Biologia dello sviluppo

Developmental biology

a.a. 2019/2020

1. Stem cells and regeneration ...................................................................................................................... 2

1.1. Cell communication and signalling .................................................................................................... 3

1.2. Pathways............................................................................................................................................ 5

1.3. Gene regulatory mechanisms ............................................................................................................ 8

2. Animal development ................................................................................................................................. 9

2.1. Cell movement and morphogenesis ................................................................................................ 14

3. Animals .................................................................................................................................................... 17

3.1. Drosophila........................................................................................................................................ 17

3.2. Xenopus laevis ................................................................................................................................. 31

3.3. Danio rerio ....................................................................................................................................... 42

3.4. Gallus gallus ..................................................................................................................................... 50

3.5. Mammals ......................................................................................................................................... 54

4. Eye development ..................................................................................................................................... 57

Some fundamental questions regarding neuronal cell type specification. ................................................. 61

5. Neural formation ..................................................................................................................................... 61

6. Left-right asymmetry ............................................................................................................................... 63

6.1. Techniques to confirm the L/R asymmetry ..................................................................................... 67

7. General techniques.................................................................................................................................. 68

7.1. Visualization

..................................................................................................................................... 73

8. Genetic manipulation .............................................................................................................................. 77

8.1. Transgenesis .................................................................................................................................... 77

8.2. Optogenetics ................................................................................................................................... 78

8.3. Gain of function techniques ............................................................................................................ 79

8.4. Genome editing ............................................................................................................................... 81

9. Applications ............................................................................................................................................. 83

9.1. Example I ......................................................................................................................................... 83

9.2. Example II ........................................................................................................................................ 84

9.3. Example III ....................................................................................................................................... 85

18/09/2019

1. Stem cells and regeneration

To develop into an organism, we need to undergo several processes like cell proliferation, specialization,

interaction and movement. A stem cell is able to develop in more than 200 different types of cells with

different characteristics like longevity and self-renewing ability: they are not fully differentiated, have an

unlimited capacity to divide and every daughter cell is a stem cell too or it can differentiate. They don’t divide

during all lifetime, but they undergo a quiescent state that allows to reach homeostasis of a tissue. If cell

divisions increase and apoptosis remains or vice versa we can potentially develop a tumour.

Stem cells are also characterized by asymmetric divisions: environmental asymmetry is driven by the cells in

the environment in which the cell is, but divisional asymmetry depends on some components of the cell that

are distributed asymmetrically in the mother thus can be inherited only by one of the daughters. A divisional

asymmetry can be observed in transparent fish embryos with fluorescent probes that show that only one of

the daughters inherits an apically located molecule.

Stem cells can have different potential to differentiate:

- Totipotency is the potential to give rise to any cell type of the body, performed by fertilised eggs

cells;

- Pluripotency, slightly restricted developmental potential, typical of cells that are found in the inside

of an early embryo; Commentato [e1]: Video early development of a mouse

- Multipotency, adult stem cells (living in gut, skin, bone marrow) that have a small differential

potency;

- Unipotency, potency to differentiate into a single cell-type, as happens to epidermis or olfactory

epithelium.

The older a cell becomes and the more its differential potency is lost. Gurdon is a scientist that in the 60s

cloned two albino frogs and that was recently awarded with the Nobel in medicine and physiology for the

discovery of the capacity of reprogramming adult cells to become pluripotent.

Another stem cells characteristic is that they can replenish other cells, in fact many cells in an organism’s

tissue have a life span of few days. For example, hair is formed by some stem cells that are conserved in a

niche near the follicle. Even gut cells are formed by stem cells that are present in a single-stem-cell niche

between two villi, that replicate and allows daughter to undergo differentiation during the migration

travelling to the villus. In this case if a mutation happens in the genome of the mother, all the daughters will

be affected: this happens with APC, an oncogene capable of starting a colon cancer. Olfactory system too has

its unipotent stem cells to replace olfactory neurons that have a brief lifespan. Blood stem cells are

multipotent and known as hematopoietic cells: they are located in the bone marrow and can generate all the

types of cells needed in the blood. The differentiation process is a multistep process,

important because in this way every step can be controlled

to obtain many types of cells from one single mother.

