Master's Research Project

MODIFIED SCRIPT

(with loose, bolded descriptions of shots and images)

SCENE 1

Shot 1 -> rooster, surrounded by antibodies.. maybe just a drawing of a rooster, i.e. a classroom drawing

It was in chickens, in the early 1950's, that bcells were first suspected as the source of antibody-mediated immunity.

Shot 2 -> zoom out from a bcell nucleus, following DNA getting pulled out and some cell events, like mitochondria dividing, nuclear pore structures

Since then, avian bcells have shed light on many molecular, and cellular events.

Shot 3 -> DNA pulled out, antibody palisade, shedding light on antigens in darkness all around

Including as those genetic modifications that make antibody retaliation to antigens possible.

SCENE 2

Shot 1 -> barnyard scene, zoom into egg and x-ray the embryo skeleton

The chicken's bcells arise in sites of embryonic hematopoiesis:

Shot 2 -> highlight the embryo's organs

The yolk sac, bone marrow, kidneys, liver and spleen.

SCENE 3

Shot 1 -> zoom in and rest in CT environment, full of dividing white blood cells

In these organs, stem cells differentiate into pro bcells,

Shot 2 -> zoom in to nucleus and rest there as DNA environment gets toony (i.e. like rooster in first shot?)

And vdj recombination, or gene rearrangement, begins.

SCENE 4

**calculator or graphs?

(calculator/slots on either side, colour coded, in middle show the IGs being highlighted, also colour coded)

Shot 1 -> no zooming past DNA, zooming out of DNA as V,D, J segments are highlighted and counter rises

In humans, there are multiple, different copies of V, D and J gene segments, for the antibody heavy chain, (highlight on IG and DNA)

and VJ for the antibody light chain, (highlight on IG and DNA)

*less confusing as heavy chain rearranges first

Shot 2 -> DNA on bottom and DNA on top; show gene rearrangements accumulating into many Igs on many bcells

In each B cell, gene rearrangement may cut and join together a unique combination of these gene segments,

resulting in different b cells possessing different functional antibody genes,

and a repertoire of b cells with diverse antibody structures.

Shot 3 -> bcells all dissolve and meld into one grey cell, V D J segments lose colour

Chickens, however, lack this multitude of VDJ gene segments to draw upon.... (rewind and zoom back in to show greyscale IG and depleted VDJ calculators)

(floating text = lack of combinatorial diversity?)

SCENE 5

Shot 1 -> zoom out of Ig on bcell surface

So even though gene rearrangement, in avian embryos, does succeed in the expression of immunoglobulins,

Shot 2 -> lots of bcells that become back-lit silhouettes

without these varied, VDJ combinations,

how do they generate antibody diversity?

SCENE 6

Shot 1 -> embryo with bursal colour cloud

The answer lies elsewhere in the embryo,

where dendritic cells are colonizing a gut associated lymphatic organ:

Shot 2 -> zoom into bursa cross section

the bursa of fabricius.

SCENE 7

Shot 1 -> dendritic cells and epithelial buds; bcells crossing into buds, start dividing and expanding the bud

Perhaps guided by these dendritic cells' chemoattractants, bcells will migrate to the bursa. (floating text = dendritic cells)

Those with surface immunoglobulin (arrow pointing out surface Ig; highlight surface Ig)

cross the bursal basement membrane, (floating text = bursal basement membrane // and/or just highlight the BBM)

and rapidly divide inside epithelial buds. (floating text = epithelial buds)

Shot 2 -> pan right to show many buds in landscape expanding with dividing bcells

With roughly 3 bcells, in each of the 10 000 epithelial buds, dividing every 10 hours, their population can reach 10 million before hatch.

SCENE 8

Shot 1 -> zoom back in to one bcell + nucleus; showing golgis exporting Igs and RER's making Igs around nucleus

And during this time, the immunoglobulin DNA is changing again.

While each bcell's newly rearranged antibody locus

Shot 2 -> Transparency showing inside of nucleus (full of DNA), in cytoplasm can still see Igs getting expressed

Is producing functional immunoglobulins

Shot 3 -> zoom into nucleus complete, zoom in to DNA, see it getting unpackaged and unwound

Their genetic homogeneity

stunts the antibody diversity needed for adaptive, specific, humoural response.

Shot 4 -> highlight pseudogenes with colour

This is where a collection of upstream pseudogenes come into play.

