Boundary Formation

Question 1

What is boundary formation?

Answer 1

1. Establishment of discrete subdivisions during development for example during hindbrain development, which is divided into segments each with a different identity. You can see the sharp boundaries

Question 2

Why are boundaries important?

What can disruption of boundaries cause?

Answer 2

2. Boundaries form signalling centres which release signals that are involved in patterning with the tissue
3. Boundary formation is also needed in the maintenance of tissue organisation in adult.
4. Disruption of boundary formation can lead to disease e.g tumour metastasis

Question 3

What are compartments?

Answer 3

Compartments: tissue domains that do not intermingle with each other.

Question 4

What is the wing imaginal disc?

What subdivided the tissue?

How do the compartments have a distinct identity?

Are there boundaries in this disk and what do they act as?

Answer 4

• The wing imaginal disc is a sheet of cells formed during the embryo which will go on to form the wing.

• Within the imaginal disc there is a boundary where which cells do not move across. This subdivides the tissue along 2 axis: the anterior-posterior axis and dorsal-ventral axis. Each of these compartments is a region of the tissue which has a distinct identity, there are TFs giving that region of a tissue a distinct regional identity.
• These compartment boundaries are signalling centres

Question 5

These compartment boundaries are signalling centres, what do they secrete?

What does the anterior-posterior boundary form express?

What does the dorsal-ventral boundary form?

Answer 5

• Anterior-posterior boundary: Dpp (TGF-beta family) – involved in patterning of tissue
• Dorsal-ventral boundary: Wg (Wnt family) – in absence of this signal the wing fails to develop
• Domains become compartments which mean they form groups of cells which don’t intermingle with their neighbours, and form signalling centres.

Question 6

Why are sharp interfaces important?

Answer 6

1. Precise organisation of cells with different regional identity. These may generate different sets of cell types.
2. Sharp interface -> straight signalling centre ->correct gradient of signal and pattern cell types. If they are not sharp patterns of cell differentiation will become disorganised.

Question 7

Give some boundaries within the nervous system development

Answer 7

• A number of compartment boundaries have been found in the anterior forebrain such as the zona limitans intrathalamica (ZLI) within the diencephalon
• The MHB
• Hindbrain segment boundaries
• Each of these demarcate gene expression domains, are compartments are signalling centres

Question 8

Describe zona limitans intrathalamica

What does it arise from?

What does it not express?

How does it change?

How is the ZLI a signalling centre?

Answer 8

• This arises in the developing diencephalon (forebrain)
• It is initially a broad wedge of tissue that is demarcated by gene expression pattern – marked by the absence of Lfgn (modulator of notch signalling)
• The ZLI does not express this gene
• The wedge then narrows to separate the different regions of the diencephalon
• The ZLI is itself a compartment (does not mix with adjacent cells)

1. The ZLI is also a signalling centre, and expresses Shh (involved in dorsal-ventral patterning of neural tube, expressed in ZLI and induces regional specific gene expression in adjacent tissue, anterior and posterior)
2. Shh induces region-specific gene expression in thalamus and prethalamus
3. Distinct gene expression in thalamus and prethalamaus due to different competence in response to Shh

Question 9

What is the MHB?

Answer 9

• This is the interface between the midbrain and the hindbrain, which is a compartment border
• Cells never cross this border (found within zebrafish)
• It is a border of expression of Otx2 (forebrain) and Gbx2 (hindbrain) transcriptional factors which regulate regional identity
• This MHB is a signalling centre that regulates cell identity (tectum and cerebellum in adjacent midbrain and hindbrain)

Question 10

What is the hindbrain segmented into?

How many rhombomeres are there?

Answer 10

• The hindbrain is segmented into regions called rhombomeres

* There is 7 of them, depending on the species which are universally found in vertebrae species

Question 11

What do the different rhombomeres form?

What genes are required to form these segments?

Answer 11

• Different types of nerves are formed in different rhombomeres
• Segmentation of hindbrain is crucial for segmentation of neurones
• Genes required for AP identity of segments: hox genes
• Other TFs are involved in the segmentation processes such as Krox20, MafB and hoxb1

Question 12

How was the hindbrain boundary discovered?

