Primary Cilla, signalling and development

Question 1

What main process are primary cilia involved in?

Answer 1

Cell signalling

Question 2

What are Axonemes?

Answer 2

Projections from cilia, project the surface of the basal body into the extracellular space and covered by a ciliary membrane which is an extension of the cell membrane with a particular molecular composition

Question 3

How are the motile cilia arranged?

Answer 3

The motile cilia have a 9+2 arrangement of microtubules. They also have accessory proteins that are needed for cilia motility. Some of these crosslink and others (pink) are motors (dynein) allow the movement of cilia

Question 4

How are the non-motile cilia arranged?

Answer 4

Non-motile primary cilia have a 9+0 arrangement of microtubules

Question 5

Name the 3 functions of motile cilia?

Answer 5

1. Cell movement: sperm (flagella)
2. Fluid movement: in airways, in the CNS, in the oviduct
3. Rotary cilia (found in the node of the developing embryo) : left-right asymmetry establishment during embryonic development. This gives rotational movement

Question 6

What does nexin do?

Answer 6

Nexin crosslinks the microtubules with each other

Question 7

What is the dynein?

Answer 7

The dynein (outer and inner) project from one pair of microtubules and come into contact with the adjacent pair of microtubules. It is there movement that leads to the sliding of the microtubules along each other. Because they are paired it produces a beating movement

Question 8

What is the function of primary cilia?

Answer 8

Sensory function such as smell and sight

Found in many receptors involved in signal perception

Wnt, notch and GPCRs

Question 9

How do cilia work in smell and sight?

Answer 9

In image 1 the olfactory neurones project these extensions into the lumen of the nasal cavity which are converted by cilia, the region where other receptors are present for other molecules.

In image 2 photoreceptors are the neurones of our eye that perceive light stimulus. The outer segment is the region that perceives light which is a modified cillum. It is called the connecting cilla and looks very similar to the primary cillum.

Question 10

What are the impacts of defects in primary cilia?

Answer 10

Cilia function is important for signalling and fluid movement in lots of tissues, therefore if we disrupt this process it can have a detrimental impact on embryonic development

Question 11

a

Answer 11

a

Question 12

Are cilia involved in establishment of left and right asymmetry?

Answer 12

Yes

Question 13

Describe the symmetry of humans

Answer 13

• Humans are not asymmetric externally, although we do have some subtle features
• Internally is arranged asymmetrically – normal arrangement is called situs solitus and flipping of internal organs is called situs inversus

Question 14

How does symmetry of the heart change?

Answer 14

• The heart will start as a symmetrical structure lying in the midline of the embryo with the aortic tree symmetric on both sides of the heart
• During development the heart will move towards the left hand side and the aortic arches will be pruned asymmetrically and differences arise on the left and right to give us the characteristics of the aortic arch

Question 15

How does symmetry of the gut change?

Answer 15

• The guy is a symmetrical structure lying in the midline of the embryo, during development there are characteristic rotations where the liver rotates to bring it round to the right hand side of the embryo. The pancreas and spleen will rotate towards the left

Question 16

How does symmetry of the lungs change?

Answer 16

• The lungs are also a symmetrical structure initially, the endodermal tube will form two buds which will grow differently to produce a 3 lobe on the right and 2 lobe lung on the left

Question 17

How is L-R asymmetry established in the mouse embryo?

Answer 17

Here we have in situ hybridisation images of the mouse embryos, these images are shortly after gastrulation, we can see the neural folding. We can also see the midline where somites will develop.

Left-right asymmetry is established by TGF-beta signalling (in this case a molecule called nodal). This is activated on the left side of the embryo. This pathway will then activate left y. This expression of molecules is transient which means it is downregulated a day or so later.

The TGF-beta signalling also activates a transcription factor called Pitx2 which is activated in the left lateral plate mesoderm and once activated will remain expressed at high levels on the left hand side of the embryo

Question 18

How is this left sided activation happening?

Answer 18

We will look at mouse development, shortly after the onset of gastrulation
Figure C – this is a transverse sagittal section through the mouse embryo, looking side on, with the future anterior side on the left and posterior side on the right. The bottom part is the embryo and the top is extraembryonic material. We can see the primitive streak, and in front of hendsons node there is the ventral node which is a transient embryonic structure which can be seen in B (orange cells)
A is a electron microscope view. VN is a group of specialised cells.
VN is involved with breaking symmetry within the embryo, it contains two different types of cilia: the conventional cilia (9+2) and the nodal cilia (primary cilia that can’t move) (9+0). The motile cilia are found within the middle part of the node and the immotile cilia are found on the outside. The moving Cilla will cause fluid to move and cause NVPs to move from right to left which encounters the immotile cilia activating signalling pathways in those cells.

Question 19

How does nodal flow cause the expression of Pitx2?

Answer 19

• The cilia all rotate in the same direction which means they are free to move when they are facing away from the cell and as they brush along the cell surface movement is impeded
• This causes a net leftward flow of fluid and particles
• This leftward flow induces other signals such as Nodal (will feedback on itself to maintain own expression) and also express lefty2 which will inhibit nodal expression and will activate the transcription factor Pitx2 on the left hand side
• Nodal will activate lefty1 in the midline of the embryo which is an antagonist of TGF-beta signalling (nodal) which prevents this left sided signal from spreading across to the right hand side of the embryo

TELLS THE EMBRYO ITS THE LEFT HAND SIDE

Question 20

How is Pitx2 a theme of LR asymmetry?

Answer 20

• Humans, mouse, fish, other mammals – nodal flow
• Chick, frogs, some mammals – no nodal flow
• Upstream effectors differ, but downstream responses are conserved that converge on the activation of pitx2
• Asymmetric ion-channel activation e.g chick and frog such being activated on one side of the embryo and not the other e.g sodium and protein pumps

Question 21

What prevents the rightward nodal signalling?

What does NVP cause in the non-motile cilia?

How do the cilla move in only one direction?

Answer 21

1. • Nodal will activate lefty1 in the midline of the embryo which is an antagonist of TGF-beta signalling (nodal) which prevents this left sided signal from spreading across to the right hand side of the embryo
2. Calcium influx which degrades TFs such as Cerl2
3. cilia rotate in one direction, when they brush the surface of the cell there movement is impeded