BIO 125 Development of Multicellular systems

What is developmental biology?

Tries to answer where babies come from.

Focus on mechanistic developmental biology

Covers from molecular to the organism scale and everything in-between

Advantages and Disadvantages of cell culture

• Large Numbers

• Homogeneous: Good for biochemical purification of factors

• Flattened cells: Stretched with large area, simplifies imaging

• Open systems: Easy to treat with drugs

Models used in biology

The fly, chick, mouse, zebrafish

Maternal contribution

Proteins and mRNA deposited in the egg by the mother during oogenesis are produced from maternal genes. Maternal genes control the embryonic development until the activation of the embryo’s genome.

Ovipary

• Development of an embryo within an egg outside the mother’s body.

• More yolk

  → fast development due to predators

Vivipary

• Embryos develop inside the mother and obtain all nutrients from mother

• Less/no yolk

What is contained in an fertilized egg?

1. DNA: haploid (n) → Fertilization → diploid (2n)

2. Yolk: to nourish the developing embryo (proteins, lipids, sugars → Energy)

3. Cytoplasm/Ooplasm: mRNAs and proteins → Instructions

What did T. Boveri discover?

He remove the nucleus from early sea urchin (Seeigel) embryos.

—> He realized that early steps of embryonic development are Chromosome indepent.

Key events depend on the cytoplasm alone.

Facts about the maternal phase? 3

1. Embryo is transcriprionally silent

2. Duration varies between species

3. This phase controls early steps of embryonic development like:

- cell division,

- cell patterning

- cellular differentiation.

Maternal Contribution

Stored mRNAs + proteins in cytoplasm of egg.

Produced from maternal genome during primary oocyte stage.

Maternal genes control embryo development until the activation of the embryos genome.

Maternal Effect Genes (Def.)

Maternal effects occur when an organism shows the phenotype expected from the genotype of the mother, irrespective of its own genotype, often due to the mother supplying messenger RNA or proteins to the egg.

Cleavage (Def.)

- Follows fertilization.

- Production of more cells.

- Embryo Volume does not increase.

- G1 and G2 (Growth phases) skipped.

WWhat are cycline-dependent kinases (Cdks) and specific cyclin required for?

For the different stages of cell division.

Once the cycline is bound to the Kinase, the Kinase-cyclin-complex is able to produce its own inhibitor. As its activity gets higher, it produces the anaphase-promoting complex which then destroys the cyclin.

Titration of cyclins and its effect on the embryo

During cleavage, the embryo switches between M and S, so only 2 cyclins are needed. -> produced from pool of maternal mRNA.
But mRNA gets degraded + cells start to become more and more —> pool is reduced.
With time, these phases slow down—> leaves more time in between for the zygotic transcription to start + transcription-silencer-factors provided by the mother become less.
The embryo not silenced anymore => change in division => normal cell division including G1 & G2.

Holoblastic Cleavage (radial) (Def.)

Entire egg undergoes cellularization.

Yolk platelets are either absent (mammals) or present as cytoplasmic inclusions that partition among cells (amphibians).

Soft yolk = astral MTs go into the yolk.

(MT=Microtubules)

(EB: Holo=Homo='Gleich')

Meroblastic Cleavage (discordial (def))

Occurs in fertilized eggs that contain a lot of yolk.

In meroblastic cleavage, the cleavage furrows form, but do not progress into the yolk.

(Partial division, egg is not divided into complete membrane-enclosed cells.

=> thick yolk -> astral MTs can’t easily penetrate yolk, no divisions 'in' the yolk.)

1. What Impact does cleavage have on the embryo?

2. Why is it important to study cleavage?

1. In most species, early cleavage patterns have an impact on subsequent developmental processes (AP and DV axes determination)

2. - Investigating embryonic cleavage/how cells divide is relevant for understanding stem cell biology, cell fate specification and cancer,…

   -Comparison between different cleavage patterns allows to understand how cell geometry and cellular organisation impact on cell division.

