Signalling pathways

Question 1

What are the different ways cells communicate?

Answer 1

1. Direct contact
   - between neighbouring cells
   - might have gap junctions in the membrane
   - cells might produce molecules that enter the other cells which can activate or inhibit responses

-cells might might complementary proteins that can bind with each other to activate response/pathway

2. Contact over larger distances
   - paracrine signalling = releasing signals in the extracellular space
   - endocrine signalling = releasing signals into blood
   - autocrine signalling

Question 2

What is the general mechanism in cell signalling (prototypic pathway)?

Answer 2

• Molecule binds to receptor (reception)
• Receptor can change conformation, become phosphorylated or dimerise (transduction)
• Response caused by transduction
• Feedback (negative - shut off pathway, positive - reinforce effects of pathway)

Question 3

How can we classify pathways?

Answer 3

-Where signal is coming from

1. Paracrine (morphogens)
   - Hedgehog, Wnt, TGFB, receptor tyrosine kinase
2. Cell-cell contact
   - Eph/ephrins, semaphorins
3. Extracellular
   - Wnt pathway
4. Mechanical cues and cell polarity pathways
   - Hippo pathway

Question 4

How is the hippo pathway involved in first cell fate?

Answer 4

• 1st cell fate = whether the cells in the embryo differentiate into trophoectoderm or embryoblasts
• This is controlled by position of cell in the embryo and its polarity - these signal via the Hippo pathway
• As the cells become specified as either inner cell mass or trophoectoderm, they start expressing different transcription factors
• ICM expresses Oct3, Oct4, Sox2, Sall2, Nanog
• TE expresses GATA4, Cdx2 and TEAD4
• TEAD4 is the key regulator of TE formation. Trophoblasts can’t express Cdx2 and GATA4 without TEAD4 expression
• TEAD4 is only activated in TE via the hippo pathway

Question 5

How was the hippo pathway discovered?

Answer 5

• First identified in Drosophila as a tumour suppressor gene
• Mutation causes overgrowth, leading to huge tumours throughout body - hence the name hippo
• Mammalian homologues were identified - these are Mst1 and Mst2

-Loss of function of Mst1/Mst2 can cause overgrowth

Question 6

How does the hippo signalling work?

Answer 6

• Main different of trophoectoderm and ICM at the 16 cell stage is their relative position in the embryo (TE is on the outside, ICM is in the middle)
• We think hippo pathway is inactivated by trophoectoderm cells having a polar apical surface. These polarised cells inactivate the kinase pathway.
• This also determines whether the cells are polar or not
• The future trophoectoderm have a free surface so become polar
• The future ICM are completely surrounded so bind to other cells so remain non-polar
• The difference in polarity is what determines Hippo pathway

In the ICM:

• Mst (encoded by Hippo gene) phosphorylates Lats (hippo pathway activated)
• Lats phosphorylate Yap
• Yap is a co-transcriptional regulator that binds TEAD4
• Phosphorylated Yap can’t trans locate into nucleus
• Hence, Yap can’t bind TEAD4 so it can’t activate transcription of trophoectoderm factors (Cdx2, GATA4)

Troophoectoderm cells:

• Mst not active (hippo pathway not active)
• Mst doesn’t phosphorylate Lats
• Lats can’t phosphorylate Yap
• Yap can translocate to nucleus and bind TEAD4
• Yap-TEAD4 complex allows transcription of Cdx2 and GATA4

Question 7

What is the Eph/Ephrin signalling pathway?

Answer 7

• This signalling pathway is important for segregation of group of cells with different identities during embryogenesis
• Plays role in axon guidance, forming tissue boundaries, cell migration and body segmentation
• Eph receptors (called Ephs) are transmembrane proteins from the receptor tyrosine kinase family
• Can only bind to either Ephrin-A or Ephrin-B, except EphA4 (receptor) which can bind to both types of ligands

-Ephrin ligands = are type A or B depending on the receptor they bind to
-Ephrin type A ligand = attached to membrane by lipid modification (GPI)
and lack cytosolic domain
-Ephrin type B ligand= transmembrane protein attached to membrane by a single transmembrane domain that contains a short cytosolic PDZ binding motif

