3. Cell differentiation Flashcards
Define differentiation
Differentiation - a process by which unspecialised cells become cell specific types (in embryos / in tissue homeostasis)
Approximately how many types of cell tissues there are in a human?
~ 200 main types
What makes cells different from one another?
- different macromolecules
- different metabolites
- different morphologies and behaviours
Housekeeping vs specialised proteins?
- Housekeeping proteins - proteins found in most cell types for shared essential cell functions (ex ATP synthase)
- Specialised proteins - proteins specific to a particular cell type (ex insulin synthesising protein in beta cells in the pancreas)
What are the main embryonic germ layers from which all tissues differentiate?
- ectoderm
- mesoderm
- endoderm
Trophectoderm (embryo tissue which forms placenta)
Why is differentiation considered progressive?
- forms intermediate (transient) states (progenitor / precursor cells)
- has a terminal differentiation point - final cell from
=> progressive specialisation, not immediate
What drives specific cell differentiation?
Differentiation is driven by interplay between cell’s lineage and its environment
During which process of embryo development are the germ layers formed?
Gastrulation
What is haematopoiesis?
Haematopoiesis - formation of blood components - differentiation of blood cells
What genetic mechanism drives cell differentiation?
Selective activation / inactivation of particular genes
Can the nucleus of a differentiated cell support development of a new organism?
Yes - nuclear transfer experiment - sheep Dolly (first cloned mammal)
What is gene constancy?
Gene constancy - all cells in a multicellular organism have a full complement genes (full genome)
=> cells differentiate because of gene activity rather than gene content (differential gene expression)
How is gene expression controlled?
By control of gene transcription - transcription factors (TF) - genes can individually be switched on/off - different gene expression patterns in different cells
What is the DNA signal sequence which is a recognition site / binding site?
TATA box - binds TATA binding proteins - transcription factors (TFIID)
What is the role of TATA box?
TATA box controls where transcription starts - binds TF
(doesn’t control when transcription starts)
What is an enhancer?
Enhancers - short regulatory nucleotide sequences recognised by TF that enhance the rate transcription
What is a promoter?
Promoters - rather long regulatory nucleotide sequences that initiate transcription - polymerases bind
Explain promoters vs enhancers
Promoters - long sequences / initiate transcription
Enhancer - short sequences / increase transcription rate
Enhancers activate promoters
What are transcription factors (TF)?
Transcription factors (TF) - proteins which activate/repress polymerase function
What are the methods by which TFs can initiate transcription?
- can act directly (directly recruit RNA polymerase to TATA box)
- can act via chromatin modification (indirectly recruit RNA polymerase: histone acetyl transferase / chromatin-remodelling complex)
What are the two indirect methods of TFs for recruiting RNA polymerase for transcription initiation?
- Histone acetyl transferase (HAT): acetylations loosens histone interactions with DNA - genes more accessible for transcription
- Chromatin-remodelling complex: chromatin modifying enzymes promote RNA polymerase binding and function by remodelling chromatin and making it more accessible
What controls gene’s transcriptional activity in a cell?
- binding sites in enhancer sequences of DNA for the gene
- presence of appropriate TFs for the gene
Give two examples of TF in differentiation
- MyoD - TF which activates expression of genes for muscle myosin II protein - in muscle cell differentiation - recognition site: E box
- GATA1 - TF which activates expression of several target genes (for α+β globin, erythropoietin receptor, heam biosynthesis enzymes, spectrin) - in RBC differentiation
Can one TF target several genes transcription?
Yes - MyoD activates transcription of many genes acting in muscle cell differentiation + represses non muscles genes
What’s the effect of MyoD knockout?
MyoD knockouts produce undifferentiated muscle cell precursors - myoblasts
How can MyoD expression pattern be investigated?
Modify embryos with MyoD mRNA staining (in situ hybridisation) - observe MyoD expression patterns where stained (muscle cell differentiation sites)
Can TF force other lineage cells to differentiate into lineage to which the TF is specific?
Yes - ex: fibroblasts (skin precursors) transfected with MyoD containing plasmid - differentiated into muscle cells
What are the correct steps in genetic analysis of TF function?
- Analysis of expression pattern (where it is expressed in developing body?)
- Analysis of loss-of-function (is the TF required for differentiation?)
- Analysis of gain-of-function (can the TF force other lineage cell differentiation into its own - is it sufficient for full differentiation?)
How TFs recognise their binding sites?
TF - high specificity - specific am. a. in recognition site - bind specific base sequence
How are TFs categorised?
TFs are categorised into families - based on 3D structure of their DNA binding domains where binding sites are located
Usually, do TFs work on their own or in combinations?
In combinations - groups of TFs at binding sites
What are the possible TF combination interactions for transcription enhancing?