Blatomeres (Def.")

Smaller cells produced by cleavage during mitotic cell division.

What happens during the Midblastula Transition (MTB)?

Phase in embryonic development (of non-mammals) when embryonic genes are Transcribed/activated for the first time.

-Activation of zygotic gene Transcription.

- Slowing down of cell cycle.

- Asynchrony in cell division.

- Increase cell motility (Deutsch: Bewegungsvermögen)

3 Characterisitcs of pre-MTB Embryonic cells:

There are three major characteristics of pre-MBT embryos.

1. All embryonic cells undergo cell division at the same time.

2. Zygotic chromatin is condensed, hypo-acetylated and H3 methylated, indicating that most of the genes are in a repressed heterochromatic state.

3. Embryos are observed to translate only maternally inherited mRNA, (i.e. that mRNA which is present in the oocyte when it is fertilised).

The mRNA is localised in different parts of the oocyte, so that as the embryo divides, it is segregated into different cells. This segregation is thought to underlie much of the differentiation of cells that occurs after MBT.

Control of MTG onset

By the titration of maternally loaded inhibitors against exponentially increasing amount of DNA.

Mammalina embryos (Blastomere orientation + further division)

The orientation of mammalian blastomeres with relation to one another is quite unique.

-After the first division, the two blastomeres divide perpendicular to each other (rotational cleavage).

- The formed blastomeres do not divide all at the same time (asynchronous) which can lead to odd numbers of cells.

- The zygotic genome is activated early during cleavage. (In the mice at the 2-cell stage, in humans at the 4-cell stage.)

Asymmetic Cell Division (Def)

Cell division in which one daughter cell receives more cytoplasm than the other during mitosis.

Asymmetric segregation of cell fate determinants that are present in the egg —> 2 daughter cells that will follow different developmental paths.

(Important not only for embryonic development but also later in life, for example, to balance stem cell self- renewal and differentiation)

Asymmetric Division in C. Elegans

After the first cell division of the egg, there's a clear anterior (AB) and posterior (P) polarity. Something happens early, that makes AB and P cells different.

—> The sperm entry point constitutes a symmetry breaking event which leads to the clear cell polarity.

PAR Proteins (Def)

PAR proteins regulate cell polarization in many different contexts in diverse animals.

—> part of an ancient + fundamental mechanism for cell polarization.

(EB: PAR=Polar)

Par Proteins: Before and During fertilization

Before fertilization:

Anterior PAR-Proteins are at membrane —> preventing posterior PARs from accessing.

Sperm entry (future posterior pole):

1. Formation of the microtubule aster happens. (Sperm brings centrosome, from which Microtubules can nucleate)

2. Microtubule inhibits PKC (anterior) from phosphorylating PAR-2.

3. Unphosphorylated PAR-2 recruits PAR-1 (posterior) to plasma membrane.

4. PAR-1 —> phosphorylates PAR-3 (anterior).

5. phosphorylated PAR-3 —> prevents anterior complex from binding to the plasma membrane at the posterior.

6. —> Anterior PARs no longer present at posterior pole. (PKC can prevent localisation of the posterior PAR at the plasma membrane).

7. Actomyosin contraction —> help movement of anterior PAR complex from posterio to anterior cortex.

(Microtubule asters Def:

Radial arrays of microtubules organized by centrosomes.

Play a fundamental role in the spatial coordination of animal cells.)

PAR proteins Function/Mechanism

Function:

PAR-Proteins are asymmetrically localized in the egg. They are conserved from worms to humans and control polarity of many cells.

Mechanism:

If one of the two groups (A/P-Pars) is mutated, the other will take over not only in the cytoplasm, but also at the membrane anterior and posterior, which suggests a competition between AB and P- PARs. They compete for access to the membrane.

--> They are each others targets: kinase of one phosphorylates the PAR of the other one + vice versa.