Answer 12

• It was shown that each of these segments forms a compartment
• Clonal analysis marking individual cells found, marked with fluorescent dye. After you mark a cell after the boundaries can be seen, it is found the progeny of that cell will never cross that boundary
• If you mark cells at an earlier cells where cells can be seen before boundary formation, cells can cross the border.

Question 13

What are boundary cells?

Answer 13

• Boundary cells are signalling centres that have a role in patterning of the tissue.
• These boundary cells have a role in patterning of neurogenesis within the zebrafish

Question 14

How can you form these sharp borders at the interface between different regions?

Answer 14

• If we look at a molecular marker such as Krox20, it is expressed in segment 3 and segment 5 of the developing hindbrain and is required for the formation of those two segments
• In the early stage of the hindbrain the pattern is not very precise, the border is not defined
• The border progressively becomes sharp over a two hr time period
• Sometimes cells can be in the wrong place, gene expression is turned off here

Question 15

What are the 2 challenges of border formation?

Answer 15

Two challenges in border formation:
• Mechanisms that regulate cell identity and not precise: variability in formation and interpretation of morphogen gradient
• During development there can be cell intermingling which can scramble the pattern e.g due to cell division and intercalation and to morphogenic movements

Question 16

What are 2 mechanisms that sharpen the border?

Answer 16

• Cell segregation (will discuss later) – prevent intermingling across the border
• Cell identity regulation

Question 17

How are the different boundaries formed within the hindbrain?

Answer 17

• An example of this occurs in the early stages of the hindbrain
• There is a gradient of RA in the hindbrain which is created by a gradient of degrading enzyme (cyp26a1)
• These enzymes are expressed in the developing hindbrain in a high anterior to low posterior gradient
• This gradient of enzyme which degrades RA sets up the gradient of RA
• Depending on how much RA is present it induces the expression of different genes such as Krox20 (r3/5) and hox1b (r4) which are expressed in different hindbrain segments
• There is also a slight overlap of gene expression at the border: so what happens is…
• These cells that expressing both of these transcription factors becomes resolved through mutual repression: see diagram

Question 18

What do cells do once they contribute towards regions that have regional identities?

Answer 18

• Using clonal analysis, cells can spread across hindbrain boundaries and therefore contribute towards regions that have regional identities
• Cells then acquire identity according to their location, location signalling must be regulating identity

Question 19

What happens when you transplant cells from one segment to another?

Answer 19

• When you transplant cells from one segment to an adjacent segment, the cell will change its identity to match its new neighbours, there’s a plasticity in cell identity
• This plasticity is lost at the later stages (after segregation mechanisms are established)

Question 20

What is identity switching using Egr2 as an example

Answer 20

• Egr2 needs to switch on enough EphA4 that cell intermingling can be occur between the hindbrain segments
• We can label cells expressing Egr2 by knocking in a stable fluorescent protein (citrine – a green protein which is fused to histone 2B). This is inserted upstream of the Egr2 coding region. So the fluorescent molecule is under the control of the enhancers controlling the expression of Egr2.
• This drives a reporter expression in segments 3 and 5
• It shows intermingling
• If we look at the expression of various molecular markers, the intermingling cells have been downregulated Egr2 and upregulated Hoxb1
• This is identity switching

Question 21

What is the mechanism of cell identity switching?

Answer 21

• The cells are responding to being at a new position on the morphogen gradient (RA)
• However, groups of cells were transplanted, and what they found was that groups of cells maintains its identity in the new location. This suggest a community effect (cells release a signal which can then turn on the same identity in the adjacent cells)

Question 22

What is RA and what forms its gradient?

How are Cyp26b1 and Cyp26c1 expressed?

When is AP lost?

Answer 22

• RA forms a AP identity
• This gradient is formed through a counter gradient of Cyp26a1 (RA degrading enzyme)
• Cyp26b1 and Cyp26c1 are segmentally expressed (not directly regulated by RA). They have a dynamic segmental expression, so initially in the anterior hindbrain then to the posterior region of the hindbrain
• AP patterning is only disrupted when loss of Cyp26a1, b1 and c1 function are combined

Question 23

How is Cyp26b1 and C1 regulated by Egr2?

