Cell Fates and Differentiation
Cell Fates and Differentiation: Mechanisms and Experiments
Stages of Development and Cell Fate Specification
Development proceeds through several key stages:
Fertilization
Cleavage Stages
Gastrulation
Organogenesis
Growth/Death (establishing internal and external final form)
Axis Formation: Establishes major body axes (e.g., head-feet, back-front).
Cell Fate Specification/Differentiation: Cells become specified (e.g., mesoderm) and then differentiate (e.g., heart cells).
Morphogenesis: Involves processes like cell division, cell state changes, cell shape changes, cell movement, cell-cell interactions, and cell death/apoptosis to establish form.
Three Primary Germ Layers formed during Gastrulation:
Ectoderm (outer layer): Gives rise to epidermal cells of skin, neurons of the brain, pigment cells (melanocytes), central nervous system, outer neural crest.
Mesoderm (middle layer): Gives rise to notochord, bone tissue, tubule cell of kidney, red blood cells, facial muscle, heart, gonads, connective tissues.
Endoderm (internal layer): Gives rise to pharynx, respiratory tube, digestive tube (stomach cell), thyroid cell, lung cell (alveolar cell), liver, lining of the gut.
Germ cells: Differentiate into sperm (male) and egg (female) cells.
Cell determination and differentiation occur in stages.
Waddington's Epigenetic Landscape of Differentiation
Differentiation: The process by which cells derived from the zygote become specialized.
Cells undergo multiple stages of commitment before reaching their final differentiated state.
Developmental Potential: Becomes progressively restricted as cells differentiate. This is often visualized as a "landscape" where cells roll down valleys, narrowing their possible fates.
Investigating the Mechanisms of Cell Differentiation: Gurdon's Experiments
Initial Hypothesis: A historical hypothesis suggested that cells lose genetic information as differentiation occurs.
Sir John B. Gurdon and Shinya Yamanaka: Awarded the Nobel Prize in Physiology or Medicine in for "the discovery that mature cells can be reprogrammed to become pluripotent."
Gurdon's Question: Can the nucleus of an adult, differentiated cell give rise to an entire organism?
Gurdon's "Somatic Nuclear Transfer" Experiment (using X. laevis frogs):
An egg cell was enucleated (its nucleus removed, often by UV irradiation).
The nucleus from an adult, differentiated somatic cell (e.g., intestinal epithelial cell) was transplanted into the enucleated egg.
The resulting cell was allowed to develop.
Gurdon's Conclusions: In terms of genetic potential, an intestinal epithelial cell nucleus is genetically equivalent to a -cell embryo (fertilized egg). This experiment refuted the idea of irreversible genetic information loss during differentiation, demonstrating that differentiated cells retain a full complement of genetic material.
Drivers of Cell Identity: Differential Gene Expression
Differences in cell identity are primarily driven by which genes they select to be expressed (transcribed and translated).
Examples: A neuron and a liver cell, despite having the same genome, express different sets of genes, leading to distinct transcriptomes and proteomes.
The average human cell expresses only of its approximately genes.
Mechanisms of Gene Regulation Influencing Cell Identity
Signaling Pathways: Can direct cellular identity by affecting any DNA-protein regulatory processes.
Signal transduction cascades: External signal molecules bind to receptors, leading to activated enzymes (e.g., adenylyl cyclase, PKA, Ras, MAP kinases) that phosphorylate downstream targets, ultimately affecting gene expression.
For example, activated PKA can phosphorylate and activate transcription regulatory proteins (e.g., CREB), leading to changes in gene expression.
Transcriptional Regulators / Transcription Factors (TFs):
These proteins bind to specific DNA sequences (regulatory sequences like promoters and enhancers) associated with genes to control their transcription.
They can act as activators (recruiting coactivators, general transcription factors, RNA polymerase II) or repressors.
Regulatory Sequences (Cis-regulatory sequences): Allow for tight control over the time and space of transcription. Examples include the Nanog cis-regulatory sequence.
Chromatin Modifications (Epigenetics):
Histone acetylation: Uncondensed nucleosomes with largely unmethylated and acetylated histone tails (e.g., H2, H3, H4 tails acetylated) are associated with active gene expression.
Histone methylation: Condensed nucleosomes with largely methylated histone tails (e.g., H3, H4 tails methylated) are associated with gene silencing.
These epigenetic changes contribute to "cell memory" by maintaining specific gene expression patterns.
Post-transcriptional and Post-translational Regulation:
Alternative Splicing: Different combinations of exons from a single gene can be spliced together to produce multiple mRNA isoforms, leading to different proteins (e.g., Protein A, B, C from the same gene).
Other mechanisms include ligand binding, covalent modification, addition of second subunits, unmasking of active sites, stimulation of nuclear entry, and release from membrane for protein activation.
