Lecture 6

Question 1

What are the two daughter cells produced by the division of the slowly dividing stem-like hypothalamic progenitor

Answer 1

One identical slowly dividing stem-like daughter and one rapidly dividing transit amplifying cell

Question 2

Describe how the different regions of the hypothalamus form

Answer 2

The initial FGF10 positive sonic hedgehog positive hypothalamic progenitors divide asymmetrically. The daughters that are displaced laterally are no longer lying above the prechordal mesoderm and thus are exposed to different signals. This causes their differentiation first to anterior hypothalamic neurons and then to posterior or mammillary neurons. The retained stem-like cells make up the tuberal region of the hypothalamus

Question 3

What happens if you knockout sonic hedgehog as the hypothalamic progenitors have formed

Answer 3

This leads to an accumulation of FGF10 positive hypothalamic progenitors as well as a failure of the anterior progenitors to form. This subsequently prevents the anterior hypothalamic neurons from developing

Question 4

If a temporal knockout of sonic hedgehog after formation leads to the failure to form differentiated anterior hypothalamic neurons what can be deduced about the role it is playing

Answer 4

Sonic hedgehog must be acting to direct the progression of the stem-like cells to differentiated (anterior) neurons

Question 5

How could you visualise the effects of sonic hedgehog knockout at the stage where the hypothalamic progenitors have formed

Answer 5

Inject a lipophilic dye such as DiI and carry out lineage tracing on the FGF10 positive progenitors in wild type and sonic hedgehog -/- embryos. Look to see the differences in the localisation of the dye after allowing the embryos to develop further

Question 6

What is the role of the early prechordal mesoderm in the formation of the pre-hypothalamic progenitors

Answer 6

The early prechordal mesoderm is expressing sonic hedgehog and nodal. These act on the cells directly above the prechordal mesoderm and cause a transcriptional upregulation of GliA and SOX2 in those cells. GliA and SOX2 work to subsequently transcriptionally upregulate sonic hedgehog in these cells causing a change in fate to the pre-hypothalamic progenitor.

Question 7

Once the pre-hypothalamic progenitors have formed how does the late prechordal mesoderm act to change the expression in these cells once more

Answer 7

The prechordal mesoderm begins to upregulate BMP7 and downregulate sonic hedgehog. The prechordal mesoderm is now co-expressing Nodal and BMP7. BMP7 acts on these sonic hedgehog positive pre-hypothalamic progenitors to induce the expression of Tbx2. Tbx2 displaces SOX2 from the sonic hedgehog enhanced in the pre-hypothalamic progenitors leading to a subsequent downregulation of sonic hedgehog in those cells.

Question 8

Fate mapping studies have shown that the FGF10 positive pre-hypothalamic progenitors above the prechordal mesoderm will give rise to the entire hypothalamus T or F

Answer 8

T

Question 9

Fate mapping experiments haven’t proved that the prospective hypothalamic cell induced by the late prechordal mesoderm is a multipotent cell why is this

Answer 9

It doesn’t prove that the cell is intrinsically able to self-renew and give rise to multiple cell fates as extrinsic factors from other areas of the developing embryo could be causing this

Question 10

Describe an experiment used to determine whether the FGF10 positive pre-hypothalamic cells are multipotent stem-like cells

Answer 10

Cut out the neuroepithelium from chick embryos at a stage in which the pre-hypothalamic progenitors are forming. Isolate the tissue from above the prechordal mesoderm verifying you have the correct tissue by carrying out in situ hybridisation for hypothalamic and telencephalic markers. If the correct tissue was obtained it will stain positive for hypothalamic markers and negative for telencephalic markers. Then culture this explanted tissue in a collaged matrix to allow the cells to grow and differentiate. After several days carry out serial adjacent sections in the tissue and use in situ hybridisation or immunohistochemistry for the various markers of the different regions of the hypothalamus. From this you can determine if the FGF10 positive pre-hypothalamic progenitors have given rise to differentiated anterior and mammillary hypothalamic neurons whilst also retaining some stem-like cells in the tuberal region.

