Module 4.4: �Modelling Convergent-Extension and Intercellular Forces with Links

Julio Belmonte

NC State University

Raleigh, NC

Friday, August 5th, 2022

• Workshop will be live-streamed, recorded and distributed

Support: NIH NIBIB-U24EB028887, NIGMS-R01GM122424, NSF-2120200, NSF-2000281, NSF-1720625, NIGMS-R01GM076692, NIGMS-R01GM077138

Zoom logistics

Screen Sharing will be disabled and microphones will be muted except in breakout rooms and for special sessions

Put any questions or needs in the zoom chat

If you need personalized attention for any reason, let us know and the moderator will put you in a zoom breakout room (where you can talk and screen share) for help

If there are any technical problems with zoom, please let us know right away

Please feel free to use the “go faster” “go slower” buttons on the Participants screen

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During exercises we will do a quick poll for completion/need more time. However, some exercises will include components with exploration beyond what we will have time to do on-line, so don’t worry if you can’t finish every exercise completely.

The exercises will be made available in google doc format and as .pdfs so you can continue working on them off-line

Videos and PowerPoints will be made available for you to review if you miss any materials

The sessions will be live-streamed on YouTube and recorded and distributed on YouTube

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Cellular movements during development

CC3D simulations w/ spring forces between cells

Epithelial Furrow Formation

Epithelial self-organization (ectopic somites)

Dias A, et al. Somites without a clock Science 343 (2014)

Plan of action

1. Overview of how links work in CC3D

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• Simple exercise to illustrate how to add links to CC3D simulations (XML)

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• Simple exercise to manipulate links from the python code

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• Create simple convergent-extension model in CC3D

How Links work in CC3D

How Links work in CC3D (Wizard)

In the wizard, the FPP (Focal Point Plasticity) Plugin is selected on the “Cell Properties and Behaviors” panel:

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Is usually a good idea to also check the “Cell Neighbors” tracer when using this plugin, specially if you plan to manipulate the FPP link from the Python code.

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How Links work in CC3D (XML)

How Links work in CC3D (XML)

In the xml code, the FPP (Focal Point Plasticity) Plugin looks like this:

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links break when distance exceeds this value

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this is the maximum number of links (of that type) per cell

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How Links work in CC3D (XML)

In the xml code, the FPP (Focal Point Plasticity) Plugin looks like this:

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These are used for the creation of new links.

When 2 cells come into contact, the creation of the new link would add a substantial amount of energy to the system, and drastically reduce the acceptance rate.

The activation energy lowers this energy barrier and facilitates the creation of a link,

and the neighbor order determines how far two cell membranes must be for link creation

Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)

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Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)
2. Call it Module4_4_1_Examples, select “Python+XML” option. Hit “Continue”.�

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�(no spaces “ ” nor dots “. ” nor slashes “/” “\” in names, no name starting with numbers)

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Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)
2. Call it Module4_4_1 _Examples, select “Python+XML” option. Hit “Continue”.
3. Make sure that “Rectangular slab” is selected, leave everything else as it is. Hit “Continue”.

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Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)
2. Call it Module4_4_1 _Examples, select “Python+XML” option. Hit “Continue”.
3. Make sure that “Rectangular slab” is selected, leave everything else as it is. Hit “Continue”.
4. Add a cell type named “MyCell” (or some other name) and hit “Add” to incorporate it to the cell type table above. Hit “Continue”.

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Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)
2. Call it Module4_4_1 _Examples, select “Python+XML” option. Hit “Continue”.
3. Make sure that “Rectangular slab” is selected, leave everything else as it is. Hit “Continue”.
4. Add a cell type named “MyCell” (or some other name) and hit “Add” to incorporate it to the cell type table above. Hit “Continue”.
5. Ignore the Chemical Field window, hit “Continue”.

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Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)
2. Call it Module4_4_1 _Examples, select “Python+XML” option. Hit “Continue”.
3. Make sure that “Rectangular slab” is selected, leave everything else as it is. Hit “Continue”.
4. Add a cell type named “MyCell” (or some other name) and hit “Add” to incorporate it to the cell type table above. Hit “Continue”.
5. Ignore the Chemical Field window, hit “Continue”.
6. On this window, select “Contact”, “VolumeFlex” and “FocalPointPlasticity”. Hit “Continue”

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Exercise 4.4.1 – Simple model with links

1. On Twedit++ create a new project (MENU -> CC3D Project -> New CC3D Project)
2. Call it Module4_4_1 _Examples, select “Python+XML” option. Hit “Continue”
3. Make sure that “Rectangular slab” is selected, leave everything else as it is. Hit “Continue”
4. Add a cell type named “MyCell” (or some other name) and hit “Add” to incorporate it to the cell type table above. Hit “Continue”
5. Ignore the Chemical Field window, hit “Continue”
6. On this window, select “Contact” and “VolumeFlex” and “FocalPointPlasticity”. Hit “Continue”
7. Configuration is complete, hit “Done”.

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Exercise 4.4.1 – Simple model with links

• The first thing you will see once this is done is a small entry under “CC3D Projects” on the left side of Twedit++ with the name of the project.
• Right click on the name and choose “Open in Player” to run the simulation

(alternatively, click on the CC3D logo ( ) right above it)

Exercise 4.4.1 – Simple model with links

• Once you play the simulation you should see something like this on the right:

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• If you do not, check the visualization settings (MENU -> Visualization) and make sure that “FPP Links” option is checked.

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• You may wish to set the cell borders to black to see the links better (click on to get there)

Exercise 4.4.1 – Simple model with links

• What is going on here?

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• Let’s look at the code.
• These are the default setting for the FPP links:

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• The <MaxNumberOfJunctions> tag is set to 1, so each cell can have at most 1 connected neighbor.