• There are 5 ephrin-A ligands (A1-A5) that interact with 9 EphAs (EphA1-8, EphA10)
• Bind with high affinity (lock and key mechanism)
• There are 3 ephrin-B ligands (A1-A3) and 5 EphBs (EphB1-4, EphB6)
• Bind with weaker affinity (induced fit mechanism)
• The interaction between ephrin ligands EphRs leads to bidirectional response - in both the receiving and signalling cell
• Binding of ephrin ligand to EphR can cause a forward signal (a response in the EphR cell), a reverse signal (response in the ephrin-ligand cell) or bidirectional signal

Forward signalling

• Phosphotyrosine mediated recruitment and tyrosine phosphorylation
• Phosphorylation of intracellular effector proteins
• These then regulate activity of Rho GTPases, hence regulate the cytoskeletal dynamics

Reverse signalling

• Ephrin-A reverse signalling involves interaction with co-rectories that can mediate attraction of repulsion of cells
• Ephrin-B reverse signalling occurs via phosphotyrosine and PDZ domain dependent pathway

Termination of pathway:
-Cleavage of ephrin ligand using proteases or endocytosis of the ligand into cell

Key point: Because both the receptor and ligand are membrane bound, the intracellular pathways can only be activated by direct cell-cell interacting

Question 8

How is the Eph/Ephrin signalling involved in axon guidance?

Answer 8

• Important for migration neuronal axons to their destination
• Controls guidance of axons through their ability to inhibit the survival of axonal growth cones
• This repels the migrating axon away from the site of Eph-ephrin activation
• The growth of cones migrating axons don’t respond to absolute levels of Eph-ephrin, but the relative levels of EphR and ephrin expression - this allows migrating axons that express EphRs or ephrins to be directed along gradients of EphR or Ephrin expressing cells towards a direction where axonal growth is no longer completely inhibited
• Although EphR-ephrin activation is associated with decreased come survival and repulsion of migrating axons, it has recently been demonstrated that growth cone survival doesn’t depend on just EphR-ephrin activation but on the differential effects of forward or reverse signalling.

Question 9

What is the notch-delta pathway (lateral inhibition)?

Answer 9

• Very important in neurogenesis
• It is used to make 2 types of cells from a homogeneous field of cells
• Directs specification of complementary fates in tissues where it is expressed
• The process is called lateral inhibition (allows cells to adopt different fates)

-Only certain cells within the field are selected and fated to become neurones

• All cells express Notch (the receptor) and Delta (the ligand)
• This means 2 adjacent cells are able to signal to each other
• Sending cell has more delta than notch
• Receiving cell has more notch than delta
• Notch is able to repress the activity of delta, therefore when notch is activated in a cell, delta is repressed
• If the levels of delta were the same in all cells, everything would be switched off and no signalling would occur
• The levels of delta vary between cells - they’re similar but not identical (this is completely random)
• The result of this is that the small difference can be amplified very quickly
• Cells with slightly higher levels of delta will activate more Notch on other cells, leading to more repression of Delta in adjacent cells
• This is lateral inhibition
• The cells that have lots of delta are the ones within the field that sill become neurones as they are able to activate more notch in adjacent cells, which will in turn repress delta in these cells
• This also happens in myogenesis, not just neurogenesis
• If there is no notch activity, there is no lateral inhibition. This is because notch represses delta, hence, with no notch, cells will produce a lot of delta so all cells become neurones.
• It’s the lack of delta expression in laterally inhibited cells that means some cells in the field do not become neurones

Question 10

What is the molecular mechanism of notch-delta pathway?

Answer 10

• Notch receptors are single pass transmembrane receptor proteins with a large extracellular domain and smaller intracellular domain
• When no ligand is bound to notch, CSL (a transcription factor in the nucleus) is complexed with repressor proteins, preventing expression of the target genes
• When a ligand (delta) binds to notch on the extracellular domain, proteolytic cleavage occurs, separating the extracellular domain from the rest of the protein - this is first cleavage
• This leads to the second cleavage, where the intracellular domain is cleaved by enzymes of the Presenilin complex
• The free intracellular domain is translocated into the nucleus and binds to the CSL complex
• This induces a conformational change that activates the CSL complex as repressor proteins can no longer bind
• The co-activator protein Mastermind is then recruited (with other co-activator factors) and binds to the complex
• This CSL complex can now initiate transcription of the target genes

Question 11

What are morphogens?