Asymmetric Accumulation of Transcription Factors: Drives cell fate decisions. The transcriptome and proteome differences between cells are mainly derived from the differential acquisition of transcription factors due to exposure to different factors.
Impact of Transcription Factors on Cell Fate
Cellular identity is wholly dependent on which transcription factors are present in the cell at a given time.
Homeotic Mutations: Drastically alter an organism's body plan, often caused by the expression of a single transcription factor in an incorrect cell type.
Cell Reprogramming: Introduction of specific transcriptional regulators can convert one cell type to another.
Example: Introducing three specific transcriptional regulators into a liver cell can reprogram it into a functional neuron.
Shinya Yamanaka's work (iPS cells): Introduced three (or four) transcription factors (Oct4, Sox2, Klf4, and sometimes c-Myc) into a fibroblast nucleus. These cells were allowed to divide in culture, resulting in induced pluripotent stem (iPS) cells. These iPS cells could then be induced to differentiate into various cell types like muscle cells, neurons, or fat cells, demonstrating pluripotency.
Example: Eve Gene Expression in Drosophila Development
Segmented Body Plan: Establishment of segments is a crucial early developmental step.
The Eve (even-skipped) protein is essential for early Drosophila development. Improper Eve expression leads to non-viable embryos.
Spatially-Restricted Expression: Eve protein (green) is expressed in specific stripes, allowing for the delineation of different body axes.
Transcriptional Control: Eve expression is controlled by four transcriptional regulators acting on cis-regulatory sequences near the Eve gene.
Eve protein is expressed in embryonic regions where transcriptional repressors are low and activators are high. This is well-understood for the enhancer responsible for generating the second segment or "stripe" of Eve expression.
Summary of Transcriptional Regulation and Cell Fates
Transcriptional regulators are effector proteins for cell signaling pathways.
Cell fate and identity are entirely dependent on the transcription factors present in a cell.
Regulatory sequences provide precise control over gene expression in both time and space.
Defining Cell Fate Terms in Development
Cell Fate: The characteristic cell type(s) that an early embryonic cell will normally give rise to.
Mapped using techniques like photo-activated fluorescent dye to track cell lineage (e.g., fate map of zebrafish central nervous system).
Potential: The full range of fates a cell is capable of expressing at any particular time.
Example: A pluripotent cell has the potential to become ectoderm (skin, nervous system), mesoderm (bone, muscle, kidney, heart, blood), or endoderm (gut, liver, lungs).
Specification: The ability of cells to adopt their normal fate when isolated from the embryo (e.g., isolated ectoderm becomes ectoderm).
At this stage, cells possess the information to become their normal fate but retain plasticity – they can still change if exposed to new signals.
Determination: The restriction in a cell's potential such that it can now only express one fate or a limited set of fates.
Experimental Definition: Using transplantation experiments (e.g., Wolpert's experiment with prospective eye region from a gastrula):
Before or at gastrula stage: If presumptive eye tissue is transplanted to the trunk of a host neurula, it can be instructed to change and forms structures typical of the trunk (e.g., somitic tissue). Cells are specified but not determined, exhibiting plasticity.
After gastrulation (later stage embryo): If presumptive eye tissue is transplanted, it develops as an eye, regardless of its new location. Cells are said to be determined, as their fate is irreversible.
A cell for which its specification is irreversible is considered determined.
Mechanisms of Cell Fate Specification
Autonomous Specification:
A cell determines its fate without requiring external input from neighboring cells.
Achieved through asymmetric acquisition of cytoplasmic molecules (determinants) during cell division.
Example: Asymmetric segregation of P granules in the C. elegans embryo, which are cytoplasmic determinants.
Conditional Specification:
Cell fate is determined by interactions among neighboring cells.
It involves Induction: An interaction where one cell or tissue (the inducer) influences another cell or tissue (the responder) to change its properties and adopt a new fate.
Mechanisms of Inductive Interactions:
Paracrine factors: Diffusion of inducers from one cell to another.
Cell-cell contact: Direct contact between inducing and responding cells.
Interaction with Extracellular Matrix (ECM): Matrix of one cell induces change in another cell.
Generating Cellular Diversity through Regulation
Combinatorial Control and Cell Memory: Simple signals can generate complex patterns of cell fate.
Cell Memory: Maintained by:
Epigenetic changes: Such as histone acetylation or methylation, which stably alter gene expression patterns.
Persistence of macromolecules: Regulatory RNAs or transcription factors can remain in the cell, influencing subsequent gene expression.
Asymmetric Cell Division: Can generate diversity in two main ways:
Type 1 (Asymmetric division): Sister cells are "born different" due to unequal distribution of cytoplasmic determinants.
Type 2 (Symmetric division followed by environmental influence): Sister cells are initially similar but become different due to influences acting on them after their birth (e.g., exposure to different external signals).
Example: Asymmetric segregation of P granules in the C. elegans embryo during asymmetric cell division.