BIOMG 1350 Lecture 16 - Cell Differentiation

Interconnectedness of Cell Signaling and Gene Expression

• Cell Signaling and Gene Expression are highly interconnected processes which together influence cell morphology and behavior to drive developmental processes.

• Cell signaling can influence gene expression and gene expression can influence cell signaling.

• Gene expression then influences cell behavior (which can also influence cell signaling or cell signaling can influence it) and then cell behavior influences morphogenesis (organization of the organism's cells).

What is the Central question for differentiation

• Central Question: How do we get many different cell types from the early embryo (a ball of similar cells)?

  • The zygote (fertilized cell) becomes a blastula and then contains 3 distinct germ layers as a result of differentiation during the gastrulation process. Blastula turns into a gastrula.

  • 3 Germ layers, mesoderm, ectoderm, and endoderm can turn into multiple different cells and this differentiation is intertwined with cell signaling and the types of signals these cells receive.

What is different between different types of cells? How do we know this to be the case?

• Two cells in an organism always have the exact same DNA (unless mutated) but they can have different gene expression and different cell signaling. Fully specialized adult cells still have all the genes necessary to make any bodily cell.

• One question scientists asked before we had a conclusive answer was whether the genome was changing during development. Or if the genome of an adult somatic cell is sufficient for the development of a normal, adult frog.

  • They were able to test this question via nuclear transplantation. They took skin cells from a frog and removed the nucleus. They then inserted this nucleus into an egg that had its own nucleus removed (de nucleated).

  • The result was the egg that contained a nucleus coming from a skin cell of the adult frog. This is an example of a move it experiment. Note the nucleus taken from the skin cells came from an albino frog whereas the egg came from a donor frog that was not albino.

  • The result was that albino offspring resulted from the donor egg with the skin cell nucleus showing that the nucleus from a fully differentiated skin cell is sufficient to direct normal frog development.

Another Example that Proves Every Cell Contains the same DNA

Other examples that proved that the genome doesn’t change during development and rather every cell's DNA matches one another was via the cloning that produced dolly the sheep (same experiment as the frog where a nucleus was inserted into an emptied egg and the cloned animal is born with exactly the same DNA as the cell donor meaning DNA was sufficient for development).

Differential Gene Expression Theory

• The genome is constant in all somatic cells.

• Only a portion of the genome in any cell type is expressed.

• Different cell types express different groups of genes that confer unique cellular characteristics.

• Unused genes that are not transcribed are not mutated or destroyed; they retain potential to be expressed.

• Differential gene expression makes cells different and there are various ways genes can be activated or inactivated or activated for specific times depending on the cell type and the function it must perform. Transcriptional regulators are the general term for the category of proteins and molecules that cause differential gene expression

What makes cells adopt different fates?

• There are two major ways to make sister cells different. One is through cell autonomous or cell intrinsic mechanism and another is through non cell autonomous (cell extrinsic) signals. Cells become different through these two forms of signaling that prompt certain genes to be activated and others to remain inactive.

• Cell autonomous (intrinsic mechanism) signaling involves asymmetric division due to cytoplasmic determinants being localized to one part of the cell.

• Cell non autonomous (cell extrinsic) signals can be further broken down into two categories. Namely, inductive signaling (all or none or morphogen gradient) and lateral inhibition.

Cell Autonomous (intrinsic mechanism) signaling

• Cell autonomous or cell intrinsic mechanism (includes autocrine signaling).

  • One way cells become different are through cytoplasmic determinants. A original cell contains cytoplasmic determinants that are asymmetrically localized to one side of the cell. As a result, when the cell divides, one cell which didn't contain those cytoplasmic determinants is different from the one that did.

  • Cytoplasmic determinants are asymmetrically localized and then are inherited by one of the two produced daughter cells.

  • Sister cells are born different and asymmetric cell division occurs.

  • An example of asymmetric cell division using this is the first division of C. elegans (roundworm) where when the roundworm is at the two cell stage, the cells are different due to presence of different cytoplasmic determinants.

