Determination, Deffrentiation And Plasticity

Question 1

Developmental commitment

Answer 1

A progressive restrictions of developmental potential.

Question 2

Specification

Answer 2

Commitment to a particular fate that can be changed if cells move to a new environment (determinants and induction)

Question 3

Determination

Answer 3

Commitment to a particular fate that cannot be changed

Question 4

Differentiation

Answer 4

Cells acquire their functional characteristics

Question 6

Differentiation; diversity of different cell types

Answer 6

• At least 250 different major cell-types in vertebrates

Question 7

Differentiation: diversity of different subtypes

Answer 7

Santiago Ramón y Cajal
(1852-1934) Spanish neurobiologist

• Many major cell types can be divided into different subtypes

Question 8

Differentiation: gene batteries

Answer 8

Morgan, working on Drosophila, was the first to:
• demonstrate that genes are carried on chromosomes and are the units of heredity

• suggest that “different batteries of genes come into action as development proceeds.”

• suggest that “initial differences in the protoplasmic [cytoplasmic] regions may be supposed to affect the activity of genes. The genes will then in turn affect the protoplasm, which will start a new series of reciprocal interactions. In this way we can picture the gradual elaboration and differentiation of the various regions of the embryo.”

Thomas Hunt Morgan (1866-1945) American geneticist and embryologist

Question 9

Differentiation: gene batteries facts

Answer 9

• Human genome contains ~20,000 protein coding genes

• Only 6-7% are transcribed by all cells - housekeeping genes required to maintain basic cellular function (e.g. metabolism, RNA & protein synthesis, cell cycle control

• Vast majority of genes are expressed tissue specifically and transcribed by only a few cell types

• These genes can be described as the terminal (differentiated) gene battery and are crucial for cell-specific functions

Question 10

Differentiation: skeletal muscle gene battery

Answer 10

Muscle-specific proteins:
Actin, Alpha 1
~90%
Myosin II
Tropomyosin (Z-disc)
Titin (27,000-33,00 AAs)
Nebulin
Creatine Phosphokinase
Acetylcholine Receptor

Question 11

Differentiation neuronal subtype gene batteries

Answer 11

Check slides

Question 12

Differentiation: neuronal gene battery

Answer 12

Neuronal specific proteins:

Rab3
Syntaxin
Synaptobrevin
Ca2+ channels
K+ channels
NT receptors
Neurexin
Neuroligin

Question 13

Differentiation: regulation of gene batteries

Answer 13

Historically several hypotheses were proposed as to why different genes are expressed in different cells:

Gene loss
Gene amplification

Question 14

Gene loss

Answer 14

Based on the observation that some invertebrate
embryos lose chromosomes during development it was proposed that cells lose genes or (permanently inactivate them).

Somatic nuclear transfer experiments demonstrate the genome of differentiated cells is fully intact

Question 15

Gene amplification

Answer 15

Based on the observation that amphibian
nuclei contain amplified copies of ribosomal RNA genes it was proposed that differentiated cells could amplify the genes they require.

However, amplified genes have not been found in most differentiated cells.

Question 16

Differentiation: differential gene expression

Answer 16

Davidson, working mainly on sea urchins proposed a theory of transcriptional developmental gene regulation (1969): (check slides)

• We now know that tissue specific transcription is indeed controlled by enhancer elements which bind tissue specific transcription factors. These transcription factors interact with general transcription factors (such as the mediator proteins) to activate transcription by RNA polymerase II.

Question 17

Differentiation: differential gene expression

Answer 17

Gene transcription is regulated through mechanisms that affect chromatin structure, including modifications of DNA and core histones:

• methylation of cytosine is associated with silencing
• methylation of bias tones is associated with activation and silencing
• acetylation is associated with activation

Question 18

Differentiation: differential gene expression
Chromatin in differentiated cells

Answer 18

Transcribed Genes:
H3K4m3
H3K9m3
H3K9ac
5-methyl Cytosine

Non Transcribed Gene:
H3K4m3
H3K9m3
H3K9ac
5-methyl Cytosine

Question 19

Determination: linking specification and differentiation

Answer 19

Davidson proposed the idea of gene regulatory
networks during which early specification events lead to the expression of key differentiation drivers:
Check slides for diagram

Question 20

Determination: (selector genes)

Answer 20

Selector genes (master regulators) regulated entire differentiation gene batteries

MyoD:

Muscle-specific proteins:
Actin, Alpha 1
Myosin II
Tropomyosin
Titin
Nebulin
Creatine Phosphokinase
Acetylcholine Receptor

Ascl1:

Neuronal-specific proteins:
Rab3
Syntaxin
Synaptobrevin
Ca2+ channels
K+ channels
NT receptors
Neurexin
Neuroligin

Question 21

Subtype specification (terminal selector)

Answer 21

Terminal selector genes directly entire differentiation gene batteries

Question 22

An entire developmental program: skeletal muscle

Answer 22

• Holoblastic cleavage
• Ingression of the mesoderm
• Mesoderm specification primarily through induction
• Somite formation
• Determination of myotome through induction leading to the expression of MyoD
• MyoD drives differentiation of myotubes by regulating the muscle-specific gene
battery

Check slides and rewatch lecture

Question 23

Plasticity: can determined cells change their fate?

Answer 23

• Determination is VERY stable
• However, he did occasionally observe transdetermination

Ernst Hadorn

Question 24

Plasticity: can the genome be reprogrammed?

Answer 24

Since all cells contain the same genome Gurdon wanted to follow on from the work of Briggs and King (the first to perform SCNT) to test if it could be reprogrammed (Spemann’s “fantastical experiment”):
• He transplanted nuclei from cells in various stages of development into enucleated cells. This is referred to as somatic cell nuclear transfer (SCNT)
• He demonstrated that the genome can indeed be reprogrammed as occasionally normal embryos developed that could even produce viable adult frogs.

Question 25

Plasticity: can the genome be reprogrammed? Yamanaka

Answer 25

• Knew that the transcription factors Oct4, Sox2 and Nanog are expressed in the inner cell mass and are required to maintain its pluripotent state. • Demonstrated that forced expression of these genes in fibroblasts transform them to embryonic stem cells. These cells are pluripotent and can be induced to form a range of differentiated cells types

Question 26

Summary and check slides/lecture

Answer 26

Differentiation involves the activation of specific cell-type specific gene batteries Development is the result of the co-ordinated action of gene regulatory networks that results in cell-type specific gene expression Difficult to change fate of determined or differentiated cell. Nuclei can be reprogrammed following nuclear transfer into eggs, but the frequency is very low. Differentiated cells can be reprogrammed following addition of master-regualtor/selector transcription factors, but the frequency is very low. May be difficult to reprogram nuclei/cells because of epigenetic changes to DNA and chromatin - can natural transdifferentiation teach us how to improve reprogramming efficiency?