Animal Models in Development

Question 1

What are the desirable characteristics of a model organism?

Answer 1

• Relevance. Representative
• Accessibility/ availability
• Experimental manipulation
• Genetics
• Cost/ space

Question 2

What are the most widely used animal models?

Answer 2

	Caenorhabditis elegans (roundworm)
	Drosophila melanogaster (fruit fly)
	Danio rerio (zebrafish)
	Xenopus laevis (frog)
	Gallus gallus (chick)
	Mus musculus (mouse

Question 3

Advantages of Caenorhabditis elegans (roundworm) as a model organism?

Answer 3

֍	Transparent
֍	Short embryogenesis (~15 hours)
֍	Genetics compact, manipulation easy
-     Invariant lineage: development proceeds along a very precise, stereotyped sequence – this has many advantages to study how specific cellular traits are established during development.
֍	Has a deletion mutant database
֍	558 cells at hatching
- Has a short reproductive cycle
֍	Origin of each cell is known
֍	Very small, can easily be maintained in lab

Question 4

Advantages and disadvantages of Drosophila Melanogaster?

Answer 4

Disadvantage: Not transparent so can’t see embryogenesis

Advantages:
• Short embryogenesis (~24 hours)
• Easily bred & maintained (14-day generation time)
- Has lots of eggs
- Characterised genome and many conserved genes
- Less complicated ethical concerns
- cheap to maintain
• Genetics – compact genome, fully sequenced
• Mutants – lots of tools created for genetic manipulation fly studied for 100 years +. Unbeatable model to study gene function

Will hatch in 24hrs, grow for a few days, when big enough to undergo metamorphosis it will become a pupa. In pupa the structures growing in larva will reorganize and assemble to give rise to all adult tissues in fly. Goes through 2 different forms, larval form and adult form.

Question 5

Advantages of Zebrafish Danio Rerio?

Answer 5

• It is a vertebrate! so closer to humans, organogenesis etc.
• Rapid development (24 hours- 2 days to hatch)
• Short generation time (3 months)
• Genetics are fully sequenced
• Mutants so can manipulate in many ways
- easy administration of drugs
- nutritionally self-sufficient
• Transparent
• Unbeatable to image development at real time
• External fertilization –> good for CRISPR

Question 6

Advantages and Disadvantages of Xenopus Laevis?

Answer 6

• Large embryos: easier to manipulate
• Develop in simple salt solution/explants
• Rapid development (~30h to hatch)
• Readily injected with RNA, labels etc.
- easy administration of drugs
- nutritionally self sufficient

Disadvantage: Poor genetics (X. tropicalis better), we don’t have many tools to manipulate their gene activity.

Question 7

Advantages of Chick gallus gallus?

Answer 7

Very big, accessible embryos, so can do a lot with them
• Experimental manipulation (grafting, dye labelling), fate mapping etc. they help understand embryonic development a lot.
- highly vascularised chorioallontoic membrane
- nutritionally self-sufficient
• Poor genetics so we rely on a lot of experimental manipulation as a result. E.g. transfection of DNA molecules that allow gene manipulation has been started, thus allowing some manipulation but not a lot.
• Embryogenesis ~4 days (21 to hatching)

Question 8

Advantages and disadvantages of mouse mus musculus?

Answer 8

• They are mammals, so a lot more similar to human embryonic development, lots of conditions in labs to maintain them
• Powerful genetic tools as they are fully sequenced, sophisticated genetic tools used to manipulate gene function, gene activity, lesions in the genome
• Good for Transgenics
- 21 day gestation
- mature quickly (6 weeks) so good model for aging

Disadvantages:
• Develop in utero, not readily accessible and manipulated
• Expensive to raise
• Ethical and legal concerns, especially because we need to kill the mother to access embryo
- skills needed to raise and work with the embryo

Question 9

What do the 3 layers of the trilaminar germ disc become?

Answer 9

– Ectoderm: skin and central nervous system
– Endoderm: digestive tube, lungs
– Mesoderm: muscles, bones, connective tissue, blood, heart, kidney, gonads

Question 10

What are the 3 mechanisms of gastrulation?

Answer 10

– Involution
– Ingression
– Invagination

Embryos from different species use one or more of these. Not all use the same methods

Question 11

Which species use invagination during gastrulation?

Answer 11

Drosophila melanogaster (fruit fly).

Question 12

Describe the process of gastrulation in the fruit fly?

