Lecture 4 - Dorsal ventral patterning of the neural tube

Question 1

How are avian embryos more beneficial to use in developmental experiments than mammal embryos?

Answer 1

• they are very similar in morphology and embryogenesis to mammals
• but they are much easier to observe and manipulate experimentally
• can be manipulated in culture or in ovo

Question 2

When does gastrula occur in the avian embryos

Answer 2

After the primitive streak stages

Question 3

How can the early mesoderm and the notochord be viewed in gastrulation in the avian embryo?

Answer 3

By marking it with brachyury

Question 4

How does gastrulation proceed in the avian embryo?

Answer 4

The node regresses posteriorly, and the notochord and the somites form anterior to the node

Question 5

What type of development do avian embryos show in neurulation/gastrulation?

Answer 5

Rostral claudal progression of development

Question 6

Outline the Rostral claudal progression of development in avian embryos?

Answer 6

• After the notochord forms, the neural plate induced from the overlying ectoderm
• the anterior part of the neural tube folds, rolls and forms a tube first, then neural tube closure proceeds in a head to tail (rostral claudal) direction
• somites, which give rise to skeletal muscle, bone and dermis, also form in a rostral claudal manner

Question 7

How is avian embryo development unlike frog development?

Answer 7

In frog development, the neural tube forms all at once, in avian embryos the anterior part of the neural tube folds, rolls and forms a tube first, then neural tube closure proceeds in a head to tail (rostral claudal) direction
-somites, which give rise to skeletal muscle, bone and dermis, also form in a rostral claudal manner

Question 8

What is the effect of the rostral claudal progression of development in avian embryos?

Answer 8

The anterior part of the embryo is developmentally more advanced than the posterior
-in the trunk region, somites will have formed and neurulation will be underway but in the posterior region behind Hensons node, the notochord and somites will not yet have formed

Question 9

How is the neural tube patterned along the AP axis?

Answer 9

• by signals from the dorsal mesoderm and the posterior mesoderm
• Hox genes provide a molecular basis for the positional identity along the AP axis of the neural tube

Question 10

Where is the effect of Hox genes patterning the neural tube particularly clear?

Answer 10

-in the hindbrain, where distinct hox genes have definitive anterior boundaries of expression that correspond to rhombomere boundaries

Question 11

What experiments have demonstrated the important of hox genes in coding the positional identity along the AP axis?

Answer 11

Posterior prevelence of Ubx, AbdA, AbdB

Bithorax mutant

Question 12

What is the dorsal ventral patterning of the neural tube from the dorsal side?

Answer 12

Sensory neurons
Interneurons
Motor neurons
Floor plate
Notochord

Question 13

When is the pattern of regional cell identity apparent along the DV axis during neurulation?

Answer 13

As the neural plate rolls up to form the neural tube, a pattern of regional cell identity becomes apparent in the DV axis

Question 14

What is the pattern of regional cell identity in the DV axis of the neural tube?

Answer 14

Where distinct populations of progenitor cells give rise to post mitotic neurons with distinct functional properties

• cells in the dorsal region of the neural tube generate sensory neurons
• cells in the ventral region of the neural tube generate motor neurons that coordinate motor input
• patterning underlies functioning neural circuits

Question 15

How do the pattern of cells along the DV axis of the neural tube form in respect to the ventral midline?

Answer 15

Distinct cells form in a bilaterally symmetrical way with respoect to the ventral midline

• At the ventral midline is the floorplate
• Motor neurons form ventrolaterally
• Sensory neurons and the neural crest form dosally
• Roofplate forms at the dorsal midline

Question 16

What are the genes that are specifically expressed in different neural progenitor populations in the neural plate and where?

Answer 16

Dorsally to Ventrally

V1, V2, MN, V3

Question 17

What are the features of the genes that are specifically expressed in different neural progenitor populations of the neural tube?

Answer 17

• code for TF
• cross regulate each others’ expression boundaries
• define the specific neural progenitor populations

Question 18

What technique can be used to identify structures like the floorplate and MN?

Answer 18

Immunostaining

-Ab recognise particular proteins specific to particular cells of the floorplate/motor neurons/notochord

Question 19

What did the experiment where an extra notochord was added to a developing neural tube structure show?

Answer 19

-Add extra notochord, resulted in two floorplates induced with motor neurons
-If put notochord in a different place, get motor neurons in that vicinity (ectopic location)
SHOWS:
that the underlying dorsal mesoderm structure (the notochord) induces floorplate and is important in patterining other cell types along the DV axis of the NT (the motor neurons)

Question 20

Aside from the notochord, what else was found to induce floorplate when it was ectopically inserted?

