Lecture 13 - Mesoderm segregation

Question 1

What does mesoderm segmentation lead to?

Answer 1

establishment of the A/P axis in the chick

Question 2

How does mesoderm segmentation lead to A/P patterning?

Answer 2

Line runs along A/P axis (more like a groove) - primitive streak - where gastrulation occurs. Arises initially at the caudal part of the embryo & extends anteriorly, until it reaches the Hensen’s node. Extension is the physical establishment of the A/P axis & site where gastrulation take place. The primitive streak & gastrulation are important in mesoderm development

Question 3

How does the mesoderm form at the primitive streak during gastrulation?

Answer 3

During gastrulation, cells from epiblast (early embryo) migrate centrally towards primitive streak. They migrate towards the primitive streak & migrate down into the groove. They then populate this space & form different cell types & form different layers of embryo.

Question 4

What happens to cells that migrate?

Answer 4

• Cells that migrate deepest become the endoderm (digestive organs)
• Cells that migrate deep & laterally become mesoderm. This means mesoderm is a germ layer that emerges during gastrulation.
• Mesoderms will not become equal & will become different structures residing in different parts of the embryo

Question 5

What are different parts of mesodermal tissues?

Answer 5

• Axial mesoderm
• Paraxial mesoderm
• Intermediate mesoderm
• Lateral mesoderm

Question 6

What determines which mesodermal region a cell will be?

Answer 6

a geographical area in which they migrate.

Question 7

What forms axial mesoderm?

Answer 7

If the cells enter primitive streak close to Hensen’s node, they will form the axial mesoderm. This gives rise anteriorly to the precaudal mesoderm, which will form the anterior CNS. Posteriorly - contributes to the notochord (signalling functions). Called ‘axial’, because it lies in the middle of the embryo.

Question 8

What forms paraxial mesoderm?

Answer 8

Paraxial mesoderm emerges from cells that migrate more posteriorly to axial mesoderm. They lie behind the axial mesoderm. We know this happens through lineages tracing experiments - dye was placed in cells & look where the cells ended up after gastrulation.

Question 9

What forms intermediate mesoderm?

Answer 9

slightly more posterior & lateral to paraxial

Question 10

What forms lateral mesoderm

Answer 10

posterior to intermediate mesoderm

Question 11

What develops from the axial mesoderm?

Answer 11

Notochord & pre-chordal mesoderm

Question 12

What develops form the paraxial (somitic) mesoderm?

Answer 12

Head & Somite (somites are small transient structures - only embryonic structures

Question 13

What does intermediate mesoderm give rise to?

Answer 13

runs along A/P axis & gives rise to kidneys & gonads

Question 14

What does lateral plate mesoderm give rise to?

Answer 14

Extra embryonic tissues & tissues within the limb, & contribute to the heart

Question 15

What is the earliest evidence of segmentation in vertebrates?

Answer 15

Somites

Question 16

What are somites segments of?

Answer 16

Somites are segments of paraxial mesoderm.

Ball shapes - are somites or segmented paraxial mesoderm. Paraxial mesoderm is more segmented as you more anteriorly. This segment occurs in a posterior to anterior manner.

Question 17

What are somites?

Answer 17

Somites are highly organized & have a distinctive structures - they are an epithelium. They are approximately the same number of cells.

There are clefs that separate the epithelial structures from each other & highly repeatable.

Question 18

How does Engrailed1 influence mesoderm segmentation?

Answer 18

Engrailed is a TF & expressed in the somites. Segmentation of somatogenesis is a highly conserved process - usually meaning it is really important. Segmentation is used throughout evolution, as it a really convenient way to maintain similar process & make repeated structures within a developing embryo, but also allows to make subtle changes in different regions of the A/P axis, if you wanted to take subtly different structures to take different positions.

Question 19

What does somite number dictate?

Answer 19

Somite number dictates the number of vertebrae.

Humans are born with 33 vertebrate - by adulthood, they have 24 vertebrae & 9 fused ones.

The human embryo has 38-45 somites, while the chick has 55, the mouse 65 & the zebrafish 33.

Question 20

What is the spine made up of?

Answer 20

A number of vertebrae - a repeated structure made along A/P axis, but vertebrae aren’t all the same. Cervical is different from lumbar.

Different species have different number & different proportions of vertebrae - but tight correlation between number of somites & number of vertebrae formed.

Some species-specific differences - in terms of number of vertebrae but also in development of vertebrae.

Question 21

How do somites form in pairs from paraxial mesoderm?

Answer 21

• paraxial mesoderm forms in a continuous manner, until the proper somite number is reached.
• the number of somites is fixed for a given species & the timing of somite formation remains constant within the species
• primitive streak is to be present until somites no longer form.
• presomitic mesoderm, although not segmented, pre-figures the future segmentation of somites

Question 22

How does elongation occur?

Answer 22

through addition to the posterior
- cell migration into the primitive streak, which happens at the posterior part of embryo, resulting in posterior elongation - addition of new paraxial mesoderm as a result of gastrulation occuring.