Non Cell Autonomous (extrinsic mechanism) signaling

• Non cell autonomous (cell extrinsic) signals that lead to differentiation come in two types inductive signaling (paracrine signaling) and lateral inhibition (juxtracrine or contact dependent signaling).

• Inductive signaling is a form of cell to cell localized signaling that either occurs in an all or none manner or signaling that occurs on the basis of a concentration gradient via morphogens.

• Lateral inhibition basically entails that cells born an equivalent cells can adopt different fates due to contact dependent cell signaling (juxtacrine signaling)

Inductive Signaling Overview (type of cell non autonomous signaling)

• Some inductive signals function in an all or none manner. This means some signals function in that they either are present and cause an effect or are not present or they either cause a response or they don't. Some inductive signals produce different responses depending on concentration.

• Inductive signals are examples of paracrine or can be juxtracrine signaling (signaling with cells nearby via direct contact or indirect contact through signaling molecules).

• Some inductive signals function in a concentration dependent manner (morphogens) or depending on concentration they have varying cellular effects.

• Inductive signals originate in certain cells and then spread such that surrounding cells are directed to new developmental fate. Certain cells are induced by others to adopt a certain fate.

All or None Inductive Signaling (type of cell non autonomous signaling)

• If a surrounding cell receives the signal then it produces an effect or the cell is directed to a new developmental fate, if it doesn't receive that signal nothing will happen.

• With this signaling type the cell can either adpot a given fate or function or not adopt it.

• Different from cncentration dependent inductive signaling.

Concentration Dependent Inductive Signaling (type of cell non autonomous signaling)

• Other inductive signaling molecules can function as morphogens in a concentration dependent manner.

• All morphogens are concentration dependent and this allows them to have various effects and work in generating organized forms in the cell.

• A morphogen is a diffusible molecule secreted from a source (signaling cell or cells).

• It forms a graded distribution (concentration gradient: higher near the source and lower further away).

• Cell fates or function is different depending on the concentration of morphogen recieved. At a far enough distance from the molecule expelling the morphogen, there is no signaling effect.

• An example of a morphogen gradient in development is the Shh morphogen that is involved in finger development among other things.

Morphogen

• A morphogen is a diffusible molecule secreted from a source (signaling cell or cells).

• It forms a graded distribution (concentration gradient: higher near the source and lower further away)

• A morphogen also produces a specific cellular response depending on its local concentration hence its role as a concentration dependent molecule in inductive signaling.

• Morphogens provide a mechanism to specify fates in a reproducible pattern.

• How morphogens work: the signaling cell releases the morphogen molecule and cells with receptors for that molecule and also depending on how much of that signal they receive can adopt different fates.

• Cell fates would be different if the amount of morphogen produced by the signaling cell has half its normal amount. Cells would more often adopt fates associated with lower morphogen concentration if this was the case.

Concentration Dependent Inductive Signaling Example

• Example of a morphogen gradient in development.

  • Shh = Sonic hedgehog, a morphogen that is involved in finger development but also functions elsewhere (brain, neural tube, hair, etc.).

  • Depending on the concentration of Shh morphogen around where the fingers grow causes cell differentiation. Higher concentration of Shh results in pointer, middle, ring finger, and lower concentrations result in the thumb. A lack of Shh results in the thumb.

  • Overexpression of the Shh gene results in polydactyly or extra fingers/toes.

  • Shh morphogen concentration is highest at the posterior or the hand (near the pinky) and decreases steadily as you go from the posterior to the anterior. The thumb recieves none of the Shh morphogen and is the furthest finger to the anterior of the hand.

Sequential Induction

• Adult tissues have many different types of cells working together.

• Sequential induction results in several cell types. Essentially a series of inductive signaling events occurs in which one group of cells induces neighboring cells to adopt a new fate. Then those newly induced cells then signal to surrounding cells, inducing additional cell fates and producing multiple distinct cell types.

