lecture 1/2

Question 1

How was Dil injection used to investigate fate mapping of the prechordal mesoderm

Answer 1

Inject Dil into cells that lie above the prechordal mesoderm in an early embryonic stage and allow for further development until distinct forebrain regions can be identified.

Question 2

What do cells that lie above the prechordal mesoderm give rise to?

Answer 2

Hypothalamus, thalamus, ventral telencephalon

Question 3

How were early markers of the nascent hypothalamus discovered

Answer 3

Analyse expression of known signals/TFs which can be found above the underlying PM - FGF10, BMP7, Nkx2.1, Tbx2 and pSmad1/5/8 all show expression over the PM

Question 4

What effect does ablation of the PM have on the markers of the nascent hypothalamus

Answer 4

Fail to induce the markers

Question 5

How was the role of the PM to induce hypothalamic markers also investigated

Answer 5

Culture neural tissue alone or with PM - only in PM did markers (FGF10) appear

Question 6

What is the role of the hypothalamus

Answer 6

Maintains homeostasis; integrates autonomic response and endocrine function with behaviour either:
Directly through the regulation of the pituitary gland
OR
Indirectly through interactions with other components of limbic system

Question 7

How can you identify the signals that come from the PM to induce the early hypothalamus

Answer 7

1. Expression studies - what signals are in the PM as it induces the hypothalamus?
2. Loss of function studies - deletion of Shh leads to failure of hypothalamic development

Question 8

Does the loss of Shh signalling mediated lack of hypothalamus development prove that Shh is required for hypothalamic induction

Answer 8

Shh acts back on PM autonomously maintaining its survival, therefore in Shh knockouts there is no PM so cannot investigate the signals the PM produces for hypothalamic induction

Question 9

What is the second loss of function study done to look at PM signals that induce early hypothalamic markers

Answer 9

Pharmacologically block Shh protein from the PM using cyclopamine and compare it to non soaked PM on naive lateral neural plate cells (ie not a tissue that would normally form the hypothalamus)
Nkx2.1 is seen in the non soaked tissue

Question 10

Why are gain of function studies using Shh in vivo hard in the context of the PM

Answer 10

Studies are easy to achieve in the posterior floor plate where Shh beads are positioned laterally of the neural tube by the somites. Somites aren’t present laterally to the PM so positioning the beads is a hard task to perform

Question 11

How is an Shh gain of function study performed in vivo

Answer 11

Neural explant is dissected and cultured in a 3D matrix with a collagen gel and Shh
The outcome does not see hypothalamic markers - Shh is not sufficient to induce the developing hypothalamus (but it is required)

Question 12

What is the role of nodal in the developing hypothalamus

Answer 12

Co-expressed with Shh in the PM. Functional studies in vitro and vivo suggest that nodal works with Shh.

Question 13

What is the impact on Shh by nodal

Answer 13

Shh and nodal don’t induce the full set of hypothalamic markers, but only induce Shh itself - a cell producing Shh can therefore be thought of as a pre-hypothalamic cell.

Question 14

What occurs immediately after Shh+ pre-hypothalamic cells are induced.

Answer 14

BMP7 is rapidly upregulated in the PM. For a brief period, Nodal and BMP7 are co-expressed in the PM

Question 15

What is the role of the co-expressed Nodal and BMP7 in the PM

Answer 15

Act together to induce FGF10 production in the Shh+ pre-hypothalamic cells: BMP7 induces pSmad1/5/8, Tbx2 and Nkx2.1 in Shh+ pre-hyp cells - creating a hypothalamic progenitor cell

Question 16

What are the characteristics of early hypothalamic progenitors

Answer 16

Multipotent, proliferative, give rise to cells that can: Grow, migrate and differentiate

Question 17

What is the role of FGF10 in hypothalamic progenitors

Answer 17

Supports survival and proliferation

Question 18

What is the role of Tbx2 (expressed in the same pattern as FGF10) in the hypothalamic progenitor

Answer 18

Supports the ability of the cells to migrate and differentiate

Question 19

What is the pattern of growth for an early hypothalamic progenitor

Answer 19

First to anterior hypothalamic neurons, the to posterior (mammillary) hypothalamic neurons. Some Fgf10+ progenitors are always retained in the so called tuberal hypothalamus where they eventually enter a very slow cell cycle (quiescence)

Question 20

How can the different hypothalamic regions be marked

Answer 20

Use markers of proliferation to look at where cells are cycling. Markers for differentiation and investigate when and where they appear

Question 21

What is the role of Shh in differentiating hypothalamic progenitors

Answer 21

Shh drives progression of FGF10+ cells to an anterior neural progenitor

Question 22

How was the role of Shh in differentiating hypothalamic progenitors identified

Answer 22

In the chick embryo: Preventing Shh signalling as soon as hypothalamic progenitors were formed. As a result FGF1-+ progenitors accumulate, anterior progenitors don’t form, anterior neurons don’t differentiate

Question 23

What specific TF is upregulated by Shh from the PM

Answer 23

Rx2

Question 24

Why is the development of the anterior hypothalamic progenitors important

Answer 24

Neurons that derive from these progenitors mediate core body homeostasis. i.e Temp, electrolyte balance, stress, food intake etc

Question 25

What is PomC

Answer 25

Neurons that regulate the feeling of satiety. Mutation in labradores is why they are always hungry

Question 26

What is the control of Shh at the level of reception

Answer 26

In the absence of Shh, Smo is prevented from entering the cilium by Ptch, which means there’s no Smo activity.
When Shh is present - It binds Ptch which removes Smo repression. Smo prevents progression of GliA to GliR, so GliA TF can move into the nucleus changing patterns of transcription. A number of other TM proteins also bind Shh (Gas1 and Cdo). These are obligate co-receptors and are also required for the alleviation of Smo repression.