e.g. evenskipped: hunchback and bicoid would activate evenskipped in a broad domain but only where giant and krüppel activity is lowest the stripe is formed.

Segment Polarity genes: equip the parasegment with polarity, forming an anterior/posterior pattern in each segment. Expression is initiated by gap and pair rule genes and maintained by autoregulatory circuits. produce signalling molecules / transducers. e.g. wingless & engrailed: neither evenskipped nor ftz is present wingless is expressed, wherever one of those pair rule genes is present engrailed is expressed.

Homeotic Genes (Hox): specify the segment identity, appear in gene clusters, regulated by gap and pair rule genes and cross-regulation. gene order on chromosome corresponds to spatial and temporal order of gene expression along the A-P axis = colinarity. homeobox = DNA binding domain, transcription factors. Mutated HOX genes -> defect body structures (e.g. 4 wing pairs [ultrabithorax HOX gene] or legs for antenna [antennapedia])

Patterning of cellular fields:

• Morphogen gradients: signalling molecules, that form concentration gradients across developing tissue. Concentration at particular location determines fate of cells in that region. E.g. Maternal gradients of bicoid (anterior) & caudal (posterior)

• Lateral inhibition: Cells with a certain fate inhibit neighboring cells from adopting the same fate. E.g. Delta-Notch inhibition

• Induction/Induction cascades: Group of cells influences the fate of adjacent cells. Induction cascades lead to sequential determination of cell fates. E.g. Lens formation

• Oscillating systems: generation of periodic patterns of gene expression or signaling. periodicity contributes to the establishment of positional information. E.g. Segmentation clock in somitogenesis

• Reaction diffusion systems: Interaction between activators and inhibitors that diffuse through tissues -> results in the formation of spatial patterns. E.g. WNT-DKK specifies hair distribution, Zebrafish stripes

Organization by cell sorting:

• Cell adhesion: Cells with similar adhesion properties tend to sort out and form distinct tissues mediated by Adhesion molecules.E.g Cadherin mediated cell adhesion and sorting leads to tissue organization

Making cells different:

• Localized determinants: positional info by establishing e.g anterior and posterior axis through asymmetric distribution. Mechanisms: Diffusion and local anchoring (Nanos), Localized protection (hsp83), Active transport along cytoskeleton (Oskar and Bicoid mRNA on microtubules)

• Asymmetric cell divisions: Daughter cells with different fates – unequal distribution of cellular components or fate determinants. E.g. Asymmetric division in Neuroepithelium

Through Cell Adhesion. E.g. Cadherin mediated:                  Different cadherin types in different cell. Some have stronger adhesion and form stronger aggregates. Strong aggregates form the center, low adhesion in the peripherie.  N-, P-, and E caherins(Cells with high adhesion maximize adhesion to each other and minimize contact to cells with low adhesion) Bsp. Neural plate epidermis

Each cell inherits a specific determinants which determines the cells development. Only the germline cells are consistent. That’s why mutation in the germ cell are inherited by the offspring and mutations in the somatic cells are not.

· Diffusion and local anchoring: The nanos mRNA diffuses in the cell, posteriorly is an anchor (oskar) that fixates nanos to the posterior site -> local translation of nanos

· Localized protection: hsp83 diffuses in the cell and gets protected by Protector protein complex otherwise it will get degraded – only proted mRna will get translated

· Active transport along cytoskeleton: Oskar and Bicoid mRNA use microtubules as transport mediated by Dynein and Kinesin, Oskar moves posterior via kinesin, bicoid moves anterior via Dynein

Symmetry Breaking (Polarity Determinants/Proteins are distributed asymmetrically within cell E.g. PAR-Proteins) -> Polarity establishment -> Determination segregation -> Spindle positioning -> Distinct daughter cells

Cell fate determining proteins in one of daughter cells

Induction:

• Group of cells influences the fate of adjacent cells.

• formation of a different tissue, induced by a signal from tissue B, the signal reaches up to a certain threshold into tissue A and leads to the formation of tissue C

• one signal induces only one new cell type with one concentration threshold.

serial induction (= induction cascade, relay mechanisms):

• From the newly formed tissue C another signal induces the formation of tissues D and E, the signal acts on tissues A and B up to a certain threshold

• e.g. formation of the lens

• signaling molecule that plays a key role in embryonic development by forming concentration gradients within developing tissues.

• concentration gradients provide positional information to cells, influencing their fate and behavior based on the concentration of the morphogen they are exposed to.

• essential for the process of pattern formation, establish the spatial organization of different cell types and structures in an organism.

• Examples: Sonic hedgehog, Bone Morphogenetic Proteins (BMPs), and Wnt proteins

stable source of signal and a limited stability of the signal leads to a balance between synthesis, diffusion and degradation to establish a stable gradient after a startup time

An inducer is extending across a field of cells and uniformly distributed, an inhibitor is then distributed from a source resulting in a gradient of inducer activity

A morphogen gradient can expand proportionally with tissue growth, it can pattern small and large scale tissue fields, an adjustment of stability, diffusion and transport rate may be needed

Field of cells that all have the same potential to differentiate- some cells inhibit adjacent cells from developing in a similar way. Individual cells within a field of cells get defined. E.g Delta-Notch forms a contact dependent signalling system between adjacent cells. Competition of Notch and Delta receptors of each cell. Delta binds to Notch on adjacent cells. Activation of Notch leads to inhibition of Delta. One cell wins.

