C2006/F2402 08

C2006/F2402 08’ OUTLINE OF LECTURE #19

Dr. Alice Heicklen, Columbia University, New York, NY

OUTLINE

1. Development Stages

2. Cell Fate Determination

3. TFs/DNA Methylation/Histone Modifications

4. Cloning Dolly

Handouts: 19A (Life Cycle & Cell Fate), 19B (therapeutic cloning & Dolly) and 19C (histone modifications affect gene expression)

Problems: This material does not match up with the development problems in the problem book (Problem Set 14). Therefore a new set of problems (Problem Set 14A) will be posted on Courseworks next week.

Vocabulary

Totipotent – can form all cells of the embryo, including placenta

Pluripotent – can form all cells of the embryo but not the placenta; a single pluripotent cell can’t form an entire individual but can form embryonic stem cells

Germ Cell – gametes

Germ layers – endoderm, mesoderm, ectoderm

ICM – inner cell mass; becomes embryo

Physiology describes how cells & organisms function

Developmental biology describes how cells and organisms are built

Class I (4/8/08): cell fate determination.

Class II (4/15/08): sex determination as an example of cell fate determination

How are different proteins turned on in different cell types?

Dr. M discusses that all cells have the same DNA but different cells express different proteins. How is differential protein expression regulated?

Embryonic Stages (19A)

Zygote->Morula->Blastocyst->Gastrulation->Embryo->Fetus->Adult

Zygote – fertilized egg

Blastomeres – cells of the early embryo

2 - 8 cell stage – totipotent; a single cell can form entire individual at 2 – 4 cell stage but not at 8 cell; removal of 1 cell at 8 cell stage still get entire organism

Morula – 16 cells until fluid filled cavity forms, inner 8 cells are pluripotent

Blastocyst – early embryo with fluid filled cavity that forms at the 32-cell stage, inner cell mass (ICM) is pluripotent and is a source of embryonic stem cells

Gastrulation – first massive cell movements

Germ Layers – endoderm, mesoderm and ectoderm

Embryo – early development stages and organ development

Fetus – size increase & organ refinement

* This is a cycle – the egg and sperm have the unique quality that they can reverse differentiation and begin the cycle again; egg and sperm are a unique type of specialization with distinctive characteristics, some, but not all, of which are in common with embryonic stem cells

Cell Fate Determination

Stem cell divides into two; one becomes a stem cell, continues to divide providing additional cells & the other cell may begin to differentiate

Different cells make these cell fate decisions at different times in the life cycle. As development progress, the body contains less pluripotent and multipotent (hair, blood & gut lining are examples of adult multipotent stem cells) progenitors.

Mesoderm can become heart, skeletal muscle, bone, cartilage, kidney, gonad, blood and blood vessel endothelium

Analogy: High School Student –> Undergraduate -> Graduate –> Doctor

You are less likely to change your fate the further you progress on this pathway

· All cells of the body contain the same DNA (with rare exceptions); what changes is the proteins that are expressed:

-Some are unique to heart or to liver or to lung

-Some are shared by more than 1 cell type: heart & liver or liver & lung

-Some are expressed in all cell types: these genes are housekeeping genes - important for the normal function of all cells: metabolism, cytoskeleton, secretion, ect.

Heart Differentiation

General: Totipotent->Pluripotent->Germ Layer->Progenitor Field->Differentiated

Heart: Totipotent–>pluripotent –>Mesoderm–>Heart Field–>Heart Muscle

Cdx2 --------------

Oct4 -------------------------------

Bry --------------

Nkx2.5 ------------------------------------

Myosin --------------------

Cardiac Actin --------------------

---- = protein expressed

Stages of Commitment

1. Totipotent – can become all cells of organism, one cell can create an individual

2. Pluripotent – can become any cell of embryo, one cell can’t form individual

3. Specification – fate is still reversible

4. Determination – fate is no longer reversible

5. Differentiation – overt changes in structure & function – looks like muscle & produces structural proteins necessary for function, e.g. muscle

