Cell Differentiation and Stem Cells

The reversibility and inheritance of patterns of gene activity
What determines the pattern of gene activity in a differentiated cell?
Two possibilites are
1) Loss of genetic material.
2) Regulatory  proteins (transcription factors, chromatin proteins?)
Transgenic studies have revealed much about gene activity and differentiation.

Control of transcription involves both general and tissue-specific regulators of transcription
First, the RNA polymerase binds to the start site of transcription in the promoter.
Then the DNA is unwound so that the template DNA can be copied.
The control regions of a gene contribute to the ability of the RNA polymerase and associated proteins to start transcription.
Cooperation of general transcription factors and RNA polymerase II is required to bind to the TATA box of the promoter.
The enhancer elements bind regulatory proteins (transcription factors).
Proteins bound at the enhancer element interact with proteins bound at the promoter region to form a transcription initiation complex and to initiate transcription at a high rate.
Enhancers are usually upstream of the transcriptional start site but can be downstream or within introns.
Enhancer regions can be located some distance away from the promoter.
Some transcription factors are required for a wide range of genes while others are only required for a small set of genes (perhaps one).
Interactions between transcription factors control increases or decreases in binding affinity.

External signals can activate genes
Patterns of gene expression are controlled by external signals  such as steroid hormone (that can enter the cell) and protein growth factors that interact with cell surface receptors to generate an intracellular signal.
Steroid hormones are lipid soluble and bind receptor proteins inside the cell.
Steroid/receptor complexes can act as  transcription factors binding steroid response elements in the DNA.
Peptide/proteins bind receptors in the cell membrane and signal transduction mechanisms cause the activation or inactivation of transcription factors (by phosphorylation of a transcription factor or release from a cytoplasmic complex).

Maintenance and inheritance of patterns of gene activity may depend on regulatory proteins and DNA modification.
In the differentiated state, some genes are active and others are repressed.
Developmentally important eukaryotic genes often have very complex control regions with binding sites for many transcription factors which can activate or repress transcription.
Continual expression of a gene may require the continual presence of a transcription factor.
The gene product may positively regulate itself to maintain its own expression.
Selector genes remain active throughout development to maintain the developmental pathway of a region.
The state of chromatin packaging can keep a gene inactive for a long period of time.
i.e. The inactivation of one of two X-chromosomes to form a heterochromatic Barr body.
Localized chromatin packaging could have similar effects on gene expression.
Methylation of the DNA can act to modify the activity of genes.

All blood cells are derived from pluripotent stem cells.
Through hematopoisis, stem cells give rise to all the blood cells of an adult mammal.
Most blood cells are short lived and must be constantly replaced.
The mammalian embryo begins hematopoiesis in
1)  the yolk sac blood islands,
2)  then the fetal liver and
3)  finally the bone marrow.
The hematopoietic stem cell generates the hierarchy of differentiation.
The pluripotent stem cell reproduces and gives rise to 1) the myloid cells (the erythrocytes (RBC) and
2) the leukocytes (WBC) - eosinophils,  neutrophils, basophils, monocytes and megakaryotes) and the lymphoid cells (B and T lymphocytes).
Initially pluripotent, the stem cell first becomes committed to either the lymphoid or myeloid lineages.
This is followed by rounds of replication and further commitment to give the final 8 cell types.
This occurs in the bone marrow and is regulated by growth factors and cytokines.

Colony-stimulating factors and intrinsic changes control differentiation of the hematopoietic lineages.
Hematopoiesis TFs have overlapping expression patterns.
c-Myb (a proto-oncogene) is expressed only in immature cells and is not lineage specific.
~20 TF's are lineage specific (i.e. GATA-2 is in all myeloid precursors but not lymphoid cells and
GATA-1 is in some myeloid and is required for RBC differentiation).
 ~20 extracellular colony-stimulating factors affect cell proliferation and cell differentiation such as the
interleukins,
erythropoietin,
thrombopoietin,
macrophage colony-stimulating factor (M-CSF)
granulocyte colony-stimulating factor (G-CSF), and
granulocyte-macrophage colony-stimulating factor (GM-CSF).