Oscillating systems generating repeat units through cycles of gene expression, the mechanism works on 3 tiers: the bottom tier is single cell oscillators where the translation of a gene expresses a protein that inhibits the gene (negative feedback) the middle tier is local synchronisation of neighbouring cells and the upper tier is global control of slowing and arrest which means the signal gradient gets slower and at some point arrested which forms a new segment. During each cycle one new segment is determined. (clock and wavefront model)

Circuit of diffusion activator and inhibitor where the activator activates itself and the inhibitor, the inhibitor then inhibits the self-activation of the activator. The Reaction diffusion system has a self-organizing property and generates patterns independent of size of the cellular field, whenever the concentration of the activator exceeds a certain level there

Example for Reaction-Diffusion System hat influences the patterning and density of hair follicles

Wnt ligands are expressed in localized regions, promoting hair follicle development, while DKK proteins act as inhibitors, restricting Wnt signaling in specific areas. spatial distribution of Wnt and DKK establishes patterns of high and low Wnt activity,

Once developed stripes do not change anymore, but over time new stripes are added.

Example for an Reaction-Diffusion System Interaction between activators & Inhibitors leads to pattern formation -> Diffusible signalling molecules establish a stable pattern

Bicoid plays crucial role in embryonic development in certain organisms (Drosophila) -> well-conserved between close related species of Drosophila, may not be conserved across all phyla

Mechanisms of embryonic development can differ among diff. organisms

· Cross regulation (e.g. gap genes, pair rule genes and segment polarity genes repressing or activating each other)

· Regulation by maternal gradients (bicoid, hunchback, caudal regulating gap genes)

· Chromatin Modification and Epigenetic Regulation

Embryos lacking Dorsal protein are homozygous for a mutant dorsal allele -> Therefore the first statement is correct

Since dorsal is a maternal gradient the second statement is also correct - If the mother lacks functional Dorsal protein due to being homozygous for a mutant allele, the embryo she produces will be dorsalized

Somatic cells: maternal gene products mRNAs & Proteins E.g Dorsal, Toll

Dorsal: Contributes the genetic information for genes involved in D-V patterning

• The somatic cells contribute to the formation of the D-V axis through the expression and localization of signaling molecules -> Toll receptor activated by spätzle (produced by ventral follicle cells) leading to nuclear localization of dorsal Protein on ventral side -> TF for ventral developement

• The germline cells also contribute to the establishment of the D-V axis by providing maternally deposited factors in the egg -> e.g. gurken which determines Anterior - Posterior but also indirectly D-V

• BMP binds to type 1 and 2 serine threonine receptor -> phosphrylation cascade

• Phosphorylation cascade: Type 2 -> type 1 -> smads (TF, regulates severall genes)

• Smad builds complex with co-smads and translocates into the nucleus

• High bmp activity dorsal, ventral low activity  -> gradient

• Sog (DPP antagonist) gradient, graded inhibition of dpp(BMP) generates a reverse gradient of dpp signaling activity

• Sog is involved in the transport from dpp to dorsal side, by binding to dpp

• Bicoid gradient – source and sink gradient (diffusion) e.g. anterior posterior dev.

• BMP gradient – time resolved inhibitor e.g. dorsal ventral dpp sog

• Dorsal gradient – nuclear translocation signal via active transport e.g. dorsal ventral dev.

• Threshold dependent activation of different genes through Dorsal gradient inhibits dpp at the ventral side

• Dorsal inhibits dpp

• Sog is distributed in a gradient and binds to dpp preventing dpp to bind to BMP receptor

• Graded inhibition of dpp via sog generates a reverse gradient of dpp signalling activity

• Sog pushes dpp from the flanks to the dorsal side (amnioserosa AS)

• Loss of function: Mutations, deletions in the gene -> no gradient -> no morphogenic activty ergo no morphogen

• Gain of function: Overexpression

• Liveimaging

• Dose response analysis

• Gene A expressed at high gradient level -> inhibition of gene C

• Gene C expressed at gradient threshold too low for gene A activation

• Gene B active when gradient is low because the gradient acts as inhibitor for B

• A fate map is a diagram or representation that illustrates the developmental destiny or fate of cells in an embryo. It shows how different regions or groups of cells will differentiate and contribute to specific tissues, organs, or structures in the fully formed organism.

• Investigation by: Tracing techiques, cell ablation (removal), transplantational studies, genetic markers and imaging/microscopy

No, the germ cells cant be assigned to the three germ layers, other cells can be assigned to the endo-, meso- and ectoderm.

Epiboly: process during embryonic development in which one or more cell layers spread over and cover another layer of cells. In Xenopus gastrulation, the ectodermal cells from the animal hemisphere spread over the vegetal hemisphere, covering the entire embryo. Radial intercalation of cells of the blastocoel roof drives epiboly.

Emboly: cells undergo movements such as invagination, involution, and ingression, leading to the formation of germ layers. Group of cells on the surface of an embryo rolls or folds inward to form an underlying layer. key process in the formation of the mesoderm & Endoderm. Invagination- sea urchin, Involution- frog, Ingression- fish

Convergent Extension: cellular movement during which cells in a tissue layer intercalate, causing the tissue to both narrow in one dimension and extend in another. Directed migration of ventral & lateral cells toward the dorsal midline results in anterio-posterior extension of the body axis

Cells that will become endoderm & mesoderm in the marginal zone move inside the gastrula through the blastopore by rolling under the lip as a coherent cell sheet -> Involution

• Surface: involution of meso- and endoderm through blastopore by rolling under the lip as coherent cell sheet

• Deeper:Tissues converge towards midline & extend along antero-posterior axis (Convergent Extension)

• Ectoderm spreads downward to cover the whole embryo ( Epiboly)

• Inward movement of Endoderm & Mesoderm begins dorsally & spreads laterally & ventrally to form a complete circle of involuting cells around blastopore

  
  

• Reorganization of the Cytoskeleton to establish polarity: Microtubule & Actin dynamic

• Polarized Membrane domains: E.g PCP pathway components like Receptors apical & planar distribution

• Cell Adhesion & ECM interactions – Integrins & Cell Adhesion molecules

Planar Cell Polarity (PCP) pathway maintains the coordinated polarization of cells within a tissue.

