Differentiation

Ahmed Ihab Abdelaziz MD, PhD

Associate Prof. Of Molecular Medicine
NewGiza University (NGU)

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 Developmental Genetics

Objectives:
Explain how a differentiated cell achieves and
maintains its mature characteristics.

Explain how we know that all genes in a

differentiated cell are still capable of function.

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Objectives:
At the end of these lectures you should understand:
That each specialized cell type contains specific proteins to suit its function.
That this is brought about by differential gene expression.
That differentiated cells contain a complete functional set of genes.
That somatic cell nuclear transfer can reprogram the genome.
That the genome is reprogrammed in induced pluripotent stem cells.
That reprogramming can be used to produce tissues for transplantation

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 General Themes in Development

Development a series of changes in the

state of a cell, tissue, organ, or organism
Underlying process that gives rise to the
structure and function of living organisms
Developmental genetics aimed at
understanding how gene expression
controls this process

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General themes
Sperm and egg unite to produce a zygote
That diploid cell divides and develops into the
embryo
Cells divide and begin to arrange themselves
Each cell becomes determined destined to
become a particular cell type
Commitment to become a particular type of cell
occurs long before a cell actually differentiates
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 Genome is a set of genes that constitute the
program of development
Unicellular organisms genome controls
structure and function of the single cell
Multicellular organisms genome controls
cellular features and the arrangement of cells

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Cell adhesion

Each animal cell makes its own

cell adhesion molecules (CAMs)
Positioning of a cell within a multicellular
organism is strongly influenced by the
combination of contacts it makes with other
cells and the extracellular matrix

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Cell adhesion
molecules (CAMs)
Cell

(c) Cell adhesion: Cell-to-cell contact conveys positional information

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Hierarchy of transcription factors

Four general phases of body formation

1. Organize body along major axes
2. Organize into smaller regions (organs, legs)
3. Cells organize to produce body parts
4. Cells change morphology and become differentiated

Differential gene regulation certain genes expressed

at specific phase of development in a particular cell type

Specific transcription factors are expressed at each

phase of body formation

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 Hierarchy of transcription factors
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Hierarchy of transcription factors

Posterior
Right 1 Phase 1: 2 Phase 2:
Transcription factors Transcription factors cause
Dorsal determine the formation the embryo to become
(ventral is of the body axes and subdivided into regions
underneath) control the expression of Evidence of that have properties of
Anterior transcription factors of segmentation individual segments. They
phase 2. also control transcription
Left
factors of phase 3.

Head
forming 3 Phase 3: 4 Phase 4:
Transcription factors cause Transcription factors cause
each segment and groups cells to differentiate into
of segments to develop specific cell types such as
specific characteristics. skin, nerve, and muscle
Limbs They also control cells.
forming transcription factors of
phase 4.

(1): Courtesy of the National Museum of Health and Medicine, Washington, D.C.; (2): Congenital Anomaly Research
Center of Kyoto University; (34): Courtesy of the National Museum of Health and Medicine, Washington, D.C.

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Phase 4 Cell differentiation
Once patterns established, cells must
differentiate to carry out roles
Studied in mammalian cell culture lines
Differential gene expression underlies cell
differentiation
Stem cell characteristics
Capacity to divide
Daughter cells can differentiate into several cell types

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Stem cell

Cellular
division
Stem cell

Red
Cellular Differentiating blood
division cell cell
Stem cell

+
Red
Differentiating blood
cell cell 12
Stem cell categories

Totipotent
Ultimate stem cell is fertilized egg
Can produce all adult cell types

Pluripotent
Embryonic stem cells (ES cells)
Embryonic germ cells (EG cells)
Can differentiate into almost any cell but a single cell
has lost the ability to produce an entire individual

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Totipotent

Fertilized egg
is totipotent. Fertilized egg
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Totipotent

Fertilized egg
is totipotent. Fertilized egg

Pluripotent

Embryonic stem
cells (ES cells) Blastocyst
are pluripotent.
Inner cell
mass

ES cells
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Totipotent

Fertilized egg
is totipotent. Fertilized egg

Pluripotent

Embryonic stem
cells (ES cells) Blastocyst
are pluripotent.
Inner cell
mass

ES cells

Pluripotent, multipotent, or unipotent

Embryonic germ
cells (EG cells)
are pluripotent.
Other fetal cells
are multipotent Fetus
or unipotent.