Par proteins function in an embryo

Par proteins have specific functions at each pole of the embryo to maintain differences and distinct anterior and posterior domains.

—>creates different cellular identities between AB and P derived cells.

Establishment of cell polarity and asymmetry crucial in embryogenesis, morphogenesis, neuronal differentiation and it's lost in cancer.

Example: PAR-3, PAR-6 and PKCs are essential to define the apical domain of an epithelial cells. Required for the formation of tight junctions —> prevent mixing of membrane proteins and leakage of fluids.

Loss of function mutations (f.e. in changes of "disheveled" regulation) in polarity regulators promote tumour growth.

How do cells acquire different functions?

- Differential Gene Expression.

Cells in our body share the same 3-Billion-BP long DNA subdivided into 30'000 protein

There are "housekeeping" genes expressed in every cell.
Cell-type specific genes expression is restricted to certain cell types.

What Evidence for genomic equivalence is there?

If you take a cell from a blastula and put its nucleus in an en-nucleated egg, every cell has the ability to develop completely. (Dolly)

What are Houskeeping Genes?

Typically constitutive genes that are required for the maintenance of basic cellular function are expressed in all cells of an organism under normal+patho-physiological conditions.

What are Gene Regulatory Elements?

Information to turn genes on/off is encoded in genome in the form of sequences called GREs. GREs control gene transcription by interacting with Transcription Factors. TFs bind to GREs and help the RNA pol. to engage with the promoter + start transcription.

How do TF "find" the right GREs and bind only to those?

DNA accessibility must be regulated.
Packing and unpacking of chromatin controls gene expression.

Heterochromatin vs. Euchromatin (Def.)

Heterochromatin:

Highly Condensed —> transcriptionally inactive

Euchromatin:

Less condensed —> transcriptionally active ("truly transcribed")

How can chromatin be modified? (3P)

- Histone acetylation

- Histone Methylation

- Methylation of DNA

What are histones?

Protein that provides structural support for a chromosome.

DNA wraps around complexes of histone proteins, giving the chromosome a more compact shape.

Play a role in the regulation of gene expression.

Histone acetylation and deacetylation

Acetyl transferase (HAT):

Acetylation of lysine in histone tails creates negative charge

→ repulsion of the negative DNA = opening up to Euchromatin.

(HATs utilise acetyl CoA as cofactor + catalyse transfer of acetyl group (-COCH3) to lysine side chains.)

Histone Deacetylase (HDAC):

Remove acetylgroups from histone Tails. Histones become less negatively charged.

→ DNA tight around histones = closed Heterochromatin.

(HDACs predominantly transcriptional repressors.)

What does the methylation of GpC islands result in?

Silencing of gene expression.

DNA methylation/demethylation of CpG islands regulates gene expression during development and differentiation.

What ultimately controls gene transcription?

Transcription factors ultimately control gene transcription.

Epigenetic modifications control gene accessibility and its modulation.

What do Polycomb group (PcG) proteins (Def.)

PcG proteins organised in multiprotein complexes that have the ability to modify histones (and other proteins f.e. stem cells) in different ways.

Often these modifications lead to chromatin compaction and gene silencing.

For maintaining cell identity.

Mutation, inactivation or increased expression of the PcG proteins leads to the misexpression of target genes and thereby to severe developmental defects (f.e. cancer).

Epigenetic regulators with key roles in development, cell differentiation and disease.

What are Epigenetic Modifications (def.)?

Modifications that modulate DNA without modifying its sequence (can occur at level of histone tails or at DNA itself)

Functions of epigenetic modifications (4P)

- Epigenetic information persists after original inductive stimulus,

providing a cellular memory.

- Epigenetic marks allow cells to remember their fate irrespective of external

stimuli and information => cell identity

- Epigenetic tags on histones and along DNA are faithfully copied during cell division.

