Lecture 11- Somite formation

Question 1

What is the site of gastrulation in chicks?

Answer 1

Henson’s Node

Question 2

In chicks, what is associated with the formation of the primitive streak?

Answer 2

A/P axis establishment

Question 3

What happens to cells over time during development?

Answer 3

They are progressively restricted and loose pluripotency

Question 4

What is formed as a result of gastrulation?

Answer 4

The mesoderm

Question 5

Describe the movement of cells during gastrulation

Answer 5

1. The epiblast cells migrate and converge towards the centre, roll over the edge of the primitive streak, involute and emerge on the inside as either mesoderm or prospective endoderm
2. During this process, the cells undergo an epithelial mesenchymal transitions (EMT)
3. Once they reside inside they will migrate laterally within the embryo

Question 6

What was identified from injecting fluorescent due along the primitive streak (fate mapping)?

Answer 6

Identified that cells were specified to various regions of the mesoderm depending on where they engraved within the primitive streak along the A/P axis

Question 7

What type of tissue do cells become within Henson’s node?

Answer 7

Axial mesoderm which forms the prechordal mesoderm and notochord

Question 8

What type of tissues do cells become slightly posterior to Henson’s node?

Answer 8

Paraxial mesoderm which give rise to somites

Question 9

What type of tissues do cells become very posterior to Henson’s node?

Answer 9

Intermediate mesoderm which form the kidneys and gonads

Question 10

What type of tissues do cells become at the most posterior end of the primitive streak?

Answer 10

Lateral mesoderm

Question 11

What provides the earliest evidence for segmentation in vertebrates?

Answer 11

Somites

Question 12

What are somites?

Answer 12

Transient structures that are laid down during embryonic development along the developing spinal cord

Question 13

What gene expression illustrates how well segmentation is evolutionarily conserved?

Answer 13

The repeating expression pattern of engrailed 1

Question 14

What dictates the number of vertebrae?

Answer 14

Somite number

Question 15

Explain how somites form in pair from the paraxial mesoderm

Answer 15

• Paraxial mesoderm forms in a continuous manner until the proper somite number is reached
• Primitive streak is the site of mesoderm formation and is to be present until somites no longer form
• As long as the primitive streak is present, cells will exit the primitive streak by forming and adding new mesodermal cells through the posterior part of the embryo
• This process leads to an elongation that proceeds from the anterior to the posterior end of the embryo
• In the anterior part of the mesoderm, segmentation occurs from the most anterior end to the more posterior end so that the anterior part of the mesoderm is the location for the most newly formed somites

Question 16

How does the number and timing of somites vary within a species>

Answer 16

The number of somites is fixed for a given species and the timing of somite formation remains constant within the species

Question 17

How do somites form from the pre-somatic mesoderm?

Answer 17

Somites bud off a band of unsegmented mesoderm

Question 18

What must cells within the pre-somatic mesoderm respond to?

Answer 18

1. Positional information
2. Mechanisms that coordinates left and right somites (for every somite on the right, there is a somite forming on its left)
3. Mechanisms that generates anterior boundary
4. Mechanisms that generate posterior boundary
5. Formation of cleft

Question 19

What model explains how the periodicity of somite formation is established?

Answer 19

The clock and wavefront model

Question 20

Briefly explain the clock and wavefront model

Answer 20

Predicts that a clock ticks in the posterior pre-somatic mesoderm and drives a molecular oscillator that dictates the periodicity and size of somites

Where cells hit the travelling wavefront, an abrupt change of property occurs leading to the decision to form somites

Question 21

What is c-hairy1 in fish and mice?

Answer 21

Fish = Her
Mice = Hes

Question 22

How was c-hairy1 identified?

Answer 22

Insitu hybridisation

Question 23

What conclusions were made regarding the expression pattern of c-hairy1?

Answer 23

Expression pattern is not continuous but instead, oscillates over time

Question 24

What are 3 characteristics of hairy/hes/her?