Question 11

What marker can be used to test for anterior hypothalamic neurons

Answer 11

Islet1

Question 12

What marker can be used to indicate posterior mammillary hypothalamic neurons

Answer 12

Emx2

Question 13

Give an example of a telencephalic marker

Answer 13

Foxg1

Question 14

Which marker is expressed by both anterior and tuberal hypothalamic progenitors

Answer 14

Six3

Question 15

Explant from the cells above the prechordal mesoderm that have been allowed to grow in a collagen matrix will stain positive for each of the three hypothalamic regions what does this tell you about these cells

Answer 15

They can give rise to different types of neurons. This information is intrinsic and doesn’t require any extrinsic signals.

Question 16

By culturing explants from the cells above the prechordal mesoderm that have been seen to stain positive for each of the three hypothalamic regions doesn’t tell you that the cells are multipotent why is this

Answer 16

This is a group of cells and could contain different progenitors that give rise to the neurons of the different regions of the hypothalamus. You would need to carry out an experiment culturing a single cell from this population to determine multipotency

Question 17

Describe the results of long-term fate mapping experiments of the FGF10 positive per-hypothalamic progenitors and what this means in relation to their character

Answer 17

These cells retain some of the DiI label used in the fate mapping and are hence known as label retaining cells. This means that they are very slowly dividing this is typical of a stem-like cell

Question 18

Describe the current model for hypothalamic development

Answer 18

FGF10 positive pre-hypothalamic progenitor cells give rise to a transit amplifying anterior progenitor which is displaced anteriorly. This TAP continues to give rise to subsequent anterior progenitors. The first set of progenitors are most displaced and the first to differentiate to neurons. The FGF10 positive cell then gives rise to posterior progenitors next which move away and differentiate too. The FGF10 positive (hypothalamic progenitor) is a hypothalamic stem cell and as it downregulates sonic hedgehog is retained as a slowly dividing stem cell.

Question 19

All hypothalamic neurons originate from an FGF10 positive hypothalamic stem-like cell that goes via sonic hedgehog positive TAP cell to commit to a particular identity T or F

Answer 19

T

Question 20

Describe the differences in signals received as the cells above the prechordal mesoderm divide

Answer 20

Cells above prechordal mesoderm are briefly co-expressing Sonic hedgehog FGF10 pSmad1/5/8 BMP7 and Tbx2. These cells then divide asymmetrically to give two daughters. One daughter is displaced from the prechordal mesoderm is no longer lying above the prechordal mesoderm and hence is exposed to different signals. The daughter that is retained is going to be exposed to higher levels of BMP than the one displaced laterally and as such it has a greater downregulation of sonic hedgehog. This downregulation of sonic hedgehog means that the daughter displaced laterally sees increased levels of sonic hedgehog signalling because of the effects of LDA. This cell will hence begin to upregulate sonic hedgehog and will become the rapidly dividing TAP cell. The daughter that remains above the prechordal mesoderm remains as a slowly-dividing stem cell

Question 21

How is it thought that the number of transit amplifying cells differentiated hypothalamic neurons and retained stem-like cells are regulated

Answer 21

Feedback loops between the differentiation and stem-like cells

Question 22

What can be said about the effects of sonic hedgehog signalling on gene transcription that indicates its role in direct these rapidly dividing TAP cells

Answer 22

Sonic hedgehog signalling often directly activates the transcription of genes that regulate the cell cycle. For example Cyclin D1 Myc Bcl-2 (anti-apoptotic) NANOG and SOX2

Question 23

Sonic hedgehog signalling often acts alone to dictate behaviour of cells T or F

Answer 23

F – it is more often the integration of multiple signalling events that dictates behaviour. Shh signalling integrates with multiple other signalling pathways (Notch Wnt) to govern cell proliferations stem cell renewal and transit amplifying progenitors

Question 24

What are the three actions of sonic hedgehog in the telencephalon

Answer 24

Spatial morphogen that is involved in patterning promotion of the cell cycle and progenitor expansion and the maintenance of Nkx2.1 expression

Question 25

Outline the roles of sonic hedgehog in the hypothalamus

Answer 25

Firstly sonic hedgehog from the early prechordal mesoderm induces the pre-hypothalamic cell. Later on sonic hedgehog acts to select anterior progenitors and then acts to amplify these progenitors before finally playing a role in the differentiation of these anterior progenitors to anterior neurons.