- Acquisition of epigenetic marks transmitted to descendants “locks”

these choices progressively (past experiences inform future choices)

If epigenetic modifications lock cells in their state, how can the transplantation of nuclei from skin or mammary cells generate a complete animal?

As soon as fertilisation occurs, extensive epigenetic reprogramming occurs to remove epigenetic marks on maternal and paternal DNA (except imprinted loci, which are protected).

Do all the cells have the same epigenetic marks?

The genome is the same in every cell, but epigenetic markers are different + linked to cell identity. Thus, they provide unique cellular signatures, a sort of cellular passport.

What is ATAC sequencing?

Is a method to identify epigenetic modifications. Assay for Transposase-Accessible Chromatin unsing sequencing. Can tell the difference between open and closed chromatin.

What’s the mechanism of ATAC sequencing?

Transposase is used that is able to insert into the genome.

The frequency of insertion is much higher in euchromatin (open) than in heterochromatin (coosed).

After insertion you amplify + sequence the DNA. Bc. inserted sequence is known, one can map the genes.

High abundance means easy accessible DNA. This doesn’t tell anything about which modification there are.

What’s the definition of ChIP sequencing?

Method used to analyze protein interaction with DNA.

ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.

What’s the mechanism of ChIP sequencing?

We have antibodies that can recognize and bind to specific epigenetic tags.

1. DNA fragments are run through a column where antibodies are attached.

2. antibodies can recognize + bind specific epigenetic tags (modifications).

3. Every fragment that has the antigen is recognized by antibody + will attach to the column.

4. will then be „fished" out.

5. Amplify + sequence the DNA and see which DNA is linked to which modification.

What’s a Mediator complex?

It’s a complex that promotes changes in the RNA Polymerase activity and activation of transcription.

What do Pioneer TFs do?

They can find motifs that are exposed on the surface of nucleosomes and access closed chromatin that is inaccessible to other TFs. They basically open up closed sections for other TFs to follow.

What are TADs (Topologically associated domains)

They are self-interacting genomic regions. DNA sequences physically interact with each other more frequently within a TAD than they do with sequences that are outside of the TAD.

What’s Feed-forward?

Feed-forward is a mechanism introducing delays, which means that in acts over long time intervals, and they are designed for reducing fluctuations. In addition, some of the targets will have positive feedback loops on MyoD expression to maintain it.

What are Insulators?

Insulators prevent inappropriate enhancer-promoter interactions.

Mutations with in insulators can cause developmental phenotype as a result of inappropriate enhancer-promoter interactions.

What’s the definition of Lineage Tracing?

Technology to understand what fate embryonic cells will become.

Connect cell in early embryos to later stages.

Cells in defined positions show tendency to develop into certain fates but mapping data show correlation not causation!

What is Fate Mapping?

For organisms that are too complex/ unable to allow complete lineage tracing.

Performed by labelling cells (groups), then asking what cell type they have become.

Mapping data show correlation not causation!

What is Gastrulation?

Organisation of cells into 3 germ layers. Extremely importnat step towards differentiation.

What are the 3 germ layers?

Ectoderm:

Cells that remain at embryo exterior after gastrulation (skin and nervous system), cells tend to have an epithelial organisation.

Mesoderm:

In the middle.

Muscles, bones, blood,cells tend to mesenchymal organisation

Endoderm:

Cells interior of embryo, gives rise to the internal organs

—> Most complex organs derived from one germ layer but have major contribution from other germ layers

What is Embryonic induction?

One cell type tells the other cell type what to do.

One group of cells (inducing tissue) directs development of another group of cells (responding tissue).

Repositioned cells can change the development of new neighbours. The response to the induction is „switch like“.

response to induction:

Tissue A converts tissue B into tissue C.

Why is precise dorsoventral patterning needed?

To determine position of different neuronal types within spinal chord.
Transplantation experiments have revealed that notochord is necessary + sufficient to induce floor plate.

What is ZPA?

zone of polarizing activity

Area that contains signals which instruct the developing limb bud to form along anterior/posterior axis.

