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

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


Describe how the different regions of the hypothalamus form

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


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

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


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

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


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

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


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

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.


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

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.


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



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

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


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

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.


What marker can be used to test for anterior hypothalamic neurons



What marker can be used to indicate posterior mammillary hypothalamic neurons



Give an example of a telencephalic marker



Which marker is expressed by both anterior and tuberal hypothalamic progenitors



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

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


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

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


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

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


Describe the current model for hypothalamic development

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.


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



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

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


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

Feedback loops between the differentiation and stem-like cells


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

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


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

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


What are the three actions of sonic hedgehog in the telencephalon

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


Outline the roles of sonic hedgehog in the hypothalamus

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.