When several TFs for the same gene regulation:
- either TF may be sufficient / doesn’t matter which binds for transcription
- all TFs required together for transcription
What are the possible TF combination interactions for transcription repressing?
When several TFs for the same gene regulation:
- repressing TF may prevent binding of activating TF (picture)
- recruit proteins that tighten chromatin - gene less accessible
What regulates TFs and how?
What:
- envronmental signals (signals from other cells: hormones, growth factors)
- developmental history (earlier TFs regulate later TFs)
How:
- controlling their activity by PTMs
- controlling their gene expression
How are TFs regulated by cell signalling?
How are TFs regulated by PTMs?
PTMs - ex: phosphorylation - phosphate alters protein conformation -> activate / inhibit TF
How growth factors can control TF activity?
Growth factors regulate TF activity by phosphorylation -> target gene activity changed
Growth factor signal activates protein kinase cascade:
1) phosphorylation of MAP-kinase-kinase kinase (activated) ->
2) phosphorylates MAP-kinase kinase (activated) ->
3) phosphorylates MAP kinase (activated) ->
4) MAP kinase enters nucleus and phosphorylates TFs - activates TFs for target gene
Explain how epidermal growth factor activates cyclin genes
Epidermal growth factors - mitogen (promotes cell proliferation)
1) Growth factors signal initiates kinase cascade - phosphorylates MYC (TF) on serine (Ser-62) + threonine -> changes MYC conformation to stable active
2) Activated MYC drives cyclin gene transcription
Explain how hormone erythropoietin (EPO) regulates GATA1 activity
1) Hormone erythropoietin (EPO) secreted from kidneys
2) EPO binds to receptors - kinase cascade
3) GATA1 (low activity) phosphorylated - conformational change -> high activity
3) GATA1 increases DNA binding
4) Stimulates blood progenitor proliferation and RBC differentiation
Low O2 conditions (ex: hipoxia) stimulate EPO secretion - more RBCs - higher efficiency
Why control of TFs is important for orderly progression of differentiation?
TF regulate gene expression which drive differentiation -> cell fate:
Different TFs regulate different steps in differentiation
Explain how leaf progenitor cells differentiate into epidermis and stomata?
Default pathway - epidermis cells
Some progenitor cells induced for stomata (guard cells) differentiation by SPCH, MUTE, FAMA TFs
What is a gene regulatory network (GRN)?
Gene regulatory network (GRN) - a set of genes which interact to control a specific cell function
How is MyoD initially activated in muscle differentiation gene regulatory network?
By co-regulation of Pax3 and Myf5 TFs (work in combination) - for initial MyoD levels - later MyoD activates its own expression
Pax3 + Myf5 - transient process
How is MyoD regulated in muscle differentiation?
Autoregulation - MyoD activates further MyoD expression - becomes independent of Pax3 and Myf5 regulation (cell memory)
How do growth factors stop MyoD expression?
1) Growth factors promote cell division -> cdk produced
2) Cdk phosphorylate MyoD and Myf5 -> proteolytic degradation -> muscle cell differentiation inhibited -> myoblast proliferation enhanced (growth factor response - cell division achieved)
What is RNA interference?
RNA interference (RNAi) - post transcriptional gene silencing - degrade mRNAs
How is Pax3 degraded during muscle differentiation?
Via RNA interference (RNAi):
- Pax3 only for early muscle development - must be down-regulated for later stages to proceed
- Pax3 mRNA degraded by microRNA (miR-1) - double stranded RNA - bound to an enzyme
Explain Waddington’s developmental landscape
Represents cell specialisation - binary fate decisions (which valley will roll into - increasing specialisation - decreasing potency to develop into any other cell type
Explain pancreas cell differentiation progress
Binary cell fate choices: specific cells from the gut can choose 1/3 cell lineages - pre-pancreatic - Ngn3 TF needed for all endocrine cells (if Ngn3 missing - no endocrine cells developed - all exocrine) - Ngn3 needed for general endocrine precursor development but not for a specific cell type in endocrine cells
What decides pre-pancreatic fate in foregut?
- Pre-pancreatic region affected by TFs - loops formed - comes closer to notochord - receives signalling - Fgf2 TF turned on -> further pancreas development
Explain erythroid/myeloid cell fate choice
Common myeloid progenitor - binary cell choice -> GATA1 / PU.1 TFs
GATA1 / PU.1 autoregulate and inhibit each other -> different blood cells differentiate - ANTAGONISTIC interactions
What are master regulators of organogenesis, give example
Master regulators - TFs which are essential for the development of all organ - without it nothing formed - if injected into wrong location - organ still formed
Example:
- Pax6 in eye development
- same TF in many animals - was present in a common ancestor
- MyoD in muscle differentiation
- Shh in anterior-posterior axis development