-No changes in shape or function until differentiation

-The difference between the first four stages is the transcription factors & signaling cascades that have been activated at each stage

Specification vs Determination

In both of these stages no overt changes are obvious

Determined - the cell has activated transcription of the genes necessary for a particular fate, the cell will no longer change its fate regardless of the signals received from the environment

Specification – if development proceeds normally, it will become the fate that tissue of the embryo normally becomes, if the environment changes, the cell fate can still be changed

Master Regulatory Genes

· Master Regulatory Genes (MRGs)– Transcription factors (TFs) that are master regulators of cell fate, they turn on all the genes necessary to confer a specific cell fate

· In between each of these steps the cell goes through steps 3-4 in the stages of commitment

· During the last step, from heart field to heart muscle, the cell progresses to step 5 in the stages of commitment, where the MRG (e.g. Nkx2.5) for the specific cell fate turns on the transcription factors (e.g. GATA-4) and the structural genes (e.g. myosin and muscle actin) necessary to confer the cell phenotype, e.g. heart muscle phenotype

· When differentiation, stage 5, occurs the MRG stays on, e.g. Nkx2.5 and Pax6 remain active even after the differentiation process is finished. However, during the progression of differentiation such as mesoderm differentiation, Bry is on during the process of specification and determination of mesoderm but then turns off once the cells become cardiac muscle. See table above

ICM vs Trophoblast

· Oct4 and Cdx2 are both on in all cells at 8-cell stage

· At the 16 cell stage, the 8 inner cells express Oct4 and the 8 outer cells express cdx2; Oct4 and Cdx2 repress each other

· Oct4 maintains proliferation and inhibits differentiation in the inner cell mass (ICM), i.e. MRG for the pluripotent cell type

· Eomesoderm is the MRG for trophoblast, i.e. turns on genes necessary to confer the cells with the characteristics of trophoblast

· Note that in trophoblast the function of inhibiting ICM fate is performed by Cdx2 and the role of activitng the genes to confer trophoblast fate, MRG, is performed by Eomesoderm. Oct4 performs both of these roles in ICM

Chromatin

· Histone modifications affect protein expression

Examples: -Acetylation opens chromatin, increasing transcription

-Methylation increase or decrease transcription depending on the amino acid methylated; H3K4 methylation increases transcription, H3K27 methylation decreases transcription

· MRG expression regulated by histone methylation (19C)

MRGs are bivalently methylated on H3K4 and H3K27 in embryonic stem cells (ES cells), i.e. gene transcription levels are poised to go either way

Nkx2.5 is methylated on H3K4 & H3K27 in ES cells, on H3K4 but not H3K27 in heart cells and on H3K27 but not H3K4 in lens cells

Pax6 is bivalently methylated in ES cells, methylated on H3K4 but not H3K27 in lens cells and methylated on H3K27 but not H3K4 in heart cells

Epigenetic Code: Histone modifications

Example:

1. ICM & trophoblast cells start off with the same histone modifications

2. Uncharacterized signal causes histone methylation in ICM cells

3. TF preferentially bind to DNA in ICM cells but not to DNA in trophoblast cells

4. Protein expression levels change in ICM cells due to the TF binding

Gene Expression

Three levels of transcriptional regulation:

o TFs

o Histone Modifications

o DNA methylation

DNA Methylation

· CGs are methylated on inactive promoters increasing chromatin condensation, decreasing transcription

· C methylation is remembered through mitosis; enzyme preferentially methylates CG where the complimentary CG (parental DNA strand) is already methylated

Cloning procedure

· Cloning Dolly (19B) – somatic nuclear transfer

· Demonstrates that DNA is intact in somatic cells; differentiation is reversible

· Dollie is not a true clone

· Hwang faked human cloning – the answer is all in the mitochondria

Pluripotent Stem Cell Sources

· Pluripotent stem cells from different sources can produce varied cell types

· Somatic nuclear transfer, i.e. Dolly, circumvents immunorejection

· Other Stem Cell Sources (19B)

· Skin + transcription factors = ES cells, circumvents immunorejection

· Advances in heart regeneration