Developmental biology 4 (Prof. Dale) Flashcards
How many cell types are there is then human body ?
Can you cite a few examples ?
~250
Goblet cells & enterocytes, fibroblast, red & white blood cells, pancreas: exocrine & ductal cells, skeletal muscle, neuron, keratinocyte, pancreas: ß-cells (can be stained for insulin)
From which which embryonic tissue is skeletal muscle made ?
How are myoblasts maintained in a mitotic state ?
Which signals triggers myocyte differentiation ?
- sk muscle –> made by myotome region of each somite –> induced by signals from adjacent tissues (Wnt signals from the neural tube)
- induced muscle precursors = myoblasts –> maintained in a mitotic state by FGF signals
- muscle specific transcription factors e.g. MyoD1 (transcription of which is induced by Wnt3a signalling) –> inhibit cell proliferation, promote cell fusion + activate transcription of genes required for differentiation of sk muscle
What is sk muscle made of ?
Large multinucleate cells called myofibres.
How is the contractile apparatus of the muscle arranged ?
What happens during muscle contraction ?
In sarcomeres, with characteristic cross-striations.
The dark band (A) is made of myosin while the light band (I) is made of actin.
Myosin and actin slide past each other during muscle contraction.
Can you cite a few muscle-specific proteins ?
Alpha-Actin, Myosin II, Tropomyosin, Titin, Nebulin, Creatine, Phosphokinase, AChR.
How many protein coding genes does the human genome contain ?
What proportion of these genes are transcribed by all cells ? To what purpose ?
~20,000
Only 6-7% are transcribed by all cells –> housekeeping genes required to maintain basic cellular functions (e.g. metabolism, RNA & protein synthesis, cell cycle control)
What are tissue selective genes ?
Tissue selective genes = genes transcribed by only a few cell-types (sometimes only one):
- globins (⍺ and ß) in erythrocytes
- insulinin ß-islet cells
- ⍺-actins in muscle cells
- crystallins in lens cells
- immunoglobulins in B-lymphocytes
What proportion of tissue selective genes are transcribed in the brain/muscle/liver/kidney/lungs/prostate ?
- ~9% in the brain
- ~5% in muscle
- ~4% in liver
- ~1% in kidney
- ~1% in lung
- ~1% in prostate
Which proteins control tissue specific transcription ?
Where are these proteins situated ?
How do these proteins interact general transcription factors ? To what purpose ?
- Tissue specific transcription –> controlled by distal control elements = enhancers –> which are bound by transcription factors (activators)
- Enhancers –> may be as much as 1 million base pairs away from the promoter + are brought to the promoter by DNA bending proteins
- Tissue specific transcription factors –> now interact w/ general transcription factors + mediator proteins ==> regulation of RNA synthesis by RNAp II
How is gene transcription regulated ?
- through epigenetic mechanisms that affect chromatin structure –> modifications of DNA + associated core histones
- methylation of cytosine in gene promoters = gene silencing
- methylation of core histones = gene activation + silencing
- acetylation of core histones = gene activation
What histone modifications can be seen in transcribed Vs non-transcribed genes ?
- transcribed genes –> H3K4m3 & H3K9ac that create “loose”
chromatin –> accessible to transcription factors - non-transcribed genes –> highly methylated DNA (5mC) + alternative histone modifications (e.g. H3K9m3) –> compact chromatin –> inaccessible to transcription factors
In D. Melanogaster, where are cells that will form the adult cuticle found ? In which form ?
In which form are abdominal segments present ?
When do these differentiate ?
In the larva as undifferentiated discs (w/ discs for the mouth parts, eye antenna, wing, haltere, legs and genitalia)
Abdominal segments –> present as nests of cells = histoblasts –> differentiate during pupal development (blue pigment stain distal elmts, the aristae of the antennal disc + wing blade of the wing disc)
Ernst Hadorn (1902-76) removed imaginal discs from larvae and cut them in two. One half he transplanted to a 3rd instar larva to see what it will form, the other to an adult female. What did he observe after 2 weeks ? What did he then do (and repeat again and again...) ?