EG cells
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Totipotent

Fertilized egg
is totipotent. Fertilized egg
Multipotent or unipotent

Adult stem cells are

multipotent (bone
marrow cells) or
unipotent (skin cells).
Pluripotent

Embryonic stem Adult stem

cells (ES cells) Blastocyst cells
are pluripotent.
Inner cell
mass

ES cells

Pluripotent, multipotent, or unipotent

Embryonic germ
cells (EG cells)
are pluripotent.
Other fetal cells
are multipotent Fetus
or unipotent.

EG cells
 Multipotent
Can differentiate far fewer types of cells
Hematopoietic stem cells (HSCs)

Unipotent
Daughter cells become only one cell type
Stem cells in skin produce only skin cells

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Hematopoietic stem cell

Bone marrow

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Hematopoietic stem cell

Cell division

Bone marrow + Hematopoietic cell

Hematopoietic that will differentiate
stem cell

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Hematopoietic stem cell

Cell division

Bone marrow + Hematopoietic cell

Hematopoietic that will differentiate
stem cell OR
Myeloid Lymphoid
cell cell

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Hematopoietic stem cell

Cell division

Bone marrow + Hematopoietic cell

Hematopoietic that will differentiate
stem cell OR
Myeloid Lymphoid
cell cell

Basophil Monocyte Eosinophil Neutrophil Dendritic cell

Red Megakaryocyte
blood
cell Platelets Osteoclast Macrophage

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Hematopoietic stem cell

Cell division

Bone marrow + Hematopoietic cell

Hematopoietic that will differentiate
stem cell OR
Myeloid Lymphoid
cell cell

T cell B cell Natural Dendritic

killer cell cell

Basophil Monocyte Eosinophil Neutrophil Dendritic cell

Red Megakaryocyte
blood
cell Osteoclast Macrophage
Platelets

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24
 Davis, Weintraub, and Lasser Identified
Genes That Promote Muscle Cell Differentiation
What causes stem cells to differentiate into a
particular cell type?

Certain proteins function as master transcription

factors

Initial experimental strategy to identify genes

expressed only in differentiating muscle cells

Narrowed down to three genes

Can any of these three genes cause non-muscle

cells to differentiate into muscle cells?
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HYPOTHESIS Muscle differentiation is induced by particular genes.

KEY MATERIALS Three cloned genes had been identified that were expressed only in differentiating muscle cell lines. The
researchers also had fibroblast cell lines, which do not normally differentiate into muscle cells.

Experimental level Conceptual level

MyoA MyoD MyoH

1 In 3 separate tubes, add each of the 3 cloned MyoA MyoD MyoH
genes, designated MyoA, MyoD, and MyoH.

DNA

1. Fibroblast cells
2 Add fibroblast cells to the tubes and incubate 2. CaPO4
in the presence of calcium phosphate (CaPO4),
which promotes the uptake of DNA into the cells.

DNA taken
Fibroblast
up by cell

3 Plate the cells on solid growth media. Allow the

cells to grow for 3 to 5 days. Cells will express
the cloned gene.
MyoD

MyoA MyoH
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Now looks like

4 Examine the cells under a microscope to determine a muscle cell
if they have the morphology of differentiating
muscle cells.

Still looks like a fibroblast

Colony labeled with myosin antibody

5 Determine if the cells are synthesizing myosin, Antibodies
which is a protein that is abundantly made in
muscle cells. This is done by adding a labeled
antibody that recognizes myosin and determining
MyoD
the amounts of antibody that bind.

MyoA MyoH

6 THE DATA

Results from step 5:

Results from step 4:
Colonies labeled with antibody
DNA added Microscopic morphology of cells DNA added that binds to myosin?

MyoA Fibroblasts MyoA No

MyoD Muscle cells MyoD Yes
MyoH Fibroblasts MyoH No

7 CONCLUSION The MyoD gene encodes a protein that causes cells to differentiate into skeletal muscle cells.

8 SOURCE Davis, Robert L., Weintraub, Harold, and Lassar, Andrew B. 1987. Expression of a single transfected cDNA
converts fibroblasts to myoblasts. Cell 51:9871000.
 MyoD was the only one to cause fibroblasts
to differentiate into muscle cells
Belongs to myogenic bHLH genes
Found in all vertebrates and activated during
skeletal muscle development

Features promoting muscle cell differentiation

Basic domain binds specifically to an enhancer
DNA sequence that is adjacent to genes that
are expressed only in muscle cells
Protein contains an activation domain that
stimulates the ability of RNA polymerase to
initiate transcription