Answer 11

• Diverse signalling molecules
• They are secreted by signalling centres of embryo
• Activates all pathways
• In the embryo, signalling molecules are often produced in a specific region and disperse thoroughout the tissue creating concentration gradients
• Cells will respond differently depending on the level of signal they detect (acquiring different fates at different concentrations)
• They provide positional information within embryo

Question 12

How are morphogens important in the neural tube?

Answer 12

• Image shoes vertebrate neural tube in chick embryo
• There are different signalling centres
• Anterior neural boundary synthesises FGF8
• Zona limit and intrathalamica and floor plate produce Shh
• Mid-hindbrain boundary and roof plate produce Wnt1 and FGF8
• Ectoderm overlying neural tube produces BMPs

-The different morphogen gradient produced by these signalling centres define dorsoventral and anteroposterior axes of developing embryo

Question 13

What morphogens are involved in the RTK, TGFB superfamily and direct pathways?

Answer 13

RTKs

• PDGF
• EGF
• FGF
• VEGF

TGFB superfamily (serine/threonine)

• TGF
• BMP
• Activin

Direct pathways

• Shh
• Wnt

Question 14

What is RTK signalling?

Answer 14

• FGF, PDGF, EGF, VEGF are associated with tyrosine kinase receptors
• They all work in the way
• Receptors exist as monomers with an extracellular binding domain, a single transmembrane domain, and a intracellular domain that is coupled to a tyrosine kinase
• They dimerise upon ligand binding leading to autophosphorylation and activation of tyrosine kinase domain
• The phosphorylated tyrosines can then act as docking sites for whole range of proteins
• In the FGF pathway, Grb and Sos bind to the phosphorylated tyrosines
• Sos is a guanine exchange factor (GEF) that activates GTPases
• Sos and Grb recruit Ras (a GTPase) in the cell membrane
• Activated Ras will activate the first serine/threonine kinase (Raf) in the transduction pathway
• Raf then phosphorylates the next kinase MAPKK which goes on to phosphorylate MAPK
• MAPK can then phosphorylate and activate other kinases, or enter the nucleus and phosphorylate transcription factors, activating gene expression

Dimerisation upon ligand binding —> Autophosphorylation of tyrosine domain—> Recruitment of Grb and Sos —> Activate Ras —> Activate serine/threonine kinase (Raf)—> Activate MAPKK —> Activate MAPK

Question 15

What are tyrosine kinase associated receptors?

Answer 15

• Similar to RTKs
• Produce a similar response
• Main difference is that the receptor doesn’t have any tyrosine kinase activity itself, instead, the intracellular domain is bound to other kinases such as JAK
• When the receptor interacts with the ligand, it dimerses, cross phosphorylation occurs and then activation occurs
• Induces signal transduction, leading to a response

Question 16

What is the JAK/STAT pathway?

Answer 16

-Tyrosine kinase associated receptor

• Activation of growth hormone receptor stimulates JAK/STAT signalling pathway
• In the absence of growth hormone, these receptors exist as monomers. Upon binding of growth hormone, conformational changes occur leading to receptor activation; dimerisation occurs.
• One molecule of growth hormone can bind to 2 receptor monomers simultaneously, therefore, they have a cooperative effect (dimerisation occurs)
• Receptor is vert closely associated with JAK
• JAK is a kinase that is anchored to cell membrane and has 2 kinase domains. Upon receptor dimerisation, JAK cross phosphorylates tyrosine residues on the receptors
• STAT (type of transcription factor) dock on the phosphotyrosines of receptor via SH2 domain
• JAK then phosphorylate the STATs, causing them to dissociate and dimerise via the SH2 domains
• The STAT dimers bind to DNA with high affinity, and regulate transcription and gene expression

Question 17

How do serine-threonine kinases work?