• Sequential induction describes how the combination of cell autonomous and cell non-autonomous mechanisms can result in many cell types from just two.

• Basically cells induce cells which induce other cells to produce layers of distinct cell types.

Lateral Inhibition (type of cell non autonomous signaling)

• The second way that sister cells become different and the second type of cell nonautonomous (cell extrinsic) differentiation.

• Lateral inhibition basically entails that cells born as equivalent cells can adopt different fates due to contact dependent cell signaling (juxtacrine signaling). It involves one cell commiting toward a particlar fate while it signals to surrounding cells to remain uncommitted.

• Lateral inhibition plays a key role in making sure specialized cells stay evenly spaced. If there are a group of equivalent cells, some cells in that group compete by inhibiting neighbors meaning only some cells predominate and differentiate into cells with a specific structure or function in the resulting animal.

• Two examples of instances where lateral inhibition is crucial is the bristle pattering on the thorax of a fly and the early feather development for a pigeon fledging.

Delta Notch Signaling Function (example of lateral inhibition)

• How this works is that Delta and Notch are both transmembrane signal proteins and when they interact, specifically Delta interacting with notch, notch's intracellular protein domain gets cleaved by a protease and that intracellular protein eventually enters the nucleus to target genes and influence cell fate. Notch suppresses delta signal as well.

• In balanced delta/notch singling in identical cells where there are an equal amount of interactions between two cells, neither cell is favored to adopt a different fate through lateral inhibition.

• In unbalanced delta notch signaling where one cell may have more notch signals than delta signals and another has more delta than notch signals, one cell adopts a new fate and cells with more active delta signals win the specialized fate. The cell with more notch is like that because Delta's effect of cleaved notch causes suppression of the delta signal.

• Positive feedback of delta signaling mediates lateral inhibition. As delta increases and notch decreases in one cell, and as delta decreases and notch increases in another cell, one cell eventually basically only has notch and another cell basically only has delta, and the delta cell becomes specified toward a fate.

How Delta Notch signaling (example of lateral inhibition) leads to pattern formation in cells.

• The whole process of lateral inhibition in delta notch signaling leading to fate determination and pattern formation is outlined below.

  • During stochastic expression or random variation in gene expression between cells resulting in some cells expressing more of a particular protein, than others causing cell competition as some cells inhibit their neighbors. The cells are unspecified at this point.

  • Then some cells predominate and become specialized while inhibiting their neighbors and this is the direct process of lateral inhibition.

  • Then pattern formation occurs as cells begin to specify while other cells remain different.

Delta Notch Signaling (example of lateral inhibition)

• One example of lateral inhibition:

  • Delta Notch Signaling Can Mediate Lateral Inhibition. It's an example of a contact dependent cell to cell signaling (juxtracrine) mediated by membrane bound ligand (Delta) and membrane bound receptor (Notch).

  • Delta/Notch can also mediate inductive signaling meaning that their interaction is a type of inductive signaling or juxtracrine signaling.

  • Cells that receive the delta signal interpret the signal as a cue to remain unspecialized. Cells that do not receive this delta signal begin the process of differentiation starting with specification.

Summary of Cell Differentiation Processes

• Cells become different through cell autonomous (cell intrinsic) or cell non autonomous (cell extrinsic) signaling with cell non autonomous signaling requiring cell to cell signaling or cell to cell communication.

• Cell autonomous signaling results in asymmetric cell division.

• Cell non autonomous signaling occurs via lateral inhibition or inductive signaling (done via morphogen gradients in concentration dependent inductive signaling, but inductive signaling can also be all or none).

• Outcome of various signaling processes with different signals and different intracellular signaling pathways is that differential gene expression occurs resulting in cells adopting different fates.

Test of Specification

• Tests whether at a given point in development are cells specified to become muscle or neuron cells (or one cell or another basically doesn't have to be muscle or neuron).

• You can run an isolation experiment where you put the cell in isolation in a neutral environment (e.g. tissue culture) and ask what the cell becomes.