ZPA induces mirror-image duplications when it‘s transplanted to anterior of a second limb. (123-> 321123)

(Limb bud: structure formed early in vertebral limb development.)

What is an organiser?

Group of cells with ability to instruct fates + morphogenesis in surrounding cells, steering their development into specific organs + tissues.

What are 3 important facts about Organisers?

- Organisers don't control differentiation/tell cells what to become.

- Organisers tell cells where they are, but don't determine cell types.

- What organisers pattern is determined by the responding tissue and not by organiser.

What is Morphogenesis?

Morphogen: Diffusible sustance that drives morphogenesis.

Morphogensis: Biological process that causes tissue/ organ to develop its shape by controlling the spatial distribution of cells during embryonic development.

How do organisers confer spatial information on surrounding tissues?

Diffusion of morphogens —>
conc. Gradients—>
graded positional info.

• Cells closer to stable gradient source receive more morphogen than cells further away.

• Gradients provide mechanism to allow positioning to influence differentiation.

‚Simple Induction' vs. ‚Morphogen Patterning' (Summary)

Simple induction:

Inducer: Secreted cues that switch cell fates.

1. Source cells secrete chemical inducer—>

trigger fate decision in responding cells.

2. Responding cells are determined as diffuser concentrations exceeds threshold.

—>Generates two cell fates maximum

Morphogen Patterning:

Organisers ‚organise‘ by being source of morphogen grad.

1. Polarised ‘organising’ cells (eg ZPA, Floor

Plate) secrete morphogens.

2. ‘Sinks‘ maintain morphogen conc. Grad.

3. Activation of different target genes with different morphogen conc:

-Gives multiple cell types

-Gradient-dependent manner.

-->Morphogens generate multiple cell fates bc. Grad differs.

What is the Heidelberg screen?

In large-scale mutagenesis screens performed in 1979-1980 they isolated mutations affecting the pattern or structure of the larval cuticle in Drosophila. The 600 mutants they characterized could be assigned to 120 genes.

They identified:

... the majority of developmental genes

...genes that caused deletion

—> with the screen they had everything they needed to understand how morphogens work.

bicoid gene and bcd gradient

Encodes homeodomain containing TF. (protein that binds DNA to regulate transcription)

Maternal bcd RNA localised at anterior pole of oocyte. Gradient of bcd protein to posterior side.

Bc. During early steps of development a single cell contains many nuclei in one cytoplsm, bcd can duiffuse without membrane barriers.
(Bcd=TF)

What are Gap gene?

Gap genes defined by effect of mutation in that gene, which causes loss of contiguous body segments -> resembling a gap in the normal body plan.

Each gap gene, therefore, is necessary for the development of a section of the organism.

How do long distance organisers work?

Bcd-type transcription factor morphogen is unlikely to pattern multicellular tissues.

Tissue patterning requires cell-cell communication mechanisms.

Multicellular tissues require another type of morphogen.

What’s Hedgehog?

Proteins of the Hedgehog (Hh) family are powerful signaling molecules that act as morphogens during development in both vertebrates and invertebrates.

What’s the Shh?

Shh expressed in regions+ at time where ZPA is active (determining AP axis)

→ Shh: sufficient to mimic ZPA ( in a concentration dependant manner)

more shh: more posterior

less shh: more anterior

Shh is also expressed in notochord + floorplate during dorsal-ventral patterning.

Shh is sufficient to mimic floor plate.

Why does transplating a floor plate to a limb results in limb duplication?

Shh expressed in notochord + floorplate during dorsal-ventral patterning.

shh mimics floor plate. Transplanting a floor plate to limb→ acting as a ZPA → duplication of a limb

What do Wnt, HH and Dpp increase?