After Cell Polarity is established Frizzled receptors are activated by signaling molecules, which leads to the recruitment and activation of Disheveled (Dsh) in the cytoplasm. Activated Dsh initiates a cascade of intracellular signaling events that result in the asymmetrical distribution of signaling components within the cell. The asymmetrical signaling results in the recruitment and interaction of proteins like Vang/Stbm and Flamingo, contributing to the establishment of planar cell polarity.

PCP pathway is regulated by Canonical WNT signalling:

Wnt ligands can activate the PCP pathway

• After fertilization cortical layer rotates towards the side of the sperm entry -> Cortical rotation establishes initial asymmetry. Corticale Rotation causes the activation of ß-Catenin on the dorsal side of the blastula and Overlap with TGF-ß/Nodal induce the formation of the Nieuwkoop Center

• Wnt/β-catenin pathway, is involved in specifying dorsal cell fates.

• The mesoderm is induced from the animal cap tissue. Nodal ( TGF-ß Signal) acts as a mesoderm inducer. Presence of ß-Catenin on the dorsal side stimulates nodal transcription -> Dorsal-Ventral gradient in Nodal related proteins -> Mesoderm is induced dorsally forms the Spemann Organizer. Maternal VegT, Vg1 activate expression of Nodal related proteins.

• A single animal cap cell or a small number of animal cap cells in contact with vegetal tissue are not induced to become mesodermal cells and do not begin to express mesodermal markers. A minimum of 200 animal cap cells must be present for induction of Muscle differentiation.

• The experiment involved transplantation of a specific region from one embryo to another, demonstrating the ability of this tissue to induce the formation of a secondary body axis. tissue from the donor's dorsal blastopore lip was transplanted into the ventral side of a recipient blastula. transplanted organizer tissue induced the formation of a secondary embryonic axis in the recipient embryo. This secondary axis included structures such as the notochord and neural tube.

Anatomically: Specialized region or group of cells that plays a crucial role in pattern formation and the establishment of body axes. E.g Amphibians -Spemann Mangold localized at dorsal blastopore lip

Molecularly: Releases signalling molecules E.g: Amphibians Chordin, Noggin, Follistatin antagonize BMP signalling

Functionally: primary function is to induce the formation of specific tissues and body axes. plays a role in establishing the dorso-ventral and anterior-posterior axes.

• Classic Organizer Transplantation: Transplanting a small piece of the dorsal blastopore lip or organizer tissue from one embryo to another can demonstrate the ability of this tissue to induce secondary axes, revealing the presence of organizer activity.

• Tissue Explants: Analyzing the behavior of tissue explants derived from different regions of the embryo to understand the intrinsic properties of organizer tissues.

• Loss-of Function Studies: Inhibiting the function of specific genes suspected to be involved in organizer activity to observe the resulting developmental effects.

• or Gain of fuction studies by Overexpression

Wnt antagonists enable the formation of dorsal & ventral structures:

Chordin, Noggin, Follistatin

Frzb also Wnt antagonist resembles Wnt binding domain of Wnt receptor Frizzled

• WNT Ligand Binding: WNT ligands bind to surface receptors E.g Frizzled

• Activation of Frizzled Receptor: WNT binding induces conformational change leading to its activation

• Disheveled Activation: Activated Frizzled receptors recruit & activate Disheveled

• Inhibition of Destruction Complex: Without WNT signal – destruction complex active (including GSK3, APC & CK1) phosphorylates ß-Catenin -> marking it for degradation. With WNT Signal Dishevelled inhibits GSK3 – preventing phosphorylation & degradation of ß-Catenin

• Accumulation ß-Catenin: unphosphorylated ß-Catenin acuumulates & migrates to the nucleus binds to TF and promotes gene expression of WNT target genes.

• Ligand Binding: Binding of BMP ligands (BMP2, BMP4…) to BMP receptors

• Receptor Complex Formation: upon ligand binding Receptor Type I & II form a complex  and Type II receptors phosphorylate & activate Type I recepors

• Activated Type I activates Smads: Phosphorylation and activation of Smads, dimerize with Co-Smads & regulate gene expression

• Chordin, Noggin & Follistatin are BMP antagonists, synthesized & localized on dorsal side establish the gradient of BMP activity via Inhibition

• BMP antagonists accumulate on dorsal side inhibit BMP activity -> High BMP ventral and No BMP Dorsal

• By using phosphor-Smad specific antibodies  -> Shows Pathway activity

• Reporter Gene constructs with fluorescent proteins like GFP etc.

Yes! TGF-β ligands typically bind to specific cell surface receptors. Different TGF-β ligands can activate distinct receptor complexes, allowing cells to discriminate between signals. Receptor Specificity. Different Intracellular Signal Transduction leads to activation of different Smads and therefore regulate different gene expression

• Formation & Signals of the Nieuwkoop Center induce the formation of the dorsoventral axis (Cortical rotation)

• Vegetal cells induce the mesoderm and the DV Nieuwkoop center induces dorsal mesoderm and the gastrula organizer

• The gastrula Organizer (Spemann) responsible for pattern formation along the DV and AP axis. Gastrulation movements, including involution and convergent extension, contribute to the establishment of the anteroposterior axis.

• Neurulation is the process during embryonic development in which the neural plate folds and transforms into the neural tube.

• Notochord induces formation of Central nervous system by signaling the Ectoderm to form thick & flat neural plate.