Answer 24

1. Are target genes for notch signalling
2. Are bLHL transcriptional repressors
3. Are very unstable

Question 25

Describe how the hairy forms an oscillatory mRNA and protein pattern

Answer 25

1. Notch induces the transcriptional activation of the gene 2. When notch is present, the gene is transcribed and the formation of mRNA (level of mRNA increases) 3. Following mRNA transcription, Hairy/Hes/Her protein is made (level of protein increases) 4. Hairy/Hes/Her protein is unstable so gets ubiquitinated and results in proteasomal degradation (level of protein decreases) 5. Hairy/Hes/Her protein are transcriptional repressors so are capable of repressing their own transcription (decreasing the level of mRNA) 6. This cycle is repeated and therefore leads to the oscillatory pattern of mRNA followed by the oscillatory pattern of the protein

Question 26

How many oscillation cycles do presomatic cells undergo before forming somites?

Answer 26

12 rounds

Question 27

What do 12 newly formed somites form in coordination with?

Answer 27

The continuous growth of the mesoderm in the posterior part of the embryo So cells are not migrating but somites are forming and the posterior part of the embryo gets pushed even more posteriorly

Question 28

At what point does oscillation stop?

Answer 28

When cells encounter the wavefront travelling in the opposite direction in the anterior part of the presomitic mesoderm, oscillations stop

Question 29

What happens to cells when they encounter the wavefront?

Answer 29

The unspecified cells become committed to form somites and oscillation slows down

Question 30

At what point doe the wavefront become the determination front?

Answer 30

Soon after the cells meet the wavefront and are committed to form somites

Question 31

Explain the 2 opposing gradients that form the determination front?

Answer 31

1. High levels of FGF8 in the most posterior part of the mesoderm with levels that decrease more anteriorly 2. The second antagonising gradient emanates at the anterior at the region of the somites and reduces posteriorly towards the presomitic mesoderm and is made of retinoic acid

Question 32

Explain how negative feedback maintains RA and FGF8 gradients

Answer 32

* FGF8 controls the expression of proteins which are responsible for shaping the gradients of RA (e.g Cyp26) * Cyp26 is a negative regulator of RA formation * FGF8 also blocks the transcription of Raldh2 which is an essential protein for RA synthesis * Therefore, where there are high levels of FGF8, there will be low levels of RA * Conversely, RA blocks the transcription of FGF8 so where there are high levels of RA, there are low levels of FGF8 transcription

Question 33

How is the determination front established molecularly?

Answer 33

* FGF8 is important for the transcription of Tbx6 (a transcription factor which is essential for the specification of the paraxial mesoderm * Tbx6 together with notch signalling controls the expression of a protein called Mesp2 * Mesp2 is precisely expressed at the level of the determination front

Question 34

How do boundaries between somites form?

Answer 34

* Boundary cells can induce somite boundary formation * Boundary cells instruct cells that are anterior to form a boundary * Posterior boundary cells are capable of instructing cells posterior to them to form a boundary

Question 35

What experiment proved that boundary cells formed boundaries?

Answer 35

Transplantation of a prospective somite boundary region into non-boundary region creates a new boundary

Question 36

What genes are expressed at the somite boundary and therefore involved in the formation?

Answer 36

Notch family genes

Question 37

How was it experimentally prove that notch signalling is involved in somite boundary formation

Answer 37

1. In gain of function experiments, scientists used lunatic fringe (involved in downstream notch signalling) and electroporated this inside the somite in a region that would not normally form a boundary 2. Found that modifying levels of notch signalling was sufficient to lead the formation of a boundary, suggesting the instruction came from varying levels of notch signalling 3. Somite derivatives are affected in the absence of notch signalling

Question 38

What are the consequences of mutation in delta/notch at the somite boundary?

Answer 38

* Defects associated with the segmentation of the axial skeleton (often ossification centres do not align) and in particular mutations in delta-like 3 * In humans, a similar mutation causes spondylocostal dysplasia (Jarcho Lewin Syndrome)

Question 39

How is the somite cleft formed?

Answer 39

* Largely dictated by the control of notch signalling on the expression of ephrins * Ephrins cause cell adhesion changes and ultimately leads to the formation of somites