- after 2 weeks he removed the imaginal disc and found that it had grown in size
- he cut it in again, transplanting half to a 3rd instar larva + half into a 2nd adult female (repeating this procedure more than 300 times over 12 years)
In most cases –> disc continued to differentiate the same tissues
Occasionally, during Hadorn’s transplant experiments, the transplanted disc formed a different part of the cuticle e.g. a wing instead of a leg.
What were these rare events called ?
Transdeterminations.
Sir John Gurdon FRS destroyed the egg nucleus (UV irradiation) of a X laevis egg and injected a nucleus isolated from a differentiated cell inside it.
What did he observe ?
- small proportion of embryos = form tadpoles + even adults
- most embryos = partial, but nuclei isolated from these and transferred to enucleated eggs –> can form tadpoles and adults
- cells from partial embryos –> can also be grafted to a host embryo + will differentiate into multiple cell types
- up to 30% of intestinal nuclei can form functional non intestinal cell types, demonstrating that the genome of differentiated cells can be reprogrammed
How does success rate of Somatic Cell Nuclear Transfer (SCNT) in X laevis vary with the stage of the embryo ?
What did Gurdon conclude from this ?
Successful SCNT in Xenopus laevi droppes as progressively later stages were used as donors.
However, even nuclei (20%) from swimming tadpoles supported normal development.
Gurdon (1962) concluded that the genome of differentiated cells can be reprogrammed to support differentiation of other cell types.
What famous experiment Keith Campbell (1954-2012) and Sir Ian Wilmut FRS perfrom in 1997 ?
Campbell & Wilmut –> fused an enucleated egg w/ a cultured mammary gland cell:
- 29 (11.7%) reached blastocyst stage + were transferred to surrogate female
- only 1 (0.3%) was born alive = female they called Dolly
What is a heterokaryon ?
Heterokaryon = a multinucleate cell that contains genetically different nuclei
If a mouse myoblast is fused with a human hepatocyte, human muscle-specific proteins are detected on the plasma membrane of the heterokaryon.
What does this demonstrate ?
That muscle-specific genes can still be transcribed in differentiated liver cells –> the mouse muscle cell must possesses factors that can activate muscle genes in human genome
What is MyoD1 ?
Who discovered and to what family does it belong to ?
What is its role (along w/ proteins from the same family) ?
- MyoD1 = transcription factor found in developing sk muscle cells
- discovered by Harold Weintraub (1945-95)
- belongs to the family of Myogenic Regulatory Factors (Myf5, Myf6, and Myogenin)
- all four proteins are required for muscle development in vertebrates + will transform fibroblasts into myoblasts when constitutively expressed
How was the role of MyoD1 established using genetic techniques ?
The gene encoding GFP –> coupled to a promoter + an enhancer bound by MyoD1
Enhancer –> drives sk-muscle-specific expression in the somites (myotome), limbs + head + acts as a genetic “switches” that regulates tissue-specific expression
What are the characteristics of Oct4, Sox2 and Nanog ?
- are specifically expressed in ICM + required to maintain its
pluripotent state - encode transcription factors that regulate gene expression in ICM
- downregulated in ICM cells as they become committed to the different germ layers
- not expressed in the trophoblast layer, which transcribes genes such as Cdx2
How did Kazutoshi Takahashi and Shinya Yamanaka confirm in 2006 that the mammalian genome is programmable ?
- Takahashi + Yamanaka –> introduced Oct4, Sox2, Myc + Klf4 (“Yamanaka factors” into fibroblasts)
- small proportion (<1%) of these somatic cells –> transformed into PSCs that resembled Embryonic Stem Cells (ESC)
- obtained PSC –> can form a range of different cell-types
Why were Sir John Gurdon & Shinya Yamanaka
awarded the Nobel Prize for Medecine or Physiology in 2012 ?
“[…] for the discovery that mature cells can be reprogrammed to become pluripotent.”