• Formation of neural folds and the elevation of such. Converging movement pushes neural folds upwards (n-Cadherin) -> Closure of neural tubes -> disconnection from epidermis

• the neural anlage, which refers to the early neural tissue or precursor to the central nervous system (CNS), initially does not have distinct anterior or posterior characteristics. Neural induction sets the stage for the development of the neural plate, and the subsequent processes of neurulation and regionalization further define the anterior-posterior (A-P) axis

• WNT signaling (and frzb as WNT inhibition) affect A-P patterning

Anteroposterior patterning in the neural plate:

—> (Spemann) Organizer - planar signals (patterning along a single layer or plane, often determining the anterior-posterior axis e.g. WNT pathway)

—> axial mesoderm - vertical signals (patterning along the vertical axis, specifying dorsal-ventral identity in tissues like the neural tube e.g. Shh specifying ventral fate)

• Sonic Hedgehog (SHH) Signaling: SHH is secreted by the notochord and floor plate cells, which are located at the ventral midline of the developing neural tube. SHH forms a concentration gradient, with higher levels ventrally and lower levels dorsally. Shh gradient determines neuronal populations along D-V axis of the spinal cord. Pathway actively repressed when no signal. Signal available binding to Patched & Smoothened -> Ci Protein made activator -> Transcription of Hedgehog genes.

• Bone Morphogenetic Protein (BMP) Signalling: BMPs are secreted by the roof plate and dorsal ectoderm, creating a gradient with higher concentrations dorsally and lower concentrations ventrally.

• Shh represses class I hox genes (pax6, pax7 ...) and activates class II hox genes (nkx2.2, nkx6.1). These hox genes interpret the shh gradient and control the  differentiation of ventral neural cell fate (V0, V1, V2, V3, MN). Shh gradient determines neuronal population along d-v axis

• Transplantation assay of Notochord. Notochord induces floor plate & ventral motor neurons. Notochord removed -> no floorplate & motor neurons. Notochord transplanted  second floorplate & motor neurons.

• Shh

• Global organization: wnt signaling —> high WNT activity on posterior, ANti-WNTs (frzb) on anterior side (forebrain)

• retinoic acid gradient along A-P axis or FGF signalling promoting posterior cell fate

• Interaction with organizer

• Regional organization: Rhombencephalon segmentation

anterior neural border

zona limitans intrathalamica

midbrain hindbrain boundary

• the process by which embryonic cells in the ectoderm acquire a neural fate (to form the neural plate) rather than differentiate as epidermis or mesoderm

• Inhibition of BMP Signaling: Chordin, Noggin, and Follistatin: Secreted proteins from the organizer inhibit Bone Morphogenetic Protein (BMP) signaling. Blocking BMP signaling is crucial for neural induction, as BMPs are potent inhibitors of neural differentiation. (“Default model “ Permissive induction, cells without BMP inhibition differentiate into Epidermis)

• FGF Signaling: Induces neural fate by inhibiting Smad phosphorylation -> Represses BMP transcription and induce BMP antagonist expression

• ß-Catenin & VegT: Nuclear distribution of ß-Catenin & vegetallocalisation of VegT determines the distribution of Chd, Nog, BMP, Fgf & Xnrs (Nodal related factors)

• developement of mesoderm around endoderm: lateral plate and Splanchnic mesoderm (Visceral mesoderm)

• (airway) smooth mucle cells (ASMC), connective tissue

-> Lungs and kidneys

• Initiation of Branching -> Bud formation, outgrowth from existing structure. Kidney: GDNF binds to Ret receptors on nephric duct. Binding initiates branching -> Uretric duct. Lung: Primary Lung buds formation via ventral Nkx2.1 expression

• Proliferation -> at the tip of the bud - extension into surrounding tissue, branched structure. Lung: Fgf10 expression at next branching event, negative-feedback loop Fgf10 -> BMP4, Shh. Kidney: GDNF & Ret Receptor binding

  
  

• Proliferation -> at the tip of the bud - extension into surrounding tissue, branched structure

• Interaction between different cell types

• ECM composition & organization influence migration & differentiation

• Apical-Basal polarity

• Remodeling & Maturation -> cell differentiate into specific cell types, branched network differntiates into functional organ

• Feed-back loops -> control branching process, Lung:

• precursor cells in the foregut region may have common developmental origins

• a specific combination of TFs form a specific organ, they overlap at some points that is why an alteration in these TF can reprogram cells to become a different organ

• Signaling pathways from the surrounding microenvironment also influence cell fate decisions. Altering the activity of specific transcription factors may impact how cells respond to signaling cues, leading to a shift in developmental fate.

  -> Siganls from Mesosderm affect TF in Endoderm several genes expressed at specific location leads to time dependent regionalization

• Premature differentiation leads to small organ size due to the depletion of the undifferentiated progenitor pool -> reduced number of cells available for further growth and development

• Impaired tissue architecture & infomplete Organ formation - rely on proliferation & differentiation of precursor cells

• Loss of regenerative Capacity -> reduction of progenitor pool limits self repair ability

Nkx2.1 induces seperation from the gut tube -> lung precursor formation

Nkx2.1 accumulates ventrally by upregulation from BMP, FGF and Wnt

Wnt signalling is upregulated by RA

Sox2 inhibited by BMP -> accumulates dorsally

Noggin at dorsal side inhibits BMP -> BMP only ventrally

-> repetitive process with self-limiting feedback loops

• Pathways involved: FGF pathway, Wnt pathway, Shh Pathway, BMP pathway

• Fgf10 from mesoderm (Receptor: Fgfr2) at tips of branches induces expression of BMP4 & Shh, Fgf10 is downregulated by Bmp4 & Shh -> limits outgrowth of individual buds, important for the development of adjacent side branches

Early Patterning:

