lecture 1/2 Flashcards
How was Dil injection used to investigate fate mapping of the prechordal mesoderm
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.
What do cells that lie above the prechordal mesoderm give rise to?
Hypothalamus, thalamus, ventral telencephalon
How were early markers of the nascent hypothalamus discovered
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
What effect does ablation of the PM have on the markers of the nascent hypothalamus
Fail to induce the markers
How was the role of the PM to induce hypothalamic markers also investigated
Culture neural tissue alone or with PM - only in PM did markers (FGF10) appear
What is the role of the hypothalamus
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
How can you identify the signals that come from the PM to induce the early hypothalamus
- Expression studies - what signals are in the PM as it induces the hypothalamus?
- Loss of function studies - deletion of Shh leads to failure of hypothalamic development
Does the loss of Shh signalling mediated lack of hypothalamus development prove that Shh is required for hypothalamic induction
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
What is the second loss of function study done to look at PM signals that induce early hypothalamic markers
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
Why are gain of function studies using Shh in vivo hard in the context of the PM
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
How is an Shh gain of function study performed in vivo
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)
What is the role of nodal in the developing hypothalamus
Co-expressed with Shh in the PM. Functional studies in vitro and vivo suggest that nodal works with Shh.
What is the impact on Shh by nodal
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.
What occurs immediately after Shh+ pre-hypothalamic cells are induced.
BMP7 is rapidly upregulated in the PM. For a brief period, Nodal and BMP7 are co-expressed in the PM
What is the role of the co-expressed Nodal and BMP7 in the PM
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
What are the characteristics of early hypothalamic progenitors
Multipotent, proliferative, give rise to cells that can: Grow, migrate and differentiate
What is the role of FGF10 in hypothalamic progenitors
Supports survival and proliferation
What is the role of Tbx2 (expressed in the same pattern as FGF10) in the hypothalamic progenitor
Supports the ability of the cells to migrate and differentiate
What is the pattern of growth for an early hypothalamic progenitor
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)
How can the different hypothalamic regions be marked
Use markers of proliferation to look at where cells are cycling. Markers for differentiation and investigate when and where they appear
What is the role of Shh in differentiating hypothalamic progenitors
Shh drives progression of FGF10+ cells to an anterior neural progenitor
How was the role of Shh in differentiating hypothalamic progenitors identified
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
What specific TF is upregulated by Shh from the PM
Rx2
Why is the development of the anterior hypothalamic progenitors important
Neurons that derive from these progenitors mediate core body homeostasis. i.e Temp, electrolyte balance, stress, food intake etc
What is PomC
Neurons that regulate the feeling of satiety. Mutation in labradores is why they are always hungry
What is the control of Shh at the level of reception
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.