Answer 17

• TGFB receptor superfamily (involving TGFB, BMP and activin) work via this serine/threonine pathway
• The receptors have a ligand binding extracellular domain, single transmembrane domain and an intracellular domain bound to a serine/threonine pathway
• The receptors are found in pairs, with a type 1 and type 2 in each pair
• The ligands arrive as dimers
• The activation of TGFB receptors activate SMAD dependent signalling pathways, some which inhibit growth whilst others stimulate it
• Initially, the TGFB ligand binds to a single type 2 receptor activating a serine/threonine kinase
• The type 2 receptor then dimerises with a type 1 receptor, activating its serine-threonine kinase
• This type 1 receptor’s serine-threonine kinase goes onto phosphorylate SMAD2 and SMAD3, activating them and causing them to associate with SMAD4
• The SMAD complex then translocates to the nucleus where it acts as a transcription factor, regulating gene expression

-Antagonists: Noggin, chordin

Question 18

What are Hedgehog proteins?

Answer 18

• Hedgehog proteins are secreted segment polarity proteins
• Mammals have 3 homologous: Sonic (Shh), Indian (Ihh) and Desert (Dhh)
• All of these proteins are involved in Hh signalling pathways in embryonic development, in processes such as digit formation in mammals
• These proteins are synthesised within ER and released via 4 mechanisms

Synthesis of hedgehog proteins:

• All are synthesised as precursor proteins
• They have N terminal and C terminal
• The C terminal adds a cholesterol molecule to the N terminal before autoproteolysis cleaves off the C terminal
• This leaves the N terminal either the cholesterol attached
• A protein called skinny hedgehog (SKI) acts on the N terminal, adding a palmitic moeity
• Once palmitic acid and cholesterol is added, it is transported out of ER to cell membrane
• Within the plasma membrane, a protein called Dispatched acts on the Hh protein
• This results in SCUBE-2 being attached to the hedgehog protein, which causes release of the protein from the cell

-Another way Hh proteins are released involve accumulation of the N terminal at the plasma membrane which leads to formation of a fat soluble multimer of N terminal of hedgehog protein

• Another way Hh proteins are released is by accumulation at the plasma membrane
• This time, the Hh protein interacts with Notum, and heparin-sulfate chains of Glypican
• This allows the Hh proteins to recruit lipophorins
• This complex can then be released from cell

-Hh can also be released by exocytosis

Question 19

What is hedgehog pathway in invertebrates?

Answer 19

• Hh signalling is important in embryological development
• Important in cell patterning and differentiation
• Important in pattern venation of wings in Drosophila
• Different concentrations of Hh protein induce different subsets of genes
• The genes are activated at threshold concentration of Hh
• Different concentrations of Hh result in different levels of intracellular signalling
• The different levels of signalling occur as different Hh concentrations have varying abilities to alter ratio of Ci repressed to Ci activated
• Ci is what mediates the transcription of Hh target genes

-There is organ and tissue specific gene induction associated with Hh signalling e.g in the wing discs of flies, we get different genes activated

• In membrane of cells responsive to Hh, there are 2 proteins - Patched and Smo
• When no Hh proteins are present, Patched inhibits Smo, causing its degradation via proteasome which reduces Smo levels

-Whilst this is occurring there is an intracellular complex of proteins consisting of Cos2, Fused (fu), suppressor of fused (SuFu) and Ci

• in the inactivated state (when no hedgehog proteins present), this complex is associated with a microtubule in the cell)
• Ci is phosphorylated by PKA, CK1A and GSK-3N
• This phosphorylation represses Ci to be repressed which increases the ratio of Ci repressed to activated Ci
• Ci in repressed form can translocate into the nucleus but can’t transcribe genes
• When the Hh proteins become available, the pathway is activated
• Hh binds to Patched with the help of Ihog and Boi
• This inhibit Patch’s ability to inhibit Smo, and causes Patch’s degradation
• This increases Smo levels
• Now an an intracellular portion of Smo can interact with the Ci complex within the cell. This portion of Smo is phosphorylated by GPRK, PKA, CK1a and GSK-3B which prevents these enzymes from phosphorylating Ci, so that Ci is not destined for proteasome
• This Ci complex undergoes a conformational change
• Cos2, Fu and Sufu bind to the intracellular domain of Smo
• This releases Ci
• Ci is now in the activated state
• It translocates into the nucleus and can start the transcription of Hh target genes
• This leads to cell patterning differentiation
• The relative levels of Ci activated to Ci repressed determines how active the pathway is
• This causes differential patterning

Question 20

What is the hedgehog signalling in vertebraes?