• the ends of the branches grow further (directed by the mesenchymal cells that send FGF signaling) the cells outside of the FGF / BMP/Shh / WNT threshold (more proximal cells) already start to differentiate

• Distal parts of each branch have proliferative progenitor populations that induce side branching. They are exposed to high levels of Fgf10 which induces Bmp4 expression

• When the side branch grows cells escape the influence of Fgf10 & BMP4 which initiates differentiation to bronchial progenitor cells

• They express Sox2 and initiate the differentiation programm into Basal, Ciliated, Clb & Goblet cells

• ASMC (airway smooth muscle cells) differentiate from mesenchyme

Late Patterning:

Alveolar Development - Sox9 expressing distal progenitor cells can differentiate into ATI & ATII cells

• Upon injury -> surviving cells expand to cover gaps -> change of cell shape induces WNT7b expression & induces Fgf10 expression in underlying mesoderm (Stem cell niche)-> cells get reprogrammed to proliferate and regenerate injured cellls

• In stem cells: fgf10 produced by mesenchyme (stem cell niche) binds fgfr2b receptor & controls Wnt7b expression -> Fgf10 -> feed forward loop

1. Pancreas develops from Pdx1 positive endoderm in the posterior foregut region through fusion of a dorsal & ventral bud that are induced by independent mechanisms.

   - Signals from the cardiogenic mesoderm induce ventral foregut-> Low dosage of Fgf signaling needed (compared to lung)

   - Signals from the notochord allow dorsal pancreas development by suppressing endodermal Shh expression. Shh is downregulated by Fgf & Activin. -> Pdx1 expression

2. Proliferation: Feed-forward loop involving Fgf10 from pancreatic mesenchyme. Sox9 initiates expression of Fgf receptor and makes cells responsive for Fgf10 signals

3. Development of branched pancreas ductal system by joining of Microlumina & following Plexus remodeling

4. Specification:

   - Early progenitor give rise to trunk & tip progenitor cells (Binary cell decision controlled by Notch pathway activity, Notch active -> Trunk cell, inactive -> Tip cells).

   - Trunk cells give rise to duct cells & endocrine precursors ( bipotent, High Notch -> Duct cells, Low Notch -> Endocrine precursors (Sox9 -> Ngn3)

   -  Tip cells give rise to acinar cell of exocrine pancreas

   -Endocrine precursors delaminate from the ducts migrate into mesenchyme cluster & form islets of Langenhans -> vascularize

Proliferation and maintenance of the pancreas progenitor cells requires FGF10 from the surrounding mesoderm

Cell types and architecture of the adult pancreas:

1. Exocrine pancreas: acinar tissue (digestive enzymes) and ducts

2. Endocrine pancreas: islets of langerhans with alpha-cells: Glukagon, beta-cells: Insulin; delta-cells: Somatostatin, PP-cells und Epsilon-cells: Ghrelin

Pancreas morphogenesis: Development of the branched pancreas ductal system

Cell type specification in the pancreas (delta - notch)

1. Specification of Pancreatic Progenitors:

   • Early in embryonic development, cells in the endodermal layer of the gut tube undergo specification to become pancreatic progenitor cells. This process is influenced by signaling molecules, including fibroblast growth factors (FGFs(10) —> upregulate to inhibit Shh), bone morphogenetic proteins (BMPs), and sonic hedgehog (Shh —> inhibited for pancreas).

2. Formation of the Dorsal and Ventral Pancreatic Buds:

   • Pancreatic progenitors give rise to two buds, the dorsal and ventral pancreatic buds, which will fuse to form the mature pancreas. The transcription factor Pdx1 (Pancreatic and duodenal homeobox 1) plays a crucial role in the specification and maintenance of pancreatic progenitors.

   • Signals from the aorta and vitellin veins further support pancreas development

3. Fusion of Pancreatic Buds:

   • The dorsal and ventral pancreatic buds fuse to form a single organ. This process is mediated by signaling centers and involves the coordinated expression of various genes, including Ptf1a (Pancreas transcription factor 1a).

4. Endocrine Cell Differentiation:

   • Within the developing pancreas, some cells differentiate into endocrine cells, which will later form the islets of Langerhans. Neurogenin 3 (Ngn3) is a key transcription factor involved in the specification of endocrine progenitors. These cells then differentiate into specific endocrine cell types, such as insulin-producing beta cells and glucagon-producing alpha cells.

5. Exocrine Cell Development:

   • Other cells in the pancreas differentiate into exocrine cells, primarily acinar cells responsible for producing digestive enzymes. Transcription factors such as Ptf1a and Rbpjl (Recombination signal-binding protein for immunoglobulin kappa J region-like) play essential roles in exocrine cell development.

6. Ductal Cell Formation:

   • Ductal cells, which form the pancreatic ducts, are also essential for proper pancreas function. Transcription factors such as Sox9 (SRY-Box Transcription Factor 9) are involved in ductal cell specification.

7. Mature Pancreas Formation:

   • The coordinated development and differentiation of endocrine, exocrine, and ductal cells lead to the formation of the mature pancreas. This process involves intricate signaling pathways, including Notch signaling, which plays a role in cell fate decisions.

8. Islet Maturation and Functional Integration:

   • Following the initial differentiation of endocrine cells, further maturation occurs as these cells integrate into functional islets. The maturation process involves the expression of additional transcription factors, such as Nkx6.1 and MafA, which are crucial for beta cell function.

