ahmed ihab abdelaziz md, phdit.ngu.edu.eg/downloads/links/week3/tuesday/2.25 differentiation...
TRANSCRIPT
Differentiation
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Ahmed Ihab Abdelaziz MD, PhDAssociate Prof. Of Molecular Medicine
NewGiza University (NGU)
Developmental Genetics
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:
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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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 in Development
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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(c) Cell adhesion: Cell-to-cell contact conveys positional information
Cell
Cell adhesion
molecules (CAMs)
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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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(1): Courtesy of the National Museum of Health and Medicine, Washington, D.C.; (2): © Congenital Anomaly Research
Center of Kyoto University; (3–4): Courtesy of the National Museum of Health and Medicine, Washington, D.C.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Hierarchy of transcription factors
4 Phase 4:
Transcription factors cause
cells to differentiate into
specific cell types such as
skin, nerve, and muscle
cells.
Phase 2:
Transcription factors cause
the embryo to become
subdivided into regions
that have properties of
individual segments. They
also control transcription
factors of phase 3.
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Evidence of
segmentation
3 Phase 3:
Transcription factors cause
each segment and groups
of segments to develop
specific characteristics.
They also control
transcription factors of
phase 4.
Limbs
forming
Head
forming
Phase 1:
Transcription factors
determine the formation
of the body axes and
control the expression of
transcription factors of
phase 2.
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Posterior
Right
Dorsal
(ventral is
underneath)
Anterior
Left
Hierarchy of transcription factors
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
Stem cell
Stem cell
+
+
Cellular
division
Cellular
division
Differentiating
cell
Red
blood
cell
Differentiating
cell
Red
blood
cell
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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Fertilized egg
Totipotent
Fertilized egg
is totipotent.
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Fertilized egg
Blastocyst
ES cells
Pluripotent
Totipotent
Inner cell
mass
Fertilized egg
is totipotent.
Embryonic stem
cells (ES cells)
are pluripotent.
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Fertilized egg
EG cells
Fetus
Pluripotent, multipotent, or unipotent
Blastocyst
ES cells
Pluripotent
Totipotent
Inner cell
mass
Fertilized egg
is totipotent.
Embryonic stem
cells (ES cells)
are pluripotent.
Embryonic germ
cells (EG cells)
are pluripotent.
Other fetal cells
are multipotent
or unipotent.
Adult stem cells are
multipotent (bone
marrow cells) or
unipotent (skin cells).
Adult stem
cells
Multipotent or unipotent
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Fertilized egg
EG cells
Fetus
Pluripotent, multipotent, or unipotent
Blastocyst
ES cells
Pluripotent
Totipotent
Inner cell
mass
Fertilized egg
is totipotent.
Embryonic stem
cells (ES cells)
are pluripotent.
Embryonic germ
cells (EG cells)
are pluripotent.
Other fetal cells
are multipotent
or unipotent.
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
stem cell
Hematopoietic cell
that will differentiate
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Hematopoietic stem cell
Cell division
Bone marrow
OR
+Hematopoietic
stem cell
Myeloid
cell
Hematopoietic cell
that will differentiate
Lymphoid
cell
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Hematopoietic stem cell
Cell division
Bone marrow
OR
+
MegakaryocyteBasophil
OsteoclastPlatelets
Macrophage
Monocyte Eosinophil Neutrophil Dendritic cell
Hematopoietic
stem cell
Myeloid
cell
Hematopoietic cell
that will differentiate
Lymphoid
cell
Red
blood
cell
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Hematopoietic stem cell
Cell division
Bone marrow
OR
+
Basophil
OsteoclastPlatelets
Macrophage
Monocyte Eosinophil Neutrophil
T cell B cell
Dendritic cell
Hematopoietic
stem cell
Myeloid
cell
Hematopoietic cell
that will differentiate
Lymphoid
cell
Red
blood
cell
Natural
killer cell
Dendritic
cell
Megakaryocyte
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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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Add fibroblast cells to the tubes and incubate
in the presence of calcium phosphate (CaPO4),
which promotes the uptake of DNA into the cells.
1. Fibroblast cells
2. CaPO4
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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.
Conceptual levelExperimental level
MyoHMyoDMyoA
DNA
In 3 separate tubes, add each of the 3 cloned
genes, designated MyoA, MyoD, and MyoH.
Plate the cells on solid growth media. Allow the
cells to grow for 3 to 5 days. Cells will express
the cloned gene.
MyoA MyoD MyoH
FibroblastDNA taken
up by cell
MyoA MyoH
MyoD
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SOURCE Davis, Robert L., Weintraub, Harold, and Lassar, Andrew B. 1987. Expression of a single transfected cDNA
converts fibroblasts to myoblasts. Cell 51:987–1000.
CONCLUSION The MyoD gene encodes a protein that causes cells to differentiate into skeletal muscle cells.
Examine the cells under a microscope to determine
if they have the morphology of differentiating
muscle cells.
Determine if the cells are synthesizing myosin,
which is a protein that is abundantly made in
muscle cells. This is done by adding a labeled
antibody that recognizes myosin and determining
the amounts of antibody that bind.
THE DATA
Results from step 4:
DNA added
MyoA
MyoD
MyoH Fibroblasts
Muscle cells
Fibroblasts
Microscopic morphology of cells
Colonies labeled with antibody
that binds to myosin?
No
Yes
No
Results from step 5:
MyoH
MyoD
MyoA
DNA added
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Colony labeled with myosin antibody
Antibodies
MyoD
MyoHMyoA
Still looks like a fibroblast
Now looks like
a muscle cell
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