• If the cell becomes a different fate than it would normally, it was NOT specified and NOT determined (at the time of isolation).

• If it becomes a normal fate it is specified but may not be determined.

Test for Determination

• Tests whether at a given point in development are the cells determined to become muscle or neuron cells (or one cell or another basically doesn't have to be muscle or neuron).

• You can run an in vitro transplantation experiment where you put a cell or tissue in a new environment with cells that are different from itself surrounding.

• If the cell still expresses its normal fate despite the surrounding environment the cell is determined.

• If the cell does not express its normal fate and instead changes to the surrounding environment cell then it is not determined.

Levels of Commitment in Sequential Order

• Cell fate becomes increasingly restricted during development through a series of irreversible commitments.

• Cell differentiation is a spectrum by which an unspecified cell becomes specified and then becomes determined before becoming differentiated.

• Commitment to a particular fate is progressive. Specified -> determined -> differentiated -> terminally differentiated.

Example of levels of cell commitment

• A blastomere could be fated to become a B-islet cell can eventually become a B-islet cell that is terminally differentiated but it must first go through a number of intermediate steps. Blastomere → endoderm cell → pancreatic cell → endocrine pancreas cell → B-islet cell.

• A blastomere when specified and determined turns into an endoderm cell (step 1), then turns into a pancreatic bud cell (step 2), then an endocrine pancreas cell (step 3) and then a B-islet cell (step 4).

  • Each step in this process is irreversible and cells have memory of the cell types they were before. Cell fate also become increasingly restricted during development.

  • Specification and determination occur at every step of the commitment journey. Blastomere becomes specified for an endoderm but it can become a different cell until its determined. The endoderm in turn can also be specified for a pancreatic bud cell but then can become a different cell type until it is determined.

Cell Fate

Cell fate or developmental fate describes what cells will normally develop into.

Differentiated Cell

• A differentiated cell is a cell that has reached a stable state that is more specialized than it was previously. This can include intermediate states along a cell's journey to its final fate. A differentiated cell cannot avoid its fate and its fate is locked.

• The step where a cell has completed a step in the developmental program. It has the molecular and functional properties of that cell type. Note that this may not be the cell’s final fate—it can go through the process of commitment again to differentiate into a more specialized cell type. A cell that has reached its final fate and has stopped dividing is terminally differentiated.

• Differentiated cells are cells that have completed a step in the developmental program by acquiring some molecular and functional properties and can no longer avoid its fate (locked fate). It can include intermediate steps along a cell's journey to its final fate.

Terminally Differentiated Cells

• Terminated differentiated cells have become fully specialized and will no longer divide. These cells are usually in the g0 phase.

• For example, some skin cells become terminally differentiated like mature keratinocytes as they lose their nuclei and die ceasing to divide further while other skin cells like epidermal stem cells continue to divide and produce new skin cells.

Commitment

• Cells may have become committed to a particular fate even though they may not look differentiated.

• There are two phases in the process of commitment: Specification (commitment to a particular fate but in a labile or reversible manner) and determination (irreversible commitment to a particular fate).

• Determination eventually turns into differentiation as differentiation is the fulfillment of determination when the cell actually adopts the fate it was determined towards.

Specified Cells

• The early, reversible stage of cell commitment in which a cell is directed toward a particular developmental fate. The cell can still change its fate if exposed to different environmental signals.

• Specified cells have a reversible fate. They might not show signs of differentiation, but its developmental fate is restricted.

• Cells can be classified as specified but not determined if they pass a test of specification but fail a test for determination. All determined cells have already been specified.

Determined Cells

• The later, stable stage of cell commitment in which a cell's developmental fate becomes fixed. The cell will maintain its fate even if placed in a different environment.

• Determined cells have an irreversible fate and the cell has irreversibly committed to a specific developmental fate.

• Cells can be classified as determined if they have undergone a test for determination. All determined cells have already been specified.