Wnt: nuclear beta-catenin

HH: GLI

Dpp: MAD

How to activate gradient of Shh

Shh ligand is present => target gene expressed

1. Shh blocks receptor ptch —> receptor smoothend is released —> no longer inhibited.

2. Smoothend —> enter into cilia, signal. —> prevent TF “Gli”from being cleaved.

(GliA) = genes expressed (note: smoothened needs cholesterol to signal)

- 1 shh gradient converted into 2 gradients that oppose each other = gives a lot of control

Cells near source:

high level of activator.

Cells far from source:

high levels of repressor

How is the Dorsoventral CNS Pattern achieved?

CNS Dorsoventral Patterned Achieved By Morphogen Gradients (Shh and BMP/Wnt)

—> Ventral cells with higher Shh signalling have increased active Gli transcriptional regulator, allows activation of lower affinity targets not active more dorsally.

What’s the definition of Cilia?

Non-motile, microtubule-based protrusions.

Play important roles in signalling.

Shh signalling takes place in primary cilia.

(Shh receptor smoothened must enter cilium to signal)

What’s the definition of Mangold-Spemann-Organizer?

Group of cells that are responsible for the induction of neural tissues during development in amphibian embryos.

MS-organizer provides evidence that fate of cells can be influenced by factors from other cell populations.

(—> This discovery significantly impacted the world of developmental biology + fundamentally changed the understanding of early development.)

How to identify the Mangold-Spemann organizer?

- Inject pools of mRNA of genes expressed in embryos with unknown function.

- Screen form RNAs that show organizer activity (axis duplication).

How is the Dorsovental Axis established by the MS organizer?

MS-Organiser is NOT a morphogen.

The morphogen is BMP-4 (dpp homolog) which can induce ventral fate everywhere + important for the dv-axis establishment.

BMP = diffusible but when left alone, it would simply fill up the whole embryo and there would not be a gradient.

Organiser = source of secreted morphogen inhibitor (chordin, noggin, follistatin)

Chordin suppresses BMP’s ventralizing signal at dorsal side.

—> allowing dorsal cell fates.

How is Organizer Gene Expression induced?
How do organizers work?

1. Organiser gene expression induced in floor plate by neighbouring notochord.

2. Organiser gene expression activates production/secretion of morphogen (here Shh).

3. Neighbour cells read and interpret morphogen-conc. of Shh and activate genes appropriate for 'position' in gradient (genes with either high or low affinity)

Is the French Flag Model really the Reality?

- In most contexts there are multiple morphogens acting simultaneously.

—> generate more complex patterns of differentiation.

- Morphogens can act in direct opposition

—> fine-tuned fates.

- Morphogen patterns in a cartesian manner but do NOT interact with each other.

- Patterns also do not show “regeneration” when morphogens are removed.

What are 2 ways to investigate Morphogen Activity?

Majority of evidence for morphogens acting in concentration-dependent manner comes from measuring output of their activity.

We can...

1. measure their impact on the final embryonic structures like digits (provide evidence for long-range effect)

2. measure their impact on the expression of target genes (provide evidence for conc. thresholds.)

Reaction-Diffusion-Model (Def.)

States that initial symmetry in embryos can be broken by the interplay between two diffusible molecules, whose interactions lead to the formation of patterns.

—> Provides valuable framework for self-organized pattern formation.

(Complex dynamic patterns can be formed when morphogen activities are interdependent. Here morphogen signalling acts as activators and inhibitors which can react while diffusing.)

FRAP (Def.)

‚Fluorescence Recovery After Photobleaching‘ allows diffusion measurement.

Bleaching leads to a black spot —> now one can measure the rate at which the molecules move as the black spot disappears again when

molecules move around.

Can be used to compare diffusion of different proteins /same protein in different genetic contexts (with or without receptor).

FLIP (Def.)

(oposite to FRAP) ‚Fluorescence Loss In Photobleaching'

A defined spot is bleached —> all molecules that pass through will bleach as well. The higher the mobility, the more rapidly the molecules will be bleached.