Answer 20

• Floorplate itself was found to induce floorplate

- Removal of the notochord prevents floorplate differentiation

Question 21

What did experiments with intermediatenerual plate show?

Answer 21

• Using explants of intermediate neural plate that would not normally give rise to motor neurons and combining this tissue with notochord, floorplate or floorplate induced media led to the expression of motor neurons
• this suggests that MN inducer is diffusible

Question 22

What is islet1 and how was it used in experiments on intermediate neural plate?

Answer 22

• islet1 is an antibody that recognises only motor neurons

- detects motor neurons induction

Question 23

What did experiments on the intermediate neural plate and the ectopic expression of floorplate and notochord illustrate?

Answer 23

That the notochord and the floorplate have an instructive role in establishing the identify and position of cell types along the DV axis of the NT

Question 24

What is Sonic Hedgehog?

Answer 24

• drosophila sonic hedgehog is a signalling molecule
• member of the family of hedgehog genes
• involved in organising pattern within the parasegment of the developing embryo
• functions later to develop the patterning of the limbs and the eye of the fly

Question 25

How does shh show conservation between vertebrates and flys?

Answer 25

• as well as being involved in patterning in the drosophila embryo
• all of the vertebrate shh have been shown to be expressed in the notochord and the floorplate

Question 26

What is the evidence that shh can act as a floorplate inducer?

Answer 26

1. Injection of shh mRNA into fly embryos leads to ectopic floorplate
2. COS cells expressing shh can induces NP explants to express the floorplate marker F3

Question 27

What are the ligands, receptors and effectors of shh signalling?

Answer 27

Ligands: shh
Receptors: Ptc/Smo
Effectors: Gli acitvator/repressor of transcription

Question 28

What genes did mutagenesis screens reveal in the hh pathway?

Answer 28

Patched (ptc)
Smoothened (smo)
Cubitus interrupitus (ci)

Question 29

What is the process of signalling via shh?

Answer 29

• in the absence of hh, patched represses smo and the multiprotein complex associated with microbtubules casues Ci to be processed into a smaller form called Ci75 which represses hh target genes
• hh signalling results in the stabilisation of a full length Ci which can move to the nucleus and function as a transcriptional activator

Question 30

What are the vertebrate homologues of cubitus interrupitus in vertebrates?

Answer 30

Gli genes

Question 31

Outline the french flag model of how morphogens act

Answer 31

• signal source at one end of the array of cells (each of which has the potential to develop into different types)
• diffusion of the signal establishes a gradient of morphogen
• morphogen gradient allows cells to aquire positional information about where they are related to the source
• cell respond approprately to the serial threshhold of morphogen concentration
• the concentration gradient could be interpreted in many ways, could generate different patterns or more threshold responses

Question 32

What evidence is there to support the idea that shh is a morphogen (it’s not, by the way, next lecture)

Answer 32

• in the ventral neural tube, motor neurons and interneurons are generated by the graded activity of shh
• cultured explants respond to different concentrations of shh protein; form more ventral neurons in reponse to high concentrations and more dorsal neurons in reponse to lower levels of shh
• which reflects their in vivo position along the DV axis of the neural tube

Question 33

Outline the model for the role of graded shh signalling in the establishment of ventral cell fates in the vertebrate spinal cord

Answer 33

• graded shh signalling establishes the ventral expression domains of a number of homeodomain transcriptional regulators in neural progentior population
• distinct boundaries are established on the basis of shh mediated repression (class I) or induction (class II) at different concentration thresholds

Question 34

What is the lateral motor column?

Answer 34

A continuous medial motor column spanning all rostroclaudal (AP) levels and contains motor neurons projecting to the axial muscles

Question 35

Where does the lateral motor column develop?

Answer 35

• develops at precise positions along the AP axis.
• spans the brachial and lumbar levels of the spinal cord but only where forelimbs and hindlimbs develop (as a result of a specific hox code)

Question 36

What does the lateral motor column contain?

Answer 36

the limb-innervating motor neurons

Question 37

How is the lateral motor column further divided?

Question 38

What is motor neuron specification down the AP axis of the neural tube dependent upon?

Answer 38

the AP expression of hox genes

• where HoxC6 is highest, the pool of motor neuron progenitors are specified as LMC (Hox C6 is highest in the brachial region)
• HoxC9 expression is highest in the thoracic region where another type of motor neuron will develop.