Globin gene expression is controlled by distant upstream regulatory sequences.
The erythrocyte requires large amounts of hemoglobin from two sets of globin genes.
Hemoglobin is a tetramer comprised of two alpha and two beta globin chains that come from two multigene clusters.
The human beta globin cluster contain five genes that expressed at different times during development and produce differnt types of hemoglobin at differnt times.
The locus control region (LCR), an element far upstream of the gene cluster confers high levels of expression of the specific developmentally correct family member.
The locus control region contains four core control regions that bind transcription factors including GATA-1.
Developmental control of expression of the different family members over time probably depends upon looping out of the intervening DNA.
Proteins bound to the LCW and to different globin gene promoters  can physically interact to promote transcription.

Differentiation of cells that make antibodies is due to changes in DNA
B lymphocytes of the vertebrate immune system recognize and respond to antigens by producing antibodies on their cell surfaces.
The Y-shaped antibody molecule is composed of 2 light and 2 heavy chains.
The antigen binding sites (at the tips of the Yís arms) are made from the variable regions of the light and heavy chains which are encoded by rearranged
Each B lymphocyte expresses just one type of heavy and one light chain to produce a specific antibody.
Rearrangement of the DNA to assemble a V-D-J heavy chain gene and a V-J light chain gene occurs by somatic recombination.

The epithelia of adult mammalian skin and gut are continually replaced.
Vertebrate skin is composed of 3 layers:
1) the dermis (mainly fibroblasts)
2) the protective outer epidermis (mainly keratinocytes)
3) the basal lamina (ECM which separates the epidermis from the dermis).
Stem cells form a population in the basal layer (next to lamina) which divide asymmetrically to form one stem cell and one committed to differentiation.
Cell adhesion molecules play an important role in skin cell development.
The basal cell layer is attached to the basement membrane by hemi-desmosomes and focal contacts.
The epidermal cells of the upper layers are connected by desmosomal junctions.
The epithelial lining of the the gut is under continuous turn-over.

A family of genes can activate muscle-specific transcription
Myoblasts are cells that are committed to forming muscle and can proliferate in culture until growth factors are removed from the medium.
This results in the stopping of proliferation and the start of differentiation with the synthesis of muscle specific proteins (actin, myosin II, tropomyosin and creatine phosphate kinase.
The myoblasts become bipolar in shape (via reorganization of the cytoskeleton) then fuse to give multinucleate myotubes.
In ~20 hours, striated muscle is apparent.
myoD expression can induce muscle differentiation by transfection into fibroblasts (which do not normally become muscle).
myoD is a key controlling gene in muscle differentiation and is expressed only in muscle precursors and muscle.
myoD family (myogenin, myf-5 and MRF-4) are basic helix-loop-helix (bHLH) DNA-binding transcription factors.
myoD is the first gene switched on in bird muscle precursors (myf-5 is first in mammals).
myoD & myf-5 are expressed in proliferating myogenic cells but myogenin is only expressed during muscle differentiation.
Each of these genes activate each other's expression.
In myoD knock-out mice, normal striated muscle is made and myf-5 is increased (a compensatory mechanism).
Mice missing both Myf-5 and MyoD lack all skeletal muscle where myogenin knockouts have heart and smooth muscle but lack most skeletal muscle.
The MyoD family of TFs activate transcription by binding the E-box (DNA sequence) in the regulatory region of muscle-specific genes as heterodimers.
MyoD/E2 (E2 is a ubiquitous TF) binds the E box much better than a MyoD homodimer.

The differentiation of muscle cells involves withdrawal from the cell cycle.
Cell proliferation and differentiation of muscle cells are mutually exclusive.
Myoblasts only differentiate after proliferation stops.
When growth factors are present, MyoD & Myf-5 are expressed and the myoblasts proliferate & do not differentiate.
However, additional signal(s) are required for differentiation.
Removal of growth factors will cause the myoblasts to withdraw from the cell cycle, fusion and differentiation follow.
The retinoblastoma protein (Rb) which can block cell growth is inactivated by phosphorylation in proliferating cells.
Dephosphorylation of Rb & blockage of cell cycle is a "differentiation decision".

Complex combinations of transcription factors control cell  differentiation.
Liver cell differentiation involves activation of HNF-4 (TF), which activates HNF-1alpha which then activates liver-specific genes such as albumin and Beta-fibrinogen.
HNF-1alpha transcription requires HNF-4 plus ubiquitous Fos & Jun and HNF-3.
TFs act as functional groups such that some contribute to the organization of general epithelium structure.
Others contribute within this context to tissue-specificity (although in other contents the same secondary factor might not have the same tissue specificity).