Answer 12

Fruit fly blastoderm is an oblong balloon of epithelial cells organised in a circle (transverse sections shown). Initially cells in the blastoderm are morphologically similar, but the ventral (bottom of balloon) cells are fated to become the mesoderm and the ones on the ends are fated to become the endoderm.
Cells become internalised inside the embryo via process of invagination. Consists of folding of epithelium along ventral portion (bottom) of the embryo so the batch of epithelium becomes internalised. They change shape, go from being straight columnar to being bottle shaped, and then continue folding and community effect leads to the process of invagination. Once they are internalised, they undergo epithelial-to-mesenchyme transition and they spread through the inside of the embryo to give rise to mesodermal and endodermal derivatives.

Question 13

Describe the process of gastrulation in the zebrafish?

Answer 13

Zebrafish at blastoderm stage. Blastoderm sits on top of the yolk cell, first step: spreading of the blastoderm cells around the yolk cell  epiboly. As epiboly is completed, the yolk sac will become completely engulfed in blastoderm. Halfway through epiboly, gastrulation begins. Cells at the edge of the blastoderm start to become internalised in the embryo by the process of involution. They will fold inside and continue moving in an opposite direction to the whole epiboly process is taking place. They will spread all underneath the cells that stay on the external side which are the epiblast cells. By the end of this whole process embryo cells have completely engulfed the yolk inside and they are already sub-divided in 2 layers, and the internal layer will go on to give rise to the mesoderm and the endoderm.

Question 14

Which species use ingression during gastrulation?

Answer 14

Chick gallus gallus and mouse mus musculus

Question 15

Describe the process of gastrulation in the xenopus?

Answer 15

Xenopus in involution. The animal pole cells of the frog blastula are going to spread to engulf the cells in the vegetal pole (green stuff) that are fated to become endoderm cells. As they engulf them, the structure appears in what will be the dorsal part of the embryo which is called the blastopore. In the blastopore there is going to be folding of the external group of cells in a similar way to what was just described in the zebrafish, by involution. The cells depicted in red will involute, moving in an opposite direction to the outside blue cells. By the end of the whole process the red cells which will give rise to the mesoderm and the green cells which will give rise to the endoderm have become completely engulfed by the cells which will become the ectoderm.

Question 16

Which species use involution during gastrulation?

Answer 16

Xenopus and Zebrafish

Question 17

Which species doesn’t undergo gastrulation in the typical sense?

Answer 17

Caenorhabditis elegans (roundworm)

Question 18

Simple comparison of embryos during gastrulation?

Answer 18

Even though their shape, organisation and morphology is looks quite different, in a way they are all very similar because they all have a dorsal-ventral, medial-lateral and anterior-posterior axis. In fish and from embryos they are bent ventrally (dorsal structures are on the outside) and the ventral structures are against the yolk. The chick is still very flat as the yolk is still comparatively very large, but the organisation is very similar in terms of axis, and the dorsal part is on the outside and the ventral part is against the yolk. Mouse: embryo is bent dorsally, the dorsal part is the ‘inside’ of the sort of cylinder, the inner sides which are bent against each other, and the ventral parts are looking to the outside. If all of the embryos were made flat, they would look very similar.

Question 19

Describe process of gastrulation in mice?

Answer 19

Epiblast cylinder, surrounded by primitive endoderm around the whole structure. Now stage at which gastrulation starts in the mouse. Ingression via primitive streak. The cells in the epiblast undergo ep-to-mes transition, along the primitive streak, whole process progresses anteriorly, the cells move away from primitive streak as they become internalised.

At the end of gastrulation the embryo still looks a bit like a cylinder but several cell types can already be distinguished. Subsequent inductive signals which are released by different tissues will eventually establish the anterior-posterior, medial-lateral and the dorsal-ventral axis.

Inside out by the end of gastrulation, need to turn inside out to get the dorsal part on the outside like the others. This happens by the twisting of the embryo, the posterior part twists around and moves towards the anterior, and eventually the embryo becomes organised with the anterior region position which was just minutes ago posterior. In the whole process of twisting, the embryo turns inside out and the outside will look out and the inside will face in. Similar thing happens in the human: silk purse folding model.

Question 20

Gastrulation essay introduction?

Answer 20

Gastrulation is the formation of a trilaminar embryo from a blastula, forming 3 distinct cell masses known as the endoderm, mesoderm and ectoderm. Although this isn’t true for all animal models e.g. Caenorhabditis elegans, the majority of species used for research today undergo gastrulation in this sense, including but not limited to zebrafish, xenopus, chick and mice. This process takes place as a chain of active cell rearrangements through movements categorised into involution, ingression and invagination.

Question 21

Which species don’t undergo EMT transition during gastrulation?

Answer 21

xenopus