Induction of HypFP cells Flashcards
Lecture 4
What does HypFP stand for?
Hypothalamic floor plate-like cells
Where are HypFP cells induced?
ventral midline of the forebrain by underlying prechordal mesoderm
How have experiments been conducted to show the prechordal mesoderm is necessary?
- LOF and GOF experiments shown no precaudal mesoderm = no HypFP due to a lack of Shh secreted by the PM
- Anterior neural plate expresses the TFs needed for Shh to bind such as Otx2
- PM also produces other signals such as Wnt agonists, Nodal and BMPs which help differentiate the forebrain into the hypothalamus
Explain the gene regulatory network maintaining HypFP cells
Central Role of SHH: Sonic Hedgehog (SHH) is the core signaling molecule, influencing transcription factors like NKX2.1, GLI3, and FOXA2.
Regulatory Transcription Factors: SIX3, RAX, SOX2, FOXA2, and NKX2.1 mediate tissue specification and respond to signaling cues.
Signaling Interactions:
Low WNT: Promotes hypothalamic development by interacting with transcription factors like SIX3 and RAX.
BMP2/BMP7: Opposed by FST (follistatin) to refine developmental outcomes.
Nodal: Activates FOXA2, contributing to patterning
Give the order of hypothalamic patterning and the TFs involved in the Gene Regulatory Network (GRN)
- FOXA2 defines midline and floorplate by SHH signalling from PM
- LOW Wnt signalling (regulated by SIX3 and RAX) promotes hypothalamic identity
- SHH expressed in ventral midline of FP , GLI genes mediate SHH effects: activation (GLI1/GLI2) and repression (GLI3)
- NKX2-1 is induced by SHH and specifies the ventral hypothalamus (prethalamic -> hypothalamic)
- SIX3 and RAX suppress excess WNT to stabilise hypothalamic identity
- BMP2/BMP7 signalling is opposed by follistatin to balance development
- More specific markers begin to appear such as ATOH7 and POMC where tuberal neurogenic progenitors (ARCH nuclei) will eventually form.
How do we know Shh plays such a vital role?
- KO studies of Shh in mice show holoprosencephaly and failure of hypothalamic development
- In 2000 the same was found in humans, observing an ultrasound where the fetus with no Shh, also had a deformed face and no nose
What type of forebrain tissue do HypFP cells come from?
Diencephalon
- the telencephalon is also above but shows no similar gene expression
Explain the process of how we found out HypFP is derived from the diencephalon
- Dissect hypothalamus and a little of the adjacent tissue from diencephalon at the developmental stage that HypFP cells are induced (called ‘HH8’)
- Perform single cell RNA sequencing - isolate + sequence individual cells and compare the gene expression profiles
- Use Principal component analysis to cluster the cells in to two major types
- By asking the bioinformatics data from the scRNA-seq you can see which known genes are expressed in each cell to annotate the clusters
- Interrogating bioinf. Data with RNA velocity allows to infer transcriptional dynamics, whether the gene is being induced or repressed
- This info can further be exploited by predicting cell fate decision via pseudotime trajectories
- This pseudotime lineage trajectory suggests HypFP cells are induced from diencephalic cells
What is pseudotime?
Helps organise single cells based on their gene expression profiles into an ordered ‘trajectory’ that reflects their progression through a biological
What does traditional fate mapping show of where HypFP cells are derived from?
- Shows most (Not all) HypFP cells are induced from prethalamic cells (Specific type of diencephalic)
EVERYTHING STILL NEEDS TO BE DONE IN VIVO
Give timestamps and the TFs found in Kim et al study with developing chick embryos
HH8 - heterogenity with pre-thal clusters (expressing Pax6), hypothalamic markers (NKX2-1) and FP rostral diencephalic ventral midline cells (FOXA2, SHH)
HH18-21 - POMC now expressed as a more specific marker
- posterior subdivision of tuberal region is dorsal to rathkes pouch and marked with RAX
HH18-21 - POMC ad NPY subpopulations are already forming where separate from one another seen via uMAP clusters