Answer 20

-Hh signalling is important for development patterning, tissue growth and mitogenesis of cerebellum and retina, tissue repair of adult neural stem cells

Difference between invertebrae and vertebrae Hh signalling pathway:

• In vertebrae’s, Hh signalling occurs in primary cilium of cell which is associated with microtubules
• GLI proteins replace Ci proteins in vertebrae (they are very similar)
• GLI proteins are more specialised (e.g GLI2 is a transcriptional activator; GLI3 is a repressor)
• Smo is intracellular (endosome) in vertebrae, opposed to membrane bound in an invertebrates
• Occurs in primary cillium which is associated with microtubules on the inside of the membrane
• In the membrane, there is Patched and GPR161
• When there is no Hh protein around, Patched inhibits Smo, preventing it from entering the plasma membrane (it stays within endosome)
• At the same time, GLI proteins are associated with Sufu
• CKI, PKA and GSK-3B phosphorylate the GLI protein
• The phosphorylated GLI proteins are guided to the proteasome and cleaved, truncating them to their repressor form
• GLI repressed proteins translocate into the nucleus and repress the transcription of Hh target genes
• When the Hh proteins are available, the pathway is activated
• Hh binds to Patched, inhibiting its ability to inhibit Smo
• Upon The binding of Hh to Patched, both GPR161 and Patched dissociate from the membrane of the cilium and are degraded by proteasome
• Smo is no longer inhibited so it can be phosphorylate by GPRK2 and CKI
• Phosphorylated Smo can then move into the plasma membrane with the help of microtubule motor
• EVC in the plasma membrane becomes closely associated with Smo
• KIF7 in the cytosol then aids with the movement of GLI and Sufu proteins
• This causes GLI proteins dissociate from Sufu and are not phosphorylated by CKI, PKA and GSK3B
• This means GLI are no longer destined for protoesome, so they exist in their active form
• They translocate into the nucleus and bind the DNA, inducing the expression of Hh target genes

Question 21

What is the Wnt/B-catenin pathway?

Answer 21

• Signalling pathway is the canonical branch of the Wnt pathway, which is composed of several branches
• The canonical branch is the best understood of the Wnt pathway branches
• It is mediated by GPCR frizzled
• Involves Wnt proteins which are growth stimulatory factors conjugated with palmitoleic acid for binding purposes
• There are 19 Wnt genes in mammals allow transport of proteins
• Wnt signalling affects a protein called B-catenin which is regulated through degradation
• The whole pathway is highly conserved and plays a key role in embryonic development, it’s particularly important in cardiac development
• Mutations in the pathway can lead to carcinogenesis
• Wnt/B-catenin pathway is known as canonical branch of Wnt signalling pathway
• The receptors is Frizzled (GPCR)
• Wnt can also bind to several other receptors on the cell surface membrane, each of which can lead to different consequences in the cell

Wnt/B-catenin pathway:

• In the inactivated state (when no Wnt is present), B-catenin is present within cytosol but bound and regulated by a large protein complex consisting of axin, CKI, GSK3, APC, Dvl and B-TrCP
• The whole complex is known as the destruction complex as it phosphorylates and ubiquinates B-catenin, trafficking it to the proteasome for degradation
• The result of this is a low level of cellular B catenin
• In the activated state, Wnt is transported in an endosome from elsewhere in the body
• Wnt binds to the membrane receptor Frizzled which subsequently phosphorylates LRP
• This induces the translocation of the destruction complex to the plasma membrane
• When Dvl binds of the LRP, it becomes activated, inhibiting the destruction complex
• This prevents phosphorylation and ubiquitonous of B-catenin, so it’s not degraded by the proteasome
• Cellular B-catenin levels increase as a result
• In the nucleus, the transcription factor TCF is found
• When the pathway is inactive, TCF is bound to an inhibitory protein called Groucho
• When the pathway is active, cytosolic B-catenin translocates into the nucleus and competitively inhibits Groucho
• B-catenin displaces Groucho and activates TCF, initiating transcription of Wnt target genes

Question 22

How is carcinogenesis involved in Wnt/B-catenin pathway?