=> Experiments prove, that morphogens do move!

What is global patterning? (Definition)

Systems operating at larger scale to organize cells into patterns+ confer positional values.

Short-range cell-to-cell communication allows:

Local sell fate differentiation:

• context-dependent differentiation

• generation of cellular diversity.

• → Allows cells to form right structures at right time+ place.

What’s Autocrine signaling?

Secreted molecules act on cell itself. Important for cell migration.

What’s Paracrine signaling?

Cell secrets diffusible molecule that can move to another cell and the receptor will receive the sign. The Gradient is not always necessary.

What’s endocrine signaling?

secreted molecules move with bloodstream.

What’s juxtacrine signaling?

Cells are next to each other+ secreted molecules not diffusible. Signal + receptor near. Homophilic/Heterophilic binding possible.

Induction, Inducing cells and responders (def).

Induction: Ability of one group of cells to affect the fate of another.

Inducing cells: Cells that produce the signals that induce other cells.

Responders: The cells that receive the signal.

Competence def.

ability of cells to respond to inducers. Depends on having right receptor.

→ Receptor sits at top of transduction pathways that “translates” a signal into a change in gene transcription.

How many signalling pathways are there?

11

Signalling pathways adopt different strategies to transduce a signal.

For Example:

- Transduction by preventing destruction (Wnt).

- Trensduction via phosphorylation (TGFβ/BMP + Growth factors signaling).

- Receptors cleavage + translocation into nucleus (Notch)

Signalling by preventing destruction (Wnt)

Wnt = secreted ligands that bind to receptors of the Frizzled family.

Wnt present:

WNT binds to receptor —> dishevelled (protein) able to prevent destruction of β -catenine.

β -catenin travel into nucleus —> interacts with TF (LEF/TCF) + drives gene transcription.

No Wnt:

β - catenin is destroyed by destruction complex (kinase and TSG “APC”).

Important for cell differentiation, polarization + migration.

The Wnt/β-catenin signalling is activated in many types of cancer + can be caused by mutations in β-catenin, the β-catenin destruction complex or over-expression of Wnt ligands (and many more).

Transduction via Phorphorylation: TGF-beta/BMP

1. Lignad binds → formation of heterotetramers (Type 1/2)

2. Type 2 Receptors phsophorylate serine-rich region of Type 1 receptor.

3. Phosphorylated Type 1 → provides docking site for Smad-receptor.

   • R-Smad2/3 for TGF

   • R-Smad 1/5/8 for BMP

4. Type 1 Receptor → phosphorylates R-smads.

5. Phosphorylation induces their dissociation from receptor → binding to Smad4 → Smad complex can enter nucleus.

6. Smad-complex binds to DNA with coactivator/corepressors → gene Expression

What do Smad 6 and 7 do?

Inhibit signalling by interacting with receptors (recruiting ubiquitin ligases) to induce receptor degradation.

Smad7 and Smad6 are targets of pathway. Their expression depends on pathway activation.

—> Negative feedback loop!

To reduce signaling

FGF/MAP-Kinase Signalling (mitogen-activated protein kinase cascade)

1. Binding of GF to receptor —> formation of receptor dimers —> receptor Tyrosine cross phosphorylation.

2. Effector Proteins (GRB2) recruited to active receptors.

3. Grb2 —> activates Sos (GEF)—> Activates Ras by GDP to GTP.

4. Active (GTP-Bound) Ras binds Raf—> phosphorylates MEK.

5. MEK phosphorylates ERK—> active ERK goes into nucleus—> modulates gene expression.
=> cellular changes

Mutations in Ras

RAS + RAF = oncogenes.

Promote cell division/ proliferation —> need to be tightly regulated.
If they gain activity it can lead to cancer.

Similarities in all pathways (3P)

Although pathways use distinct signalling components + strategies, they also have similarities.

1.Positive and negative feedback loops

2.Redundancy

3.Discriminating ligand identity