Developmental Biology 8

Front 1

Skeletal Muscle Cell Differentiation

Back 1

Muscle cells are derived from the somites
myoblast
cultured myoblasts: differentiate when growth factors withdrawn
Synthesize muscle specific proteins

Front 2

Structural Changes in Muscle Cells

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Cytoskeletal changes result in bipolar cells
Cells align and fuse to form multinucleate myotubes
Differentiation continues and striated fibers are formed
integrins are required in vivo for cell fusions

Front 3

Genes Required for Muscle Cell Differentiation

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Pax 3
myoD
mrf4, myf5, myogenin

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Pax3

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homeobox gene that is expressed throughout somite then becomes more restricted: dermomyotome, then limb muscle cells

Front 5

myoD

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transcription factor gene is expressed in muscle cells and their precursors: key controlling factor, binds the E-box

Front 6

mrf4, myf5, myogenin

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belong to the same family as myoD
all influence muscle differentiation
myogenin only expressed late in differentiation
Some redundancy is evident

Front 7

Pathway of Skeletal Muscle Differentiation

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External signals (unknown) initiate the pathway
myoD and myf5 promote their own expression
Growth factors promote proliferation, inhibiting differentiation
Myogenin is a differentiation factor

Front 8

Differentiation involves Withdrawal from the Cell Cycle

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Proliferation and differntiation of myoblasts are mutually exclusive
Presence of MyoD and Myf5 not sufficient for differentiation
Molecular link to the cell cycle: MyoD and Myf5 not sufficient for differentiation
Molecular link to the cell cycle
This link necessary so that sufficient cells are produced to make a functional structure, before differentiation begins

Front 9

Molecular link to the cell cycle

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MyoD and Myf5 are phosphorylated by cyclin-dpendent kinases
More degradation when phosphorylated
Other proteins present at high concentrations in dividing cells inhibit MyoD and Myf5 activity
The myogenic factors also work to slow cell-cycle progression

Front 10

Muscle cell Differentiaion is Reversible

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Dedifferentiation: the loss of differentiated characteristics and re-entry into the cell cycle
Mouse Mox1 is a homeobox-containing transcriptional repressor
Mox1 is normally expressed in undifferentiated cells
In tissue culture experiments using mouse myoblasts: Ectopically express Mox1 in myotubes, the myotubes break down into individual cells, these cells can differentiate into other cell types under appropriate conditions
A similar dedifferentiation appears to occur in regenerating tissues in amphibians

Front 11

Some Cells can be Replaced in Adults

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muscle cells and neurons do not divide after differentiationg
Skeletal muscle cells and some neurons can be replaced
Specific population of muscle stem cells exists
Satellite cells proliferate and differentiate into new muscle cells if the muscle is damaged
Can be visualized by the expression of specific genes

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Neural Stem Cells

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Only two locations in the CNS for adult neurogenesis: Subventricular zone of the lateral ventricle; new neurons migrate to the olfactory bulb, Subgranular zone of the hippocampus: new neurons form w/in the dentate gyrus of the hippocampus
Gilial cells also generated

Front 13

Neural Crest Cell Differentiation

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Neural crest cells give rise to a large number of cell types
neural crest cells regarded as mesectoderm

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Neural Crest Fate Map

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In general, anterior NC cells form anterior structures and posterior NC cells form posterior structures
Exception is perhaps the parasympathetic ganglia of the gut

Front 15

Developmental Potential of NC Cells

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Developmental potential of NC cells is revealted by transplant experiments: NC cells are multipotent
Tissue culture experiments also indicate NC cells are multipotent
differentiation of NC cells is determined largely by their location

Front 16

Model for Neural Crest Cell Differentiation

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Cells higher in the linage have more developmental potential
Some intermediate progenitors have the ability for self renewal

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Neural Crest Cell Differentiation Factors

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Different factors provided by the cells in the different environments that neural crest cells migrate through
Notch-Delta signaling also involved