Answer 23

• Egr2 knockdown increases cyp26b1/c1 expression in r3 and r5
• Therefore Egr2 underlies lower cyp25b1/c1 (high RA) in r3, r5 than r2, 4 and 6
• High cyp26 decreases RA autonomously and in adjacent cells
• R3, R5 cell mingling into R2, r4 and r6 encounters low RA which may underly identity switching

Question 24

Identity switching by RA mediated community effect

Answer 24

On image

Question 25

Summarise formation of regional domains

Answer 25

1. Graded signal induces rough pattern of two domains with different identity 2. Mutual repression of transcription factors creates strict complementarity of identity 3. Fuzzy border becomes sharp (cell segregation and identity regulation) 4. Boundary signalling centre forms at the interface 5. Signalling from boundaries underlies patterning within regional domain

Question 26

What 3 mechanisms prevent mixing of cells across borders?

Answer 26

* Differential cell-cell adhesion * Differential cortical tension * Contact repulsion (contact inhibition of cell migration)

Question 27

How was cell-cell adhesion discovered? What is cell sorting driven by?

Answer 27

Cells were taken from early amphibian embryos from two different species which had different pigmentation, they took cells of the neural epithelium and endoderm tissue. They dissociated these cells into single cells, mixed and reaggregated them. They found the neural cells segregate from endoderm cells and neural crest cells aggregate in the centre. They thought that this was driven by direct cell movement. But, cell sorting is driven by differential cell adhesion mediated by cadherins.

Question 28

What are cadherins? What do they they mediate? What do the cadherins bind to?

Answer 28

* Large family of transmembrane proteins * Mediators of homophilic cell adhesion (when the same cadherin family member is expressed in adjacent cells the extracellular domain of the cadherin will interact with the same cadherin from the adjacent cell to mediate homophilic binding (strong)) , some also heterophilic interactions * These cadherins bind to actin filaments and proteins in cells that interact with the cytoplasmic domain of the cadherin. This is important for cell to cell adhesion. * Strong adhesion requires link to actin cytoskeleton

Question 29

What differences in cadherins cause cells to segregate away from each other?

Answer 29

Cell populations will sort from each other if they: • Express different cadherins • Express different level of the same cadherin Cell sorting is driven by differential sorting – cells with strong stickiness will separate and join together, cells with weaker stickiness will join together (Oil sticks to oil and water sticks to water analogy) Cells maximise their contacts with cells with which they have the strongest mutual affinity (qualitative and quantitative) Differential cell affinity and surface (interfacial) tension

Question 30

How is cadherins6 involved in cell sorting in the forebrain?

Answer 30

* Electroporation has been used here to introduce a DNA expression vector using an electric current expressing a green fluorescent protein into this region of the developing forebrain * If this is done in a wild-type mouse embryo what you find is you label the cells both in the cortex and lge * If you also use this green fluorescent protein and express cadherin 6, the cadherin 6 cells segregate into the lge (where cadherin 6 is being endogenously expressed) * If there is a cadherin 6 mutant the segregation does not occur

Question 31

In classical models, cell segregation and border formation are due to differences in global properties of the two cell populations (amount of homophilic adhesion, cortical tension) What other mechanism can cause cell to segregate?

Answer 31

Local signalling between the two cell populations induces a distinct property at the border: adhesion, tension, repulsion: * Contact repulsion * Decreased adhesion * Increased tension

Question 32

Describe how tumour cells show contact repulsion Describe Cortical tension at cell interfaces

Answer 32

Collision of tumour cells with other cell type: • Tumour migration arrested, reinitiates in new direction Or • Tumour cell continues to migrate, invade other tissue Cadherins that are mediating homophilic cell-cell adhesion (E-cadherin). These interact with catenin in the cytoplasmic domain that interact with the actin-myosin 2 network which can contract upon phosphorylation. This then generates tension on the outside of the cell, because they cells are pulling upon each other.

Question 33

How is there tension at the border?

Answer 33

Cells in epithelium have adhesive contacts Cortical actomyosin linked to cell adhesion molecules Actomyosin contraction -> cells pull on each other, creates tension Image The evidence came from observing there is a higher amount of actin at the boundary than there is away from the boundary, and also myosin at the AP border boundary When there is a myosin 2 mutant, instead of forming a flat interface there is a wiggly interface.