Answer 22

• If APC (adenomatous polyposis coli) (regulates B catenin levels) is mutated, the destruction complex unable to bind to B-catenin
• As a result, even in the absence of Wnt, the pathway is active so B-catenin can accumulate in the cytosol
• It therefore translocates into the nucleus and upregulates Wnt target genes
• These genes lead to growth and proliferation of cells, causing formation of a tumour

Question 23

What is the non-canonical Wnt signalling pathway?

Answer 23

• Wnt pathways that are not mediated by B-catenin are known collectively as the non-canonical Wnt pathways
• There are 2 pathways: Planar cell polarity (PCP) pathway and the Ca2+ pathway

Planar cell polarity (PCP) pathway:

• Wnt signalling via Frizzled receptors directs assymetric cytoskeleton organisation and cell polarisation by causing modification of the actin cytoskeleton
• Dsv initiates 2 independent pathways that induce the activation of 2 GTPases Rho and Rac
• Rho activation leads to the activation of a Rho-associated kinase called ROCK
• Rac activation induces Jun Kinase activity
• Rho and Rac then alter cell cytoskeleton, polarising and mobilising the cell

Ca2+ pathway:

• Activation of Wnt signalling pathway via Frizzled receptors can also result in the release of intracellular Ca2+
• The frizzled co-receptors Knypek and Ror2 are activated in this pathway
• Other activated intracellular messengers in the pathway are G-proteins, PLC and PKC
• PLC cleaves PIP2 into IP3 and DAG
• Production of DAG and IP3 result in activation of PKC, the calcium, calmodulin-dependent Protein Kinase type 2 (CaMKII)-TGF-beta-activated kinase 1(TAK1)-Nemo-like kinase (NLK) pathway, and the calcineurin phosphatase, which dephosphorylates and activates the transcription factor NFAT

Question 24

How are Wnt levels regulated?

Answer 24

• Wnt levels are regulated by secreted Wnt antagonists

- These are: Dickkof (Dkk1), Wnt inhibitory factor 1 (Wifi1), Secreted frizzled related protein (Sfrp)

Question 25

What is activin (TGF-B superfamily)?

Answer 25

• Activin is member of the TGFB superfamily
• It is the inducing signal for mesoderm in Xenopus embryos
• In Xenopus blastocyst, the cells in the vegetal pole of the blastula will eventually form the endoderm; those at the animal pole will become the ectoderm and some mesodermal derivative

By using grafts, we have found that:

• Region closest to animal pole (animal cap) becomes ectodermal cells
• Vegetal cells give rise to undifferentiated vegetal tissue
• The animal cells ventral and dorsal to the animal cap gives rise to mesodermal derivatives
• The dorsal cells of animal pole mainly give rise to muscle cells; ventral ones give rise to blood cells

When explants from animal cap and vegetal pole are taken and put next to each other, these observations were made:

• A portion of the cells from the animal cap gave rise to mesodermal derivatives such as muscle and mesenchyme
• This indicated that the vegetal tissue was the source of a mesoderm inductive signal
• This is because when the animal cap is cultured alone, the animal cap cells only gave rise to ectodermal derivatives
• When cultured with vegetal cells, animal cap cells gave rise to mesoderm derivatives and ectodermal derivatives
• Further experiments were done where varying regions of vegetal portion of blastula were combined with the animal cap
• It was found that dorsal and ventral vegetal regions had different inductive properties
• Dorsal vegetal cells were found to induce muscle and notochord from animal cap (these are dorsal mesodermal derivatives)
• Ventral vegetal cells were found to induce blood and associated tissues from animal cap cells (these are ventral mesodermal derivatives)
• Eventually it was found that activin was the signal for the mesoderm induction and that it worked in a concentration dependent manner
• Dorsal mesodermal derivatives seem to develop in higher concentrations of activin
• Concentration of activin is much mightier in dorsal region of embryo than ventral region

Question 27

Why is activin important?

Answer 27

• It is the mesoderm inducing signal
• Works in a concentration dependent manner
• Concentration of activin is higher in dorsal region (producing muscle and mesenchyme) than ventral region
• Part of the TGFB superfamily
• Also important in humans

Question 29

What is BMP?

