Lecture 3 - gene regulation Flashcards
Early embryo development stages
- Single fertilisation
- Cell division - blastocyst forms
-
Gastrulation - form 3 germlines
Ecto/Meso/Endoderm - Organogenesis
Why do we want to compare embryos?
Early stages are very similar across species.
Mouse models have been useful in research
Early CNS formation
- Neural Plate
- Folds to form Neural Groove
- Then Neural Tube
What stage are we at in a 3-4 week embryo?
3 Vesicle stage
What stage are we at in a 5 week embryo?
5 Vesicle Stage
Dorsoventral patterning
Mainly controlled by opposing signaling gradients of WNT/BMP from the roof plate, and SHH from floor plate cells
Shh pathway is strongly associated with…
Development of the neural tube, patterning of the ventral structures and ventral forebrain, neuronal differentiation, proliferation
WNT-signalling controls…
Neural tube rostrocaudal patterning
* progresive caudalisation from fore to hindbrain
What does neuroectodermal expression of Dickkopf1 (Dkk1) do?
Antagonises and inhibits Wnt signalling in the ANTERIOR
What signalling component is highly expressed in the anterior?
Sfrp1
What is the role of notch signalling?
Maintains neural progenitors/stem cells in developing brain
What aspects of neurogenesis does notch signalling impact?
Morphology, migration, synaptic plasticity, maintenance of mature/immature neurons + radial glia , and dendrite development.
What does notch signalling promote and inhibit?
Inhibit: Neurons and Oligodendrocytes
Promotes: Astrocytes
What is essential for a “prepattern” of neural induction?
Fibroblast growth factor (FGF) signals from precursors of organiser prior to gastrulation
How does FGF work?
Activates Sox3 and early response to neural induction
How is the process of transcription initiated?
RNA polymerase binds upstream of the gene on its promoter
* 1 TF binds to one of these promoter sequences, initiating a series of interactions.
What are Transcription factors (TFs)?
Regulatory proteins that activate (inhibit is rare) transcription of DNA by binding to SPECIFIC DNA sequences
How are TFs categorised?
They have highly conserved DNA binding domains which catagorise them into families (MADS box-containing proteins, SOX proteins, and POU factors)
Key transcription factors involved in determining specific neural fates
Homeodomain proteins and basic helix-loop-helix (bHLH) TFs
* provide regional identity (A-P and V-D axes)
Basic-helix-loop-helix (bHLH) Transcription Factors
Ensure that appropriate no. of specific neuronal and glial cell types are produced
* bind E-box motifs with the consensus sequence CANNTG.
What neural lineages are bHLH genes expressed in?
Neurogenin, Neurod, Atonal and Olig families
What does Neurod1 do?
Differentiation of inner ear sensory neurons and granule cells in the cerebellum and hippocampus
What does Neurod2 and 6 do?
Both required for formation of callosal connections in the Cerebral Cx.
What does Bhlhe22 do?
Differentiation of neurons in several CNS domains, incl. dorsal horn of the spinal cord, dorsal cochlear nucleus in the brainstem and retinal amacrine cells
Homeoproteins role in CNS development
Essential for formation of borders
What significance does OTX2 and GBX2 have in mice brain development?
There is a sharp border between these proteins. If it is altered:
* More OTX2 > larger midbrain
* More GBX2 > larger hindbrain
What does areal development refer to?
Formation of distinct functional brain regions
What does laminar development refer to?
Formation of distinct layers within each brain region, creating a layered structure known as a “lamina.”
What is Pax6 and what does it do?
TF which promotes development of rostro-lateral regions
What is Emx2
TF which promotes caudal-medial regions
In what axis does Pax6 and Emx2 create an opposing gradient. How?
Anteroposterior axis
* EMX2 directly represses PAX6 expression and vice versa
What effect did removing Emx2 expression in mice have?
Zone of Pax6 expression is enlarged
* Increase in primary motor (M1) and sensory (S1) areas of the anterior cortex.
What effect did removing Pax6 expression in mice have?
The primary visual (V1) cortical area is increased.
What are HOX genes?
Conserved family of homeodomain TFs with roles in (A-P) patterning and the early NS development
What regulates HOX genes?
Expression of RA, FGF, WNT
Outline where Hox1-5 and Hox4-11 are expressed
Hox1-Hox5: expressed in the hindbrain
Hox4-Hox11 detected in the spinal cord
SOX2
TF expression marks the CNS from the earliest developmental stages.
SOX4
Promote neuronal differentiation both in the adult and embryonic neural progenitors
SOX5
Controls the sequential generation of distinct corticofugal neuron subtypes by preventing premature emergence.
SOX9
Differentiation of neural stem cells (NSCs) into glial cells, rather than neurons.
Transgenesis
Introducing new gene into mouse genome to overexpress specific protein
Gene knockout
Deleting specific gene
Conditional knockout
Allow gene deletion in specific cell types at specific developmental stage using Cre-loxP system
Point mutations
Introduce specific changes in gene to mimic human genetic mutations associated with neurological disorders
Where is Twist1 expressed?
In Neural Crest Cells (NCCs)
What happens when you knockout Twist1 only?
Not too much morphological difference
Twist1 AND Chd7 knocked out?
Extreme morphological difference, indicating they are in the same pathway of development
Twist1 AND Chd8 knocked out?
Neural tube does not close properly, nerves are not formed properly, not enough forming.
What happens when Otx2 is knocked out in mouse models?
Change in the amount and location of expression of telencephalon markers
Morphology is also changed
What human cells can we use to study NDDs?
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)
Brain organoids
Take hSCs and put in tissue culture
Make embryoid bodies and induce them into neural cells
RNA sequencing
Reverse transcribe RNA fragments > DNA fragments > create library and compare to human genome
Bulk RNA sequencing
Measures the average gene expression level across all cells in a sample
Single cell RNA sequencing
Examines the gene expression profile of individual cells, when you want to study the diversity of cell types within a tissue
What are the limitations of single cell RNA sequencing
Input has to be a single cell suspension, time, cost, loss of data.