Front 18

Melanocyte Differentiation

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Steel factor (aka SCF or stem cell factor) is the ligand produced by fibroblasts
The receptor Kit encoded by the white spotting gene is expressed by "melanoblasts"
Activation of Kit is required for melanocyte differentiation
defects in this pathway result in mice with strange coat colors

Front 19

Programmed Cell Death (apoptosis)

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Cell death during development (apoptosis) is different from death to to damage (necrosis)
Known for decades that cell deaths can be prevented by survival factors
It was a surprise that the deaths were a result of cell suicide
Requires RNA and protein synthesis: an active process
Involved in the development of the nervous system, digit formation, tissue growth and cancer

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Programmed Cell Death in C. elegans

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ced-1 and ced-2 were the first cell death genes identified; required for the phagocytosis of the dead cells
These genes also critical for further screening for cell death genes
Screens resulted in the identification of important genes in the pathway: ced-3, ced-4, ced-9
identification of gens specific for cell death that were working in the dying cell was a surprising discovery

Front 21

Apoptosis Pathways

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Mammals: BID/BIM --> Bcl-2, Cytc ---> APAF1 --> caspases

Front 22

Programmed Cell Death Proteins

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Pathway centered on CED-3: Caspase protease that triggers the cellular changes leading to death
CED-4 is an adapter that activates CED-3
Mutations in either gene cause cells that normally die to live

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CED-9

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normally prevents cell deaths
Mutations in ced-9 cause cell deaths in multiple cells that normally do not die
Rare ced-9 gain-of-function mutations cause cells to live: no cell deaths
CED-9 mammalian homolog is BCL-2 which was identified through its involvement in B cell lymphoma

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EGL-1

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a cell death activator
Removes CED-9 inhibition which causes cell deaths
Acts in response to an apoptotic signal

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Nuclei of Differentiated Cells can Support Development

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Examine the ability of the DNA in a differentiated cell to support development using nuclear transplantation; cloning
Xenopus cells are large and easy to work with

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Cloning Using Blastula Cell Nuclei

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Experiments are more successful using blastula cell nuclei
Can obtain many adult frog clones; they have the identical genetic constitution
Even further success using the blastula nuclei from the clones
Conclusions: Genes required for development are not irreversibly altered, Gene activities depend on the factors present in the cytoplasm in the cell

Front 27

Cloning Experiments

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Similar cloning experiments have been done in many species
Dolly the lam was the first mammal cloned
Reasons for cloning: Transgenic animals are used to generate drugs or proteins used in medicine: easier to clone these animals, Therapeutic cloning
The later the developmental stage of the cell the less successful the experiment is
In general success rate is very low, epigenetic modifications not removed, not imprinted properly

Front 28

Cell Fusion Experiments

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Can expose the nucleus of one cell to the cytoplasm of another by cell fusion
many different types of differentiated human cells have been used in fusions with rat muscle cells
]Expression of human muscle genes is induced

Front 29

Differentiation and Transdifferentiation

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Some cells in regenerating tissues have the ability to dedifferentiate and redifferentiate into another cell type
Transdifferentiation: the change of one differentiated cell type into another A(in tissue culture)
Another demonstration of the potential reversibility of gene activity patterns
Examples: Epithelium to lens; chromatin cell to neuron: liver to pancreas

Front 30

Embryonic Stem Cells

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ES cells can differentiate into a particular cell type in the proper culture conditions: ES cells maybe be the key to regenerative medicine, Goal: restore the structure and function of damaged or diseased tissues, Use cells generated from ES cells in cell replacements

Front 31

Using ES cells for Therapy

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Problem: ES cells can't be used directly:
Teratocarcinomas, tumors containing a mix of differentiated cells, still cause immune reactions, ethical reasons: must destroy a human blastocyst
use adult stem cells: must determine ways to get these cells to differentiate into the required type
Devise a protocol for therapeutic cloning: same ethical problems