Lecture 3

Question 1

Why is it that with no sonic hedgehog from the prechordal mesoderm leads to cyclopia

Answer 1

The eye fields fail to split

Question 2

Knockout itself doesn’t provide direct evidence that you need sonic hedgehog to induce the hypothalamus. What experiments have been done that do prove this

Answer 2

By preventing sonic hedgehog coming from the prechordal mesoderm when the embryo is alive. This can be done by soaking a bead with a sonic hedgehog antagonist to see if you still get hypothalamic progenitors. Similarly experiments where you culture naïve neural plate tissue with the prechordal mesoderm to see if this tissue begins to express hypothalamic markers.

Question 3

What is significant about sonic hedgehog action in the ventral forebrain in terms of its ability to induce its own expression

Answer 3

Sonic hedgehog from the prechordal mesoderm in the ventral forebrain requires the additional Nodal signal in order to induce sonic hedgehog positive pre-hypothalamic progenitors. This is in contrast to the spinal cord where sonic hedgehog from the notochord alone is able to induce sonic hedgehog expression by the floor plate cells

Question 4

Sonic hedgehog from the prechordal mesoderm is necessary for hypothalamic development T or F

Answer 4

T

Question 5

It isn’t possible to carry gain of function studies with sonic hedgehog soaked beads in the forebrain because these beads wont be held in place. How else can we examine the sufficiency of sonic hedgehog alone in inducing the definitive hypothalamic progenitors

Answer 5

In vitro studies exposing neural tissue to sonic hedgehog. This doesn’t results in the expression of hypothalamic marks as nodal is absolutely required as well

Question 6

What is the effect of the action of nodal and sonic hedgehog signalling by the prechordal mesoderm

Answer 6

Induction of sonic hedgehog expression in pre-hypothalamic cells

Question 7

Nodal and sonic hedgehog from the prechordal mesoderm induce hypothalamic marker expression in the cells above it T or F

Answer 7

F – they only act to induce sonic hedgehog expression itself not the markers of hypothalamic progenitors

Question 8

What is the term given to the sonic hedgehog positive cells induced by sonic hedgehog and nodal from the prechordal mesoderm

Answer 8

Pre-hypothalamic cells

Question 9

If a cell it responding to sonic hedgehog signalling what sort of gene products would you expect it to be expressing

Answer 9

Ptc-1 and Gli-1

Question 10

The cells that are receiving the sonic hedgehog signal from the prechordal mesoderm are also then expressing it T or F

Answer 10

T

Question 11

Following induction of the pre-hypothalamic cell what rapid change(s) in expression are seen in the prechordal mesoderm

Answer 11

Immediately after Shh is induced and pre-hypothalamic cells appear BMP7 is rapidly upregulated in the prechordal mesoderm. Hence for a brief period Nodal and BMP7 are co-expressed in the prechordal mesoderm. This is unlike the floor plate of the spinal cord

Question 12

What is the combined effect of both Nodal and BMP7 expression by the prechordal mesoderm

Answer 12

These signals act together to induce the expression of FGF10 by the newly formed pre-hypothalamic cell

Question 13

What are the downstream targets as a result of BMP7 signalling in the pre-hypothalamic cell

Answer 13

BMP7 signalling leads to the phosphorylation of Smads1/5/8. pSmads1/5/8 then upregulate Tbx2 and Nkx2.1 in the sonic hedgehog positive pre-hypothalamic cells

Question 14

In what ways are the pre-hypothalamic cells alike the neural crest cells of the spinal cord

Answer 14

There are multipotent proliferative and migratory

Question 15

<p>How many different types of neurons are there in the hypothalamus</p>

Answer 15

<p>~5x10⁴</p>

Question 16

What is the role of FGF10 expression by the pre-hypothalamic cells

Answer 16

Supports survival and proliferation

Question 17

What is the role of Tbx2 expression by the pre-hypothalamic cells

Answer 17

Supports the ability of these cells to migrate and differentiate

Question 18

The tbx2 and FGF10 expression patterns in the pre-hypothalamic cells differs T or F

Answer 18

F – they are expressed in the same pattern

Question 19

How can we prove that FGF is required to drive proliferation in these cells

Answer 19

Use an FGF inhibitor such as SU5042 to block FGF signalling. Then look at markers of cell cycle to see the effects on proliferation

Question 20

What could you use to look at proliferation in cells

Answer 20

A marker of the cell cycle such as anti-pH3 which indicates M phase or EdU which can be used to indicate S phase

Question 21

How can we assay the effects of tbx2 on differentiation

Answer 21

Knockdown its expression via siRNA

Question 22

Once the prechordal mesoderm has established an early multipotent hypothalamic stem-like cell describe how this gives rise to the different regions of the early hypothalamus

Answer 22

Hypothalamic progenitors grow and differentiate first to anterior hypothalamic neurons then to posterior/mammillary hypothalamic neurons. Some FGF10 positive progenitors are always retained in the so-called tuberal hypothalamus. These cells eventually enter a very very slow cycle and are retained as stem-like cells

Question 23

Which signalling factors act to specify the hypothalamic stem like cell seen in the tuberal hypothalamus

Answer 23

Sonic hedgehog and BMP

Question 24

FGF10 positive cells in the hypothalamus will show strong staining indicative of proliferation T or F

Answer 24

T – this will be in the tuberal region

Question 25

The cells in the anterior hypothalamus will strain positive for EdU T or F

Answer 25

F – this is a marker of proliferation whereas the anterior hypothalamic cells will have begun to differentiate

Question 26

Sonic hedgehog drives the progression of FGF10 positive hypothalamic progenitors to an anterior neuronal fate T or F

Answer 26

T

Question 27

What would happen if we experimentally prevent sonic hedgehog signalling as soon as the hypothalamic progenitors form at the same time as injecting a dye to fate map the cells

Answer 27

After leaving the embryo to develop we would observe an accumulation of FGF10 positive progenitors as well as a failure of anterior progenitor formation and their subsequent failure to differentiate

Question 28

Sonic hedgehog is acting as a spatial morphogen in the early hypothalamus to direct the 3 cell fates depending on the concentration of the ligand the cells are exposed to T or F

Answer 28

F – sonic hedgehog is acting to select a differentiation pathway and drive the progression of FGF10 positive hypothalamic progenitor cells to anterior fates

Question 29

What transcription factor is upregulated as a result of sonic hedgehog signalling in the anterior hypothalamic progenitors

Answer 29

Rx2

Question 30

What neurons do the anterior hypothalamic progenitors ultimately give rise to

Answer 30

Neurons that mediate core body homeostasis

Question 31

What are the two main differences between induction of the ventral spinal cord and induction of the ventral forebrain in terms of the action of shh?

Answer 31

Sonic hedgehog from the prechordal mesoderm in the ventral forebrain requires the addition of a nodal signal to induce its own expression in pre-hypothalamic cells. Whereas in the spinal cord, sonic hedgehog alone from the notochord is capable of inducing its expression in the floor plate cells. Secondly, following induction of the pre-hypothalamic cells by the PM, it then rapidly upregulates BMP7. In contrast, sonic hedgehog from the notochord only induces its own expression in the floor plate cells of the spinal cord, BMPs are not expressed by the notochord but are restricted to dorsalised expression.

Question 32

<p>Below is a table containing a list of markers of forebrain structures. Decide which ones are expressed by each</p>

Answer 32

<p>See completed table below</p>

<p></p>

Question 33

What marker are the differentiated anterior and mammillary hypothalamic progenitors expressing?

Answer 33

Cyclin-depedent kinase inhibitor 1C (p57 or Kip2)