Notochord Ablation or Notochord tissue transplantation to different localization:

• Surgical removal/ablation or relocalization of the notochord (tissue) in chick embryos

• compare pancreas developement between control group and manipulated chick

• analyze development of pancreatic buds in both groups -> Examine pancreatic buds, especially dorsal pancreas bud -> Immunostaining for expression markers like Shh & Pdx1

• If removal of the notochord results in a significant alteration in dorsal pancreas development compared to the control group -> evidence that signals from the notochord are necessary for the induction of the dorsal pancreas bud in the chick embryo

Inhibition /Overexpression of Notochordal Signaling

• pathway inhibitors or blocking antibodies specific to notochord-derived signaling molecules

Tissue Recombination Experiments:

• isolate the Pdx1 positive endoderm and combine it with different mesodermal tissues

Removal or transplantation of the surrounding mesodermal structures and compare the effects on the mouse

genetic or chemical blocking of Signaling Pathways from the mesoderm

Live imaging techniques

Context-Dependent Signaling:

• Target genes of Fgf signaling can be cell type specific – therefore distinct transcriptional response

• Expressions of Receptors, Inhibitors and Activators of FGF10 signalling varies and there might be Crosstalk with other pathway that influence downstream gene expression

• Sox9 initiates the expression of the Fgf receptor and makes cells responsive for fgf10 signals – feed forward loop -> (disruption leads to respicification of pancreas progenitors into liver progenitors)

• Could result in overall decrease in Pancreatic size since Sox9 drives proliferation & survival of pancreatic progenitor cells -> Since Sox9 is involved in Cell type specification it could lead to altered tissue architecture, irregular cell organization, and disrupted cellular arrangements

• Neurogenin 3 (Ngn3) is a key transcription factor involved in the specification of endocrine progenitors. These cells then differentiate into specific endocrine cell types, such as insulin-producing beta cells and glucagon-producing alpha cells.

knock out phenotypes would have:

• Loss of Endocrine Cell Differentiation

• Decreased Beta Cell Mass (insulin producers —> type 1 diabetes) and alpha cell mass (glucagon)

• Islets of Langerhans altered, which contain clusters of endocrine cells, may exhibit altered morphology

Since Ngn3 is particularly important for the specification of endocrine precursor cells -> Could result in absence of endocrine cells -> deficiency in Hormone producing cells

No hormone production like Insulin could lead to Diabetes Phenotype

Isles of Langerhans cells may not develop since they are made of endocrine cells

On the other hand cells would preferably differentiate into ductal cells, increased cell production of these cells

Lineage Tracing:

• Utilize lineage tracing techniques to mark and track the descendants of Ngn3-positive cells over time —> genetic reporter system under control of Ngn3 promotor

Generate transgenic mouse lines that express reporter genes (e.g., green fluorescent protein, LacZ) under the control of the Ngn3 promoter

Specific marker such as immunohistochemistry or immunofluorescence on the Ngn3 pos cells

knock out mice —> inhibit or delete Ngn3 and see which cell do not deveope later

• Development of 3 different transient kidneys that develop from intermediate mesoderm: Pronephros ( no function), Mesonephros (embryonic kidney), Metanephros (permanent kidney)

• Formation of the nephric duct which elongates through proliferation -> Chemotaxis towards FGF8 produced in tail bud region

Metanephros development:

• GDNF binds to Ret receptors on nephric duct. Binding initiates branching -> Uretric duct formation

• Negative Regulators in adjacent cells of uretric bud inhibit GDNF (FOXC1) & Ret receptor expression (SPRY1)

• GDNF also controls further branching morphogenesis

• Wnt signals essential for nephron formation & branching morphogenesis of collecting ducts ( WNT11 tip positive feedback loop GDNF & RET, WNT9b acts on underlying mesenchyme -> mesenchym condensation -> WNT9b (stalk) -> ß-Catenin-> FGF8 -> WNT4. 3 Progenitor pools exposure to different concentrations of WNT9b in the stalk -> differentiation -> fusion with duct -> Nephron patterning with Glomerulus

GDNF functions at different times of development:

• Prior to Ureteric Bud Formation: Initiation of Bud fails and no collecting duct system is formed

• During Ureteric Bud Growth: Branching of uretric bud -> Inactivation would cause reduced collecting duct complexity with limited numbers of branches

• After Bud Branching: Further Growth and Maturation compromised

  Experiments: Fusion to gene switch (adding of substrate -> gene switched off), adding GDNF inhibitors during different stages, Knockout of GDNF gene during different stages of development

• Wnt -> frz -> dishevelled -> destruction complex inactive -> beta catenin into nucleus (transcription)

• Overexpression/Inhibition of Wnt ligands Adv: distinct phenotype Disadv: wnt signalling is everywhere -> off target effect limited specificity -> can effect multiple tissues & pathways

• Fusionprotein with gene switch -> makes Wnts visible + modulating amount (kp ob das geht)

• β-Catenin Staining

• fluorescence in situ hybridization

• immunohistochemistry: antibody staining

• reportergene with responsive molecule e.g. beta-galactosidase

• GDNF Staining

• GDNF Reporter mice/construct

• fluorescence in situ hybridization

• immunohistochemistry: antibody staining

• reportergene with responsive molecule e.g. beta-galactosidase

Oncogenes: Tumourigenesis due to dysregulated gene expression and/or gene product activity ->gain of function mutation, often in a dominant way (E.g. Ras,Src)

Tumorsupressorgenes: Tumorigenesis by loss of gene expression and/or function – loss of function mutations usually recessive (E.g. RB, p53)

Oncogenes: Ras (H-Ras, K-Ras), Raf, Src (v-Src), Bcr-Abl, Myc

Tumorsuppressor genes: BRCA1/2, Rb, p53, p16, p21

gain of function (oncogenes hyperactive) —> point mutations, insertions, translocations

loss of function (inactivation of tumor supressor genes) —> premature stop codons, epigenetic silencing, loss of genes/chromosomes

