Ahmed Ihab Abdelaziz MD, PhDit.ngu.edu.eg/downloads/links/week3/Tuesday/2.25 Differentiation...

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.

1

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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2

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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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5

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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