Answer 29

• Member of TGFB superfamily
• Involved in induction of neuroectoderm, which occurs slightly later than gastrulation and mesoderm induction
• The neuroectoderm is derived from the ectoderm, along with epidermis
• The epidermal fate is induced by BMP4 which is expressed throughout the ectoderm
• It is only active in the ventral portion of ectoderm
• This is because in the dorsal portion, chordin, noggin and follistatin inhibit BMP4
• Chordin, noggin and follistatin are produced by blastopore
• This causes development of the neuroectoderm

Question 30

What is the Hedgehog signalling (Hh)?

Answer 30

• Originally discovered in Drosophila
• Hh mutants produced a larva devoid of the naked cuticle - similar appearance to hedgehog

-Hh has roles in vertebrae development: patterning of limb buds

• Our limbs are highly assymetric along all axis; proximal parts of limb are very different to distal parts; there’s a marked different between the anterior and posterior parts
• These asymmetrics are established by secreted molecules expressed in specific parts of the developing limb bud
• In the proximodistal axis, the assymetrics are established by FGF secreted by apical ectodermal region
• These FGFs diffuse out and establish the different fates of the structures we find along the axis of the limb
• Anteroposterior assymetry is caused by Shh which is secreted by cells in the zone of polarising activity
• The Shh diffuses through the extracellular space, creating a gradient
• In the dorsoventral axis, Wnt7a is secreted from the dorsal ectodermal covering of the growing limb bud is responsible for causing asymmetry
• From there, it diffuses into the mesenchyme of limb bud and establishes a gradient along the dorsoventral axis
• An important concept is that the 3 signalling centres (apical ectoderm region, zone of polarising activity, dorsal ectoderm) work together to develop patterning of limb
• FGFs feedback to ZPA reinforcing Shh, which feeds back to AER, stimulating more FGF (positive feedback)
• Wnt7a stimulates AER to increase FGF secretion

Question 30

What did the determination of different determination centres in the limb bud show?

Answer 30

• Our limbs are highly assymetric along all axis; proximal parts of limb are very different to distal parts; there’s a marked different between the anterior and posterior parts
• These asymmetrics are established by secreted molecules expressed in specific parts of the developing limb bud
• In the proximodistal axis, the assymetrics are established by FGF secreted by apical ectodermal region
• These FGFs diffuse out and establish the different fates of the structures we find along the axis of the limb
• Anteroposterior assymetry is caused by Shh which is secreted by cells in the zone of polarising activity
• The Shh diffuses through the extracellular space, creating a gradient
• In the dorsoventral axis, Wnt7a is secreted from the dorsal ectodermal covering of the growing limb bud is responsible for causing asymmetry
• From there, it diffuses into the mesenchyme of limb bud and establishes a gradient along the dorsoventral axis
• An important concept is that the 3 signalling centres (apical ectoderm region, zone of polarising activity, dorsal ectoderm) work together to develop patterning of limb
• FGFs feedback to ZPA reinforcing Shh, which feeds back to AER, stimulating more FGF (positive feedback)
• Wnt7a stimulates AER to increase FGF secretion

Question 30

How do Shh, TGFB and Wnt work together to pattern the neural tube?

Answer 30

-As the neuroectoderm folds to become the neural tube, it forms an alar dorsal plate (sensory) and basal ventral plate (motor)

• The location of different neurones along the dorsoventral axis determine their fate (ie motor or sensory)
• Their fate is controlled by interactions between Shh, TGFB and Wnt
• The notochord patterns the ventral neural tube - it’s a source of Shh
• It also induces Shh expression from ventral floor plate of neural tube
• This means there is a Shh gradient from ventral (higher Shh gradient) to dorsal (lower Shh gradient)
• The gradient leads to the development of different types of neurones
• The dorsal neural tube is patterned by TGFB superfamily members (specifically BMPs)
• The TGFB are expressed in the overlying ectoderm
• These diffuse out to form a gradient with high concentration dorsally and a low concentration ventrally
• Wnt and Shh have antagonistic effects on neural tube dorsoventral patterning by regulating Gli3R:Gli3A ratios
• Wnt is expressed in the dorsal roof plate of neural tube. The Wnt pathway induces dorsal fates by inducing Gli3 expression.
• Gli3 works as a repressor, unless it is activated by Shh
• The ratio of Gli3R:Gli3A depends on the relative levels of Wnt and Shh activity