• No needs mutation in Oncogene & Tumor suppressor gene -> Otherwise tumor suppressor buffers over proliferative cell -> Example Leberflecken

• Predisposition for Cancer can be inherited e.g the recessive character of tumor suppressorgenes can be inherited E.g. BRCA1 Mutations

• The Cancer itself, the tumors can not be inherited ( you also need 2 mutated copies to sustain cancer for recessive genes)

• Retinoblastoma is a type of cancer that occurs in the retina, Mutations in the rb gene responsible for this type of cancer specifically affect retinal cells (only in the eye) leading to the development of tumors in the retina

• tumor cells are not lytic

• Acute transforming retroviruses –  Produce a dominant acting oncoprotein (v-ONC) that is derived from a cellular proto-oncogene (C-onc), more rapid mechanism

• Slowly transforming retroviruses – transformation by insertional mutagenesis in the promotor of proto-oncogenes, induce cell transformation gradually

• Bcr-Abl is a fusion oncogene that generates a constitutively active kinase with new properties

• ATP competitive Inhibitors like Imatinib block active centre of Bcr-Abl and inhibit its tyrosine activity therefore disrupts signaling pathways crucial for cancer cell survival

• Difference to other chemotherapies is the specificity of targeting cancer cell growth with reduced impact on normal cells

• targeted therapeutic approaches can be designed to specifically inhibit the aberrantly activated signaling pathway. RTK Inhibitors that bind the kinase region of the RTK thus inhibits oncogenic signalling pathway.

• Antibodies that specifically target the receptor

• Local invasion: Tumor cells break through basal lamina & enter stroma

• Intravasation: Cells invade the blood vessels

• Dissemination: Cancer cells travel through the blood stream

• Arrest in Microvessels: Cells adhere to blood vessel wall & arrest

• Extravasation: Cells exit the blood stream & enter the tissue

• Micrometastases: Cancer Cells form small clusters

• Colonization: Formation of Macro metastases by proliferation -> Tumor is formed

• Apical-basal polarity

• cell-cell-adhesion (tight junctions, adherens junctions, desmosome, gap junctions)

• cell-matrix-adhesion (anchorage to the basal-lamina)

• Basal lamina ( Laminin, Collagen IV, Integrin, Perlecan, Nidogen)

• E-cadherin is an adherens junction component in epithelial cell-cell adhesion. The transmembrane component forms homophilic interactions with each other and links to the actin cytoskeleton via catenins. Ca2+ dependent.

• Downregulation of E-Cadherin in Cancer leads to the disruption of Cell-cell adhesion contributing to the disintegration of adherens junctions and the breakdown of tissue structure. Downregulation associated with Epithelial- Mesenchymal Transition (EMT) -> Tumor cells can disseminate more easily and metastasize. (Or via Proteases MMPs)

transmembrane proteins that form intracellular connections to cytokeratins in hemidesmosomes. Form the extracellular link to basement membrane components (Intermediate filaments). Are Ca2+ dependent. Mediate cell-ECM interactions – aberrant integrin signaling contributes to tumor progression & metastasis

Specialized extracellular matrix that underlies epithelial tissue. Main components are Collagen Type IV, Laminins, Nidogens, Perlecans & Integrins

• Transcriptional downregulation by transcription factors like SNAIL

• Mutations

• Proteolysis (Degradation by metallo proteases )

• downregulation of gene activity (epithelial-mesenchymal transition [EMT])

Protease secretion like metalloproteases allow ECM removal & degradation therefore loss of adhesion -> Enable Tumor to break through the basal lamina, disconnect Cell-cell adhesion & create channel for further invasion (ECM degradation) -> Metastasis formation

Substrates for MMPs: Integrins, Collagen IV, Laminin, pro-MMP,

Change in gene Expression leads to:

• Loss of epithelial features -> weakend cell-cell junctions & disruption of epithelial tissue

• Gain of motility -> ability to move through ECM

• Gain of invasiveness -> Penetration through Basal lamina

• Protease secretion (MMP-2, MMP-9) -> allow ECM components (E-cadherin) removal therefore loss of adhesion

• Precision oncology: Inventory of mutations that are present -> Which potential targets are present and therefore tailor-made & personalized therapy

• Bipotential gonad with Müllerian & Wolffian duct

• Gonad primordium can form ovaries & Testes depending on chromosomes - Wolffian duct maintained in males, Müllerian duct in females. Develops adjacent to Mesonephros

• XY -> Sox9 induction in Gonad primordium -> Sertoli & Leydig cells -> Testosterone from Leydig cells acts on Wolffian duct, AMH leads to regression of Müllerian duct -> Male genitalia

• XX -> WNT signaling activity leads to Estrogen production-> Müllerian duct forms female genitalia

• Even though the Pseudokinase does not exert any kinase activities it could still retain its Protein-Protein Interactions and act as scaffolds, or adapters to form certain protein complexes needed for other signalling processes -> Therefore still plays a role in regulating signalling cascades and pathways.

• Could act as allosteric regulators like inhibitor and activator of other kinases

• targeting proteins to other compartments.

• act on gene expression as transcriptional regulators or influence the activity of other TFs.