Question 23

What is pre-somitic mesoderm?

Answer 23

12 somites worth of tissue, before segmentation occurs in the anterior most region. Segmentation occurs in the anterior part - and a new pair of somites emerge - every 90 mins in chick. They have the same side - A/P boundary & a cleft in between them & are a repeated unit.

Question 24

How to explain the periodicity & reproducibility of somite formation?

Answer 24

Cells within pre-somitic mesoderm must respond to:
- positional information
- mechanism that coordinates left & right somites
- mechanism that generates anterior boundary
- mechanism that generates posterior boundary
- formation of cleft

Question 25

How is the periodicity of somite formation established?

Answer 25

The clock & wavefront model Predicts that a 'clock' ticks in the posterior pre-somitic mesoderm & drives a molecular oscillar that dictates the periodicity of somites. Where cells hit the travelling wavefront, an abrupt change of property occurs leading to the decision to form somites Said that clock starts in posterior, bit wavefront comes from anterior. Wavefront moves in opposite direction. Said that when these meet, then somites need to change from pre-somitic mesoderm into somite. 0-60 mins going anteriorly, but back to posterior by 90 mins. High/low expression of gene correlates with how long it takes to create somite. (C-Hairy expression)

Question 26

Describe Hairy/Hes/Her proteins

Answer 26

Hairy/Hes/Her protein half-life & negative feedback loop is critical for the oscillatory pattern of its mRNA -Hairy/Hes/Her are target genes of Notch signalling - Hairy/Hes/Her proteins are bHLH transcriptional repressors. mRNA isn't stable & has a short half-life. This protein is able to repress its own expression.

Question 27

How do presomitic mesoderm cells form somites?

Answer 27

Presomitic mesoderm cells undergo 12 cycles of oscillations before forming somites

Question 28

What does the position S-1 (pre-somitic region just before somite) mark?

Answer 28

The position S-1 marks the specification of boundary formation - determination front. When cells encounter the wavefront travelling in opposite direction, oscillations stop When cells encounter the determination front, the oscillations stop.

Question 29

Where is the determination front positioned?

Answer 29

At the interface of 2 opposing gradients RA (retinoic acid) & FGF8 determine the position of the wavefront. RA is produced by the developing neural tubes, as well as the somites already produced - diffuses from anterior to posterior. FGF8 produced in the posterior & diffuses anterior. Intersection of the 2 becomes the determination front - both found in low concentration

Question 30

What maintains the reverse relationship between RA & FGF8 gradients

Answer 30

negative feedback regulations

Question 31

What is FGF8 & Notch signalling required for?

Answer 31

Mesp2 expression (Notch signalling is oscillatory) Mesp2 is only transiently expressed - first in S1 region & blocks Dll, which is required for Notch activity. It also activates Ripply2, forming a feedback loop by inhibiting its own expression. S-1 region has lower levels of Notch signalling, next to low levels of Notch signalling, forming a boundary between formed somite & somite that is going to be formed.

Question 32

How can boundary cells induce somite boundary formation?

Answer 32

- Transplantation of a prospective somite boundary region that allow them to make a boundary - Boundary cells instruct cells that are anterior to form a boundary Cells located in anterior of S-1 region have specific properties that allow them to make a boundary. Took quail cells from anterior part of mesoderm & transplanted it into the middle S-1 region of a chick - boundary shouldn't be forming. Saw that cells found themselves at another anterior boundary - S-1 cells therefore determine where the boundary is.

Question 33

Where are Notch family genes expressed?

Answer 33

Notch family genes are expressed at the somite boundary Gene expression of different Notch components - suggested that levels of Notch activity between A/P boundaries driving change.

Question 34

How is Notch signalling involved in somite boundary formation?

Answer 34

Cells taken from middle region & transfect those cells with constitutively active notch. This led to a new somite boundary being made. Therefore, it is Notch signalling that determines where a boundary is formed.

Question 35

What is the role of Mesp2 in linking A/P identity to boundary formation?

Answer 35

Mesp2 induces the expression of ephA4 in the anterior part of the cell. When ephrin & eph interact, they induces transcriptional changes, which is going to change adhesion properties, conversion from mesenchymal to epithelial cells & induce mechanical changes, which are going to change the shape of that tissue. The equilibrium in gene expression between A/P boundary, induces expression of ligand/receptor pairs, which send a whole set of signals to the cells on each side of the boundary, saying we should disengage from each other, become an epithelium & become a somite.

Question 36

What is somitogenesis?

Answer 36

epithelialization of somites

Question 37

What is the determination front?

Answer 37

Forms at the interface of 2 opposing gradients

Question 38

What is segmentation?

Answer 38

Oscillation slows down as cells mature. Acquisition of anterior & posterior identity Notch1 & D11 in posterior Notch2 & D13 in anterior

Question 39

What is the onset of oscillator?

Answer 39

Activation of c-Hairy1 & Lunatic Fringe

Question 40

What is gastrulation?

Answer 40

Acquisition of paraxial mesoderm identity (BMP activity)