Neural crest cells  give rise to both the chromaffin cells of the adrenal medula (which secrete epinephrine) and to sympathetic neurons (which secrete norepinephrine).
Differentiation of chromaffin cells requires a high concentration of glucocorticoid hormones which are synthesized by the adrenal cortex.
Glucocorticoids inhibit neuronal differentiation and promote maturation of the chromaffin cells.
Neuron differentiation is induced sequentially by fibroblast growth factor (FGF; to induce neuron formation) and nerve growth factor (NGF; to induce  survival of the neurons).
Differentiated chromaffin cells in culture can undergo transdifferentiation into sympathetic neurons is glucocorticoids are removed and FGF is added.

Neural crest diversification involves signals for both specification of cell fate and selection for survival.
Several growth factors direct neural crest cell's fates.
Glial growth factor promotes differentiation of glia and suppresses neuron differentiation.
BMP-2 promotes neuronal differentiation.
Two type of spinal ganglia, the dorsal root sensory ganglia (with cholinergic neurons) and the sympathetic ganglia (most are adrenergic) develop from the neural crest.
Early dorsal ganglia explants can give rise to sympathetic neurons but early sympathetic neural explants never give rise to dorsal sensory neurons.
A factor (probably brain-derived neurotrophic factor:BDNF) produced by the embryonic neural tube causes the sensory cells to survive.
Possibly, the sympathetic ganglia which develops removed from the neural tube does not require BDNF to survive but the sensory ganglia do.
The vertebrate development, apoptosis (programmed cell death) is crucial in the formation of the nervous system.

Programmed cell death is under genetic control.
Cell death genes are highly conserved from nematode to mammals (and humans).
ced-3 & ced-4 are pro-apoptotic and are required for the normal death of the 131 cells that die during development of C. elegans.
Without these genes, the cells survive and develop to resemble their sister cells (worms survive for several weeks).
ced-9 is antiapoptotic and loss of function mutations will cause many cells to die while gain of function mutations will result in no cell death.
The human homolog, Bcl-2, is an oncogene that encodes a mitochondrial membrane protein.
ced-3 and it's homologues are proteases that initiate a proteolytic cleavage cascade which results in cell death.

Nuclei of differentiated cells can support development of the egg
UV-radiation directed to the animal pole destroys the nuclear DNA of the Xenopus egg.
Nuclei from other cells can then be injected into the enucleated eggs.
Nuclei from adult skin, kidney, heart and lungs (and tadpole intestinal cells) can develop into adults (low rate of survival).
Transplantation of blastula cells have a high rate of success (1 blastula nuclei into several eggs will produce clones of identical frogs).
Therefore developmental genes are not altered during development.
The transplanted genes act as the original nuclear genes would.

Gene activity can be changed by cell fusion of differentiated cells
When transplantation experiments are difficult, then cell fusion experiments can expose the nucleus of one cell to the cytoplasm of another.
Fusion of chicken red blood cells (inactive but present nucleus) with human cancer cells leads to reactivation of chick-specific gene expression.
Therefore, human cells have cytoplasmic factors that can activate chicken genes.
Fusion of human (non-muscle) cells with rat muscle cells induces human muscle gene activity.
Clearly, the gene expression of cells that are differentiated are controlled by cytoplasmic factors which can be altered.
Cell differentiation can be reversed.

The differentiated state of a cell can change by transdifferentiation
Full differentiation is normally stable.
However, cells can be altered in regenerating tissues.
With transdifferentiation in the newt, a lens can be regenerated from the dedifferentiation of iris pigmented epithelium or cartilage from dedifferentiated limb muscle tissue.
Transdifferentiation occurs in the culture of embryonic chick retina under certain culture conditions where the pigment disappears and lens-specific proteins are made.
In jellyfish cell culture, digestion of the extra-cellular matrix results in the transdifferentiation of striated muscle to smooth muscle then nerve cells.
Alteration of steroid hormone and polypeptide growth factors that specify chromaffin cells can lead to transdifferentiation into sympathetic neurons.