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• Most will have a pair, but since the coupling is random, some cell may end up with no pair.

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• Try changing this parameter to 2, 3 and higher to see their effects.

Exercise 4.4.1 – Simple model with links

• Because this is a 2D simulation made of cells of the same size, there is no gain of connections beyond 6.

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• Let’s look at the code.
• These are the default setting for the FPP links:

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• Right now, the target distance between connected cells is set to be equal to the cell diameter (7 pixels), so there is not any visual effect on the tissue.

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• Let’s play with that parameter. Choose a connectivity level (1-6) and first decrease the target distance. Then increase it (slowly).

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• For better results, try do it directly on the player.

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Exercise 4.4.1 – Simple model with links

• Decreasing the target distance below the expected center-to-center distance of cell-pairs leads to a slight condensation of the tissue.

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• Increasing it beyond that can lead to a roughening of the tissue outer boundary.

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• It also leads to the creation of crossed FPPlinks inside the tissue.
• This happens because the links do not break unless they exceed the MaxDistance value.

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• And if the target value far exceeds the expected center-to-center distance of cell-pairs, the resulting effect can be very dramatic

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Exercise 4.4.2 – Model with 2 cell types

• Let’s now move to a slightly more complex exercise.

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• Create a simulation with FPP links, and two cell types (G and P).

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• Do not allow cells of different type to have links.

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• Make one pair of cells to have very short links, and the other longer links. Check what happens.

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• Can you come with a set of FPP link properties to induce cell type mixing?

(TIP: make the MaxDistance close to 7 to induce frequent link remodeling)

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• Can you come with a set of FPP link properties to induce some cell type sorting?

(TIP: make the MaxDistance close to 7 to induce frequent link remodeling)

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Exercise 4.4.2 – Model with 2 cell types

• LambdaFPP GG = 15
• TargetDistance GG = 1
• MaxDistance GG = 7
• MaxNumberNeighbors GG = 6

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• MaxNumberNeighbors GP = 0

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• LambdaFPP PP = 10
• TargetDistance PP = 1
• MaxDistance PP = 7
• MaxNumberNeighbors PP = 3

• MaxNumberNeighbors GG = 0

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• LambdaFPP GP = 10
• TargetDistance GP = 1
• MaxDistance GP = 7
• MaxNumberNeighbors GP = 3

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• MaxNumberNeighbors PP = 0

Controlling FPP links from the Python

• In the previous examples we set the FPP links properties at the XML and let the simulation evolve from the initial conditions.

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• But in some situations, we might want to have full control of when and where the links will be created.

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• This is possible from the Python level.

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• In order to do so we must first set the <Local/> tag in the XML code inside the plugin.

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• You may also want to set all <MaxNumberOfJunctions> to zero to ensure that links can only be created from the python code.

Controlling FPP links from the Python

• Once you turn on the <Local/> tag in the XML, you are able to, from the Python level:
• create new links
• update existing links
• delete existing links
• get information about current links

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• All these function are accessible from the “CC3D Python” menu:

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Controlling FPP links from the Python

• In the next exercise we will use two of those functions:

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first cell

second cell

Brief (and oversimplified) recap of development

Two conceptual models of convergent-extension of tissues

John Wallingford

Two conceptual models of convergent-extension of tissues

John Wallingford

Kong, Wolf, Grosshans,

Mech Dv 2027

Two conceptual models of convergent-extension of tissues

John Wallingford

Kong, Wolf, Grosshans,

Mech Dv 2027

Ray Keller

Exercise 4.4.3 - Simple model of convergent-extension

Create simulation with a group of cells of the same type

1. Volume
2. Contact
3. FPP links
4. Cell Neighbors

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Every <T_freq> MCS (after some initial time), cells should:

1. Delete all FPP links
2. Create new ones

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Creation of links must follow these rules:

1. links must be between neighbors
2. neighbors must be to left or right of the cell

(up to some max angle <MaxAng>)

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You may want to use:

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and:

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Exercise 4.4.4 - Intermediate model of convergent-extension

Create simulation with a group of cells of the same type

1. Volume
2. Contact
3. FPP links
4. Cell Neighbors

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Every <T_freq> MCS (after some initial time), cells should:

1. Delete all FPP links
2. Create new ones

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Creation of links must follow these rules:

1. links must be between neighbors
2. neighbors must be some global direction <ANG>

(up to some max angle <MaxAng>)

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Exercise 4.4.4 - Advanced model of convergent-extension

Create simulation with a group of cells of the same type

1. Volume
2. Contact
3. FPP links
4. Cell Neighbors

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Every <T_freq> MCS (after some initial time), cells should:

1. Delete all FPP links
2. Create new ones

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Creation of links must follow these rules:

1. links must be between neighbors
2. neighbors must be cell specific direction

(up to some max angle <MaxAng>)

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Exercise 4.4.6 - Super Advanced model of convergent-extension

Create simulation with a group of cells of the same type

1. Volume
2. Contact
3. FPP links
4. Cell Neighbors

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Every <T_freq> MCS (after some initial time), cells should:

1. Delete all FPP links
2. Create new ones

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Creation of links must follow these rules:

1. links must be between 1^st or 2^nd neighbors
2. neighbors must be cell specific direction� (up to some max angle <MaxAng>)
3. cells cannot have more than <N> links

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Module 4.4 Questionnaire

Support: NIH NIBIB-U24EB028887, NIGMS-R01GM122424, NSF-188553, NSF-186890, NSF-1720625, NIGMS-R01GM076692, NIGMS-R01GM077138

Please take a minute or two to let us know about your experience with this module by filling out the brief zoom survey

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Feel free to provide additional comments and suggestions in the slack or by email to us as well (haydenfennell@gmail.com)

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