• Atg8/LC3-I – cytosolic form -> Conversion to LC3-II to initiate formation of autophagosome

• Atg8/LC3-II – membrane bound form

• Interesting for Research because they play a key role in the regulation of autophagy – stress mechanism that allows cells to adapt to nutrient, glucose deprivation -> Since autophagy dysregulation is involved in the progression of various diseases they are potential drug targets

• Distinction: LC3-II has lipid extensions -> higher molecular weight  (Gel electrophorese) Ratio of LC3-II to LC3-I shows progression of autophagy

• Prokaryotes: Binding of Operator region on gene (Consensus sequence) -> prevents RNA Polymerase binding -> No Transcription initiation

• Eukaryotes: Binding of Promotor region of a gene in case of repressors -> binding of Silencer site

• Protein-Protein Interactions with TF that act as co-repressors

• Interaction with chromatin structure, Chromatin remodeling

  
  

• Tissue specific promotor for nervous system

• eukaryotic promotor which is neuron specific

• eukaryotic 3’ UTR for Stop codon

• docking site for TetR -> tetO

• Fusion of TetR with Activator domain

• GFP reporter gene

-> TetR binds as activator to docking sequence in promotor -> Transcription of GFP reporter gene in neurons

-> Mutation of TetR (not losing DNA binding ability after Tc binding) -> addition of Tc in cell -> DNA binding -> activating transcription

• Tissue specific promotor

1) Need C.elegans neuron specific promotor

either: mutation in APP encoding gene + ER secretion signal -> misfolded Protein -> induces UPRER

or: spliced xbp1 -> induces stress response directly (spliced because IRE1 not active, no stress signal)

2) GFP fusion to Promotor activated by hsp4 (hsp4 expression only induced by ER stress) -> Reporter gene expressed in all cells + only when ER stress (Neurons -> Intestine)

a)

• Main target of mTORC1 is ULK1. If there are plenty of nutrients mTORC1 is active and inactivates ULK1 by phosphorylation. Phosphorylated ULK1 cannot activate Autophagy Initiation.

• Low nutrient levels -> mTORC1 activity inhibited by AMPK – prevents ULK1 phosphorylation. Release of the Ulk1 complex -> Activation of ULK1 via AMPK phosphorylation-> activates multiple downstream proteins initiating Autophagy and formation of Autophagosome

b) Dephosphorylated - binds and inhibits Translation Initiation Factor eIF4E -> no binding of eIF4E to 5’ cap -> no translation

Phosphorlation of 4EBP by mTORC1 - cannot inhibit eIF4E -> Translation active

a) Multiple variations (alleles) within a population ( at least 1 %) at one specific locus. E.g SNPs (Single nucleotide Polymorphisms), Insertion/Deletion Polymorphism, Tandem Repeats (Microsattelites), Copy number variations (CNV)

doesn’t always affect function: - variations in non-coding regions, regions that do not effect the proteins function, Silent-mutations.

b) Gene Knockout - Advantage: distinct Phenotype, Disadvantage: Gene Knockout might be lethal

Gene Silencing- Advantage: Reversible, Disadvanatage: Challenging across tissues

a) Nucleus: Nuclear localization signal (NLS, typically positively charged Aa residues like Lysine & arginine) is recognized by nuclear import receptors (Importins) and binds the NLS containing cargo protein in the cytosol and facilitates translocation through nuclear pore complex

b) ER signal sequence (rich in hydrophobic AA, L,V,I,F…) is recognized by the signal recognition particle (SRP) and halts protein synthesis. SRP guides ribosome complex to ER where the complex binds SRP receptors. SRP receptor is released and complex changes onto a protein translocator. The synthesis resumes and peptide is transferred across membrane

c) Mitochondria: Targeting signal with mitochondria targeting sequence (MTS). Signal sequences are amphiphilic a-helices with positive charge on one side and nonpolar on the other. Signal sequence recognized by outer membrane receptor (TOM). Associated protein translocator transports signal sequence across outer Mito. Membrane to IM space- signal sequence recognized by  2nd Translocator in inner membrane (TIM) which transports protein across both membranes

d) Exocytose ??

a) Mitochondrial localization signal in ATFS-1 is probably located at the N-terminus -rich in positively charged amino acids, creating an amphipathic helix -> interaction with negatively charged mitochondrial outer membrane.

Nuclear Localization Signal (NLS): rich in basic AA (Lysine, Arginine)

b) predominant entering of either Mitochondria or Nucleus

when ATFS-1 is imported into Mito. -> it gets degraded and if it enters Nucleus the TF activates UPRmt by transcription activation

c) Post-Translational modifications or Protein-interactions that mask nuclear signal -> Mitochondria direction

d) Import machinery via Translocase on outer membrane (TOM) & inner membrane (TIM) transports ATFS-1 acrosss mitochondrial membranes -> allowing Mitochondrial matrix protease (LONP-1) to interact with ATFS-1 -> Degradation

e) Accumulation in the Cytoplasm leads to translocation in the nucleus & triggering of nuclear stress response genes

a) F1 generation is not homozygous mutated for daf-2 or daf-16 therefore you do not get one of the 2 phenotypes. Functional copy of either one of the alleles compensates loss of function in the other

b) The F1 generation will inherit one allele from each parent. Possible F1 Genotypes: Daf-2/daf-16, daf-2/+ (WT allele of daf-16), +/daf-16 (WT allele of daf-2), +/+ (WT)

Inheritance of daf-2 allele, daf-16 allele or respective WT alleles -> 4 possible genotypes

c) Possible genotypes:

• WT phenotype- 2/+, +/2, +/+, 16/+, +/16

• Daf-2( dauer constitutive) phenotype: 2/2

• Daf-16 (Dauer-defective) phenotype: 16/16

d) Compared to Daf-2 & daf-16 mutants that generate opposing phenotypes you get unrelated phenotypes for Unc-2 & daf-16 crossing and therefore get a combination off unc-2 & daf-16 phenotype. The underlying genetic mechanism for the daf-1, daf-16 cross is called Epistasis where mutations of 1 gene supresses the effect of mutations in the other gene therefore one phenotype prevails over the other.

a) Construct Cas9 expression plasmid with a tissue specific promotor that drives expression in C.elegans – Include marker (Fluorescent or drug) to identify & selecting transgenic worms expressing Cas9 – microinjection of plasmid