(floating text = pseudogene symbols)

Shot 5 -> pan right to nuclear pore where AID is coming in... golden and purtty

(floating text = AID (activation -> fades to A, Induced fades to I etc.. leaving AID floating above protein)

In a series of events, thought to be triggered by activation induced, cytidine deaminase,

A protein also active in class switch recombination, and somatic hypermutation,

SCENE 9

Shot 1 -> same as in animatic, toony

(floating text = DSBR theory of gene conversion)

A cytidine base is converted to a uracil.

These are then detected, and excised, by uracil dna glycosylase,

Leaving dna open to be targeted and cleaved by an apurinic endonuclease.

If such a nick were to occur on both strands

repair machinery will surround a single strand, and search out nearby, homologous pseudogenes.

This pseudogene, is used as a template to extend the broken DNA strand.

And can occur on both nicked ends.

Extended strands may hybridize and ligate,

forming a double holiday structure

when these two holiday junctions resolve,

the immunoglobulin gene is converted, leaving the pseudogene intact.

(floating text = pseudogene, converted AB gene)

SCENE 10

Shot 1 -> same as in animatic, toony

This may occur multiple times, using different pseudogene donors, in each bcell.

Gene conversion can thus sprinkle several to hundreds of pseudogene bases into antibody loci.

SCENE 11

Shot 1 -> chick hatches

Now, the chick hatches, and begins its lifelong acquaintance with antigens

Shot 2 -> back-light chick, antigens glow yellow, see gut anatomy from the side, zooming into bursa

Which can travel through the gut, and into the bursal lumen.

SCENE 12

Shot 1 -> follicle landscape, zooming into one follicle

Here, the epithelial buds, containing bcells, have changed into follicles

(floating text = epithelial buds -> change into follicles)

Shot 2 -> show antigens being transported into medulla

Follicle associated epithelial cells transport antigen into an inner medulla

(floating text = follicle associated epithelial cells)

Shot 3 -> change textures and brightness to highlight areas, popping sounds and bursts of light with gene conversion

And bcells migrate again, crossing the follicle's cortico-medullary junction into an outer cortex.

(floating text = cortico-medullary junction and outer cortex)

Rapid cell division and gene conversion, happen primarily in this outer cortex.

(floating text = gene conversion)

SCENE 13

Shot 1 -> show colourful bcells migrating out of follicle cortex, zooming out...

Successful bcells may migrate out of the bursa, while others undergo apoptosis.

Shot 2 -> bcells migrating out of bursa, colourful on greyscale toon chick

In this manner chickens derive a diverse bcell repertoire,

using methods rare in humans.

SCENE 14

Shot 1 -> long zooming out from: inside nucleus showing gene conversion -> exocytosis and expression of colourful Igs in a plasma cells -> follow Igs to cause a degluttination reaction on a mast cell... or dimers across mucous cells and secreted into intestine to attack bacteria

Avian B cells (B for bursa), with their capacity for gene conversion, continue today as a powerful research model:

advancing the fields of genetics, cell biology, and immunology.

OLD SCRIPT

It was in chickens, in the early 1950's. that bcells were first suspected as the source of antibody-mediated immunity.

Since then, avian bcells have shed light on many molecular, and cellular events.

Such as those genetic modifications that make antibody retaliation to antigens possible.

The chicken's bcells arise in sites of embryonic hematopoeisis.

The yolk sac, bone marrow, kidney liver and spleen.

In these organs, stem cells differentiate into pro bcells.

And vdj recombination begins.

This dna cutting, and joining together, of heavy and light chain gene segments,

Is diversified by drawing on multiple, v,d, and j copies in humans,

providing 2 640 000 possible combinations

but is limited by the paucity of v,d, and j segments in the chicken genome.

Rearrangement therefore produces surface immunoglobulins,

but falls short of producing a diverse b cell repertoire.

Meanwhile dendritic cells have colonized a gut associated lymphatic organ

the bursa of fabricius.

It is here that bcells migrate, while they are undergoing gene rearrangement,

And perhaps guided by dendritic cell chemoattractants,

2-4 bcells, with surface immunoglobulin,

will cross the bursal basement membrane,

into epithelial buds.

And undergo mass cell division.

With roughly 3 bcells, in each of the 10 000 follicles, dividing every 10 hours, their population can reach 10 million.