Question 34

How can tension be measured?

Answer 34

* Determine the amount of tension by cutting cell-cell bonds with laser, measure speed and amount of cell displacement * They found there is 2.5x more tension at the cell of the border than away from the border * This requires Rho Kinase which phosphorylates myosin light chain * Computer simulation show this difference in tension can prevent mixing * Conclusion: Myosin 2: dependant tension can prevent mixing across borders (but not known what causes myoll contraction at border)

Question 35

Describe Eph receptor tyrosine kinases and ephrins

Answer 35

* Eph receptors are the largest family of receptor tyrosine kinases in vertebrates, up to 16 family members depending on the vertebrae species * They are transmembrane receptors, so they have an extracellular domain and a transmembrane domain and a cytoplasmic tyrosine kinase domain. * When a ligand binds, they become clustered which will activate the tyrosine kinase phosphorylation leading to signalling within the cell (forward signalling) * The ligands are called ephrin’s, which are membrane bound, such as A which are anchored in the membrane by a lipid attachment (GPI linkage) which interact with EphA receptors * Also have Ephrin B ligands are also transmembrane proteins which bind to Eph B receptors but also to EphA4. When they become clustered they become phosphorylated by cytoplasmic tyrosine kinases that leads to reverse signalling * You get forward signals in Eph receptor cells and reverse signalling in Eph expressing cells leading to bi-directional signalling.

Question 36

How does Eph-ephrin signalling segregate cells and maintain borders?

Answer 36

Eph receptor and EphrinB activation regulates multiple pathways including Rho family GTPases, MAPK and Dishevelled Some of these pathways regulate the actin cytoskeleton and cell adhesion Three mechanisms: • Decreased adhesion • Increased tension • Contact repulsion Eph-Ephrin signalling can lead to responses that could restrict cell migration

Question 37

Describe how Adam 10 (metalloproteinase) is physically associated with Eph family members that leads to decreased adhesion

Answer 37

Adam 10 (metalloproteinase) is physically associated with Eph family members and upon activation of these EphB receptors -> Adam 10 is activated which cleaves the E-cadherin from the surface leading to decreased cell-cell adhesion across the interface

Question 38

How does Eph-ephrin signalling lead to increased Cortical tension?...so actomyosin contraction

Answer 38

There is a higher amount of actin at the rhombomere boundaries where we know the eph receptors are involved in cell segregation preventing intermingling. If you block actomyosin using drugs that blocks myosin function it decreases the sharpness of the borders. This shows there is a critical role of cortical tension in maintaining the sharpness of the hindbrain boundaries. This occurs downstream of Eph receptor signalling

Question 39

How does Eph-ephrin interaction stop the mesoderm invading the endoderm?

Answer 39

This is the border between the ectoderm and mesoderm in xenopus embryos. They found the migrating mesoderm cells are expressing various Eph receptors, the ectoderm are expressing ephrins which activate those Eph receptors on the mesoderm. This activation leads to cell repulsion. The mesodermal cells will migrate over the ectoderm. They initially have an adhesive interaction which is required for them to grip onto the ectoderm and migrate forwards. Then through Eph signalling they are repelled, which is crucial for preventing the mesoderm from invading the ectoderm

Question 40

Is heterotypic repulsion (tension) more efficient than differential adhesion in driving cell segregation?

Answer 40

Yes

Question 41

What are intestinal crypts? How is Eph-ephrin signalling involved in this?

Answer 41

* These tissues are undergoing rapid cell proliferation all the time. This is because the gut is a harsh environment and is constantly shedding differentiated cells in the gut which are then replaced. * This replacement occurs through stem cells, found within intestinal crypts. At the base of these crypts is a population of slowly dividing stem cells * The main signal driving the proliferation of these stem cells is Wnt signalling, Wnt upregulates EphB receptors and downregulates ephrinB. * As the cells move away from the Wnt signalling they downregulate EphB and upregulate ephrinB. So there is complementary expression of Eph receptors and Ephrins * EphB gene knockouts lead to mis location of proliferating cells and differentiated cells