Cell And Developmental Biology Lecture 13

Front 1

1) Hans Driesch experiment with sea urchins, and what does it support the ideas of?

2) Pre-implantation genetic diagnosis.

3) Ian Wilmut and gene constancy.

Back 1

1) Hans Driesch (1892): cells in the early (sea urchin) embryo can be separated from each other and individual cells giverise to fully developed sea urchins. Thismeans that each cell in the early embryomust containall the necessary information for normaldevelopment:totipotent. This supports the ideas of“regulative” development. Regulative development generally occurs in early gastrulation when cells are induced to form different structures according to the cell-cell signaling interactions in a specific area of the embryo that lead to the conditional specification of a cell's fate.

2) Pre-implantation geneticdiagnosis: following in vitro fertilization a singlecell is removed from an embryo and istested for gene mutations; if there is no evidenceof a defect, the embryo isimplanted to give rise to a healthy baby. Regulative development ,and the totipotency of the cells, allows the rest of the embryo to compensate for the missing cell.

3) Ian Wilmut(1996): Dolly the sheep.

A fully differentiated adult cell wasused to generate the first cloned mammal. This shows thatnothing in the genome is lost when cells becomespecialised/differentiated.“Gene constancy”

Front 2

1) What are somites?

Back 2

1) In vertebrates, all skeletal muscle in the body is derived from the somites. Somites are segmented blocks of paraxial Mesoderm (On either side of the dorsal neural tube, with notochord underneath). New somites form from the anterior end of the pre-segmental plate mesoderm at regular intervals.

Front 3

1) Somitogenesis?

Back 3

1) When the somite is first formed, the cells areunspecified. Later, regions of the somites become committed to forming only certaincell types. The parts of the somites are:

The scleratome:forms in the ventral medial part of the somite. The cells of the scleromeundergo mitosis, lose their epithelial structure and become mesenchymal cells. This part of the somite willdifferentiate as chondrocytes andgive rise to the ribs and vertebrae.

The dermamyotomeforms in the dorsal part of the somite and goes on to form:

The dermatomewhich is the dorsal most layer of the somite and will give rise to the mesenchymallayer of the dorsal skin (the dermis)

The myotomewhich will give rise to the deep muscles of the back andother muscle cells migrate from the ventral lateral part of the myotome toform the musclesof the bodywalland the limb.

Allskeletal muscle in the vertebrate body is derived from the somites.

Front 4

1) What specifies the skeletal muscle cell lineage?

Back 4

1) The skeletal muscle cell lineage isspecified in a subset of somite cells.

Signalling molecules are secreted bynearby structures such as theneural tube, notochord, dorsalectoderm and lateral plate mesoderm.These signals act together to activatemyogenesis in the dorsal medialsomite cells.The signals do this by activating theexpression of regulatorygenes that direct cells down the skeletal musclelineage.

Front 5

1) Myogenesis: skeletal muscle development? Myoblasts? Role of growth factors in vitro? Key observation?

2) Liver and muscle experiment?

Back 5

1) •Myoblasts are undifferentiated, proliferativecells within the somitecommitted to forming skeletal muscle.

•Myoblasts can be isolated from embryos andkept in culture where they proliferate; but willdifferentiate as muscle when growth factors are removed .

•Growth factors in the media allow myoblasts toproliferate.

•Removal of growth factors allows myoblasts todifferentiate.

•When myoblasts differentiate they:

(i)stop dividing

(ii) fuse to form multinucleated myofibre and (iii) express contractile proteingenes

When myoblasts stop dividing and fuse, they activatethe expression ofa very large number of different genesall at the same time.

-->Points to a common regulator.

2) When a human liver cell is fused with a mouse muscle cell, the expression of human, muscle specific genes is detected. This means the human nucleus, that was expressing liver genes, is now expressingmuscle genes.This suggests that there is something inthe muscle cell that can activatetranscription of muscle genes.Points to a dominant regulator of myogenesis.

Front 6

1) Experiment with mouse fibroblasts to show the activation of a single loci can...

Back 6

1) Experiment: A mouse fibroblast cell line was treatedwith a drug that hypomethylates DNA.50% of these cells converted to myoblasts.Hypomethylationremoves methyl groups from DNA; these methyl groups are usually found inregions of DNA that are not transcribed and removing them activates geneexpression.The number of fibroblasts that convert tomyoblasts(50%) is consistent with the activation of a single locus. Points to a dominantregulator ofmyogenesis.

Experiment:A comparison of the mRNAs present in thefibroblasts to the mRNAs present in the 5-aza-myoblasts, identified acDNAcapable of converting fibroblasts to myoblasts: itwas named MyoD.

5-azacytidine is the hypomethylating agent.

Front 7

1) MyoD?

Back 7

1) MyoD wasthe first identified member of a family of bHLHtranscription factors that regulate the expression of skeletalmuscle genes. The other members are myf5, myogenin and MRF4.They bind specific DNA sequences (called E-boxes) to activate gene expression. The bHLH transcription factors coded for by themyogenic regulatory genes are collectively referred to as the MRFs(myogenic regulatory factors).These regulators are expressed very earlyand specifically in the skeletal muscle cell lineage in all vertebrates.

Front 8

1) When are MyoD, myf5, myogenin and Mrf4 expressed?

2) Binding of the 4 MRFs?

Back 8

1) MyoD and myf5 are expressed very early in somitecells. Myogenin and Mrf4 are expressed later. TheMRF genes are expressed exclusively in cells in the myogeniclineage.

•MyoD and Myf5 are muscle determination genes.

•Proliferating myoblasts express MyoD and Myf5prior to differentiation

•Myogenin regulates myoblast fusion and muscledifferentiation.

2) •There are 4 Myogenic RegulatoryFactors (MRFs)

MyoD,

Mrf4

Myf5

Myogenin

•MRFs are basic helix-loop-helix transcription factors.

•MRFs heterodimerize withotherbHLHtranscription factors called E-proteins (such as E12)

•This dimer binds to anE-box , a regulatory sequence found in the enhancers of targets genes.

•An E-box consists on a sequence of“CANNTG”

•The genes transcriptionally activated byMRFs include contractile protein genes like actin, myosin, troponin…etc…as wellas MRFs.

MRFs auto- and cross-regulate MRF expression (bybinding E-boxes).

•A mouse knock-out, lacking myoD andmyf5 has no skeletal muscle (ie they are essential for muscledevelopment).