Differentiation. Ahmed Ihab Abdelaziz MD, PhD Associate Prof. Of Molecular Medicine NewGiza University (NGU)

Transcription

1 Differentiation Ahmed Ihab Abdelaziz MD, PhD Associate Prof. Of Molecular Medicine NewGiza University (NGU) 1

2 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. 2

3 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 3

4 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 4

5 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 5

6 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 6

7 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 7

8 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cell adhesion molecules (CAMs) Cell (c) Cell adhesion: Cell-to-cell contact conveys positional information 8

9 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 9

10 Hierarchy of transcription factors Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Posterior Hierarchy of transcription factors Right Dorsal (ventral is underneath) Anterior Left 1 Phase 1: Transcription factors determine the formation of the body axes and control the expression of transcription factors of phase 2. Evidence of segmentation 2 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. Head forming Limbs forming 3 Phase 3: Transcription factors cause each segment and groups of segments to develop specific characteristics. They also control transcription factors of phase 4. 4 Phase 4: Transcription factors cause cells to differentiate into specific cell types such as skin, nerve, and muscle cells. (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. 10

11 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 11

12 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Stem cell Stem cell Cellular division + Stem cell Cellular division Differentiating cell Red blood cell + Differentiating cell Red blood cell 12

13 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 13

14 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Totipotent Fertilized egg is totipotent. Fertilized egg

15 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Totipotent Fertilized egg is totipotent. Fertilized egg Pluripotent Embryonic stem cells (ES cells) are pluripotent. Blastocyst Inner cell mass ES cells

16 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Totipotent Fertilized egg is totipotent. Fertilized egg Pluripotent Embryonic stem cells (ES cells) are pluripotent. Blastocyst Inner cell mass ES cells Pluripotent, multipotent, or unipotent Embryonic germ cells (EG cells) are pluripotent. Other fetal cells are multipotent or unipotent. Fetus EG cells

17 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Totipotent Fertilized egg is totipotent. Fertilized egg Multipotent or unipotent Pluripotent Adult stem cells are multipotent (bone marrow cells) or unipotent (skin cells). Embryonic stem cells (ES cells) are pluripotent. Blastocyst Inner cell mass Adult stem cells ES cells Pluripotent, multipotent, or unipotent Embryonic germ cells (EG cells) are pluripotent. Other fetal cells are multipotent or unipotent. Fetus EG cells

18 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 18

19 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hematopoietic stem cell Bone marrow 19

20 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hematopoietic stem cell Cell division Bone marrow Hematopoietic + stem cell Hematopoietic cell that will differentiate 20

21 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hematopoietic stem cell Cell division Bone marrow Hematopoietic stem cell + OR Hematopoietic cell that will differentiate Myeloid cell Lymphoid cell 21

22 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hematopoietic stem cell Cell division Bone marrow Hematopoietic stem cell + OR Hematopoietic cell that will differentiate Myeloid cell Lymphoid cell Red blood cell Basophil Megakaryocyte Platelets Osteoclast Monocyte Eosinophil Neutrophil Dendritic cell Macrophage 22

23 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hematopoietic stem cell Cell division Bone marrow Hematopoietic stem cell + OR Hematopoietic cell that will differentiate Myeloid cell Lymphoid cell T cell B cell Natural killer cell Dendritic cell Red blood cell Basophil Megakaryocyte Platelets Osteoclast Monocyte Eosinophil Neutrophil Macrophage Dendritic cell 23

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25 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?

26 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 1 In 3 separate tubes, add each of the 3 cloned genes, designated MyoA, MyoD, and MyoH. MyoA MyoD MyoH MyoA MyoD MyoH DNA 2 Add fibroblast cells to the tubes and incubate in the presence of calcium phosphate (CaPO 4 ), which promotes the uptake of DNA into the cells. 1. Fibroblast cells 2. CaPO 4 Fibroblast DNA taken 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

27 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4 Examine the cells under a microscope to determine if they have the morphology of differentiating muscle cells. Now looks like a muscle cell Still looks like a fibroblast 5 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. Colony labeled with myosin antibody MyoD Antibodies MyoA MyoH 6 THE DATA Results from step 4: DNA added Microscopic morphology of cells Results from step 5: DNA added Colonies labeled with antibody 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 Expression of a single transfected cdna converts fibroblasts to myoblasts. Cell 51:

28 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