Controllable parameters are different in any stage of the

differentiation, to maintain an equilibrium state.

Stem cells carry mutations with high frequency because

they often replicate so it is not rare that a stem cell

originates a tumour because mutations can also enhance

the proliferation rate. They can also undergo an epithelial-

mesenchymal-transition typical of tumors, receiving signals

that stimulates to lose contact with the extracellular matrix

and enter the bloodstream to travel along it until the

inverse process happens and a metastasis form. Invasive tumours can be individuated with keratin signature,

also with antibodies that are specific for one type of cells. In this way, imaging can spot metastasis and where

they come from.

Necrosis is a non-controlled cell death due to traumas or lack in oxygen, that can result in an inflammatory

response. Differently, the controlled death (apoptosis) is a kind of suicide happening in a cell that doesn’t

liberate dangerous material in the environment. This second way of death is aimed to removal of damaged

cells, cells that are no longer needed or potentially dangerous, abnormal or misplaced cells. Apoptosis

happens during the limb sculpturing in the mouse or in tadpole tail degradation when becoming an adult

frog. This process can be stimulated by the contact with a K lymphocyte that starts the caspase mechanism.

This process is fundamental in embryo development, to obtain a correct organism and is carried out by

microglia in brain, that removes damaged neurons.

If too many cells die heart attacks or strokes may happen, and if too few cells die autoimmune disease,

lymphocyte cancer or cancer in general can occur.

Regeneration is limited in humans: it happens in liver, muscles and bones but not in limbs and after a spinal

cord injury. It is studied in some animal models like salamander and others. Hydra regenerates all its

complexity of shapes and polarity from a cell aggregate thanks to the Wnt mRNA, fundamental to produce

an extremity. Planaria can regenerate its head or near all of its structure if it is cut in pieces. Exploring mRNAs

that are expressed in the cut extremity we can individuate what are the genes that are more expressed during

regeneration, to investigate if human genome has those genes too. Zebrafish is active in fin regeneration.

There are 4 different types of regeneration, always stimulated by inducers that are given or present in a zone:

- Stem cells mediated regeneration, in which the actors are stem cells;

- Epimorphosis, in which cells that are in the environment can dedifferentiate and differentiate again

to one type of cell;

- Morphallaxis or transdifferentiation, in which cells go from a differentiate stage to another

differentiate cell without dedifferentiation;

- Compensatory regeneration in which one cell can regenerate cells of the same type in the same

environment.

Hopes in stem cells are to generate tissues and organs to cure dangerous pathologies, even if many countries

apply severe restrictions to use of these techniques.

Name different properties of stem cells. What are the different properties of stem cells, where can the

different type be found? How do scientists try to find genes important for regeneration? What is apoptosis

and why is it important for regeneration? 24/09/2019

Currently, there isn’t any naturally occurring trans-differentiation process (cells change their type without

dedifferentiating), and even oesophagus related processes are turned out to be false. Trans-differentiation

via dedifferentiation instead happens, for example in pancreas’ cells α and β. It is possible to induce the

dedifferentiation and differentiation of a cell, but the problem of induced pluripotent stem cells is that they

are still manipulated.

1.1. Cell communication and signalling

Cell interaction includes signalling needed to communicate between cells. Certain ways of signalling are in

common between pathways. Cells develop in the surrounding environment, in contact with their immediate

cellular neighbourhood and depending on their tissue identity and their position in the body. Developing cells

receive signals from each of these locations, and they, in turn, signal to the cells around them.

Categories of cell-cell signalling:

- Cytoplasmic connection between cells (free diffusion);

- Cell-to-cell contact mediated signalling;

- Cell contact independent signalling, including local signalling or long-distance signalling.

Signalling between cells happens with cell junctions (gap junctions in animal cells or plasmodermata in plant

cells) or recognition mechanisms. Small molecules like cAMP and ions can move from one cell to another. To

see that movement between cells are allowed, labelling of these molecules have been performed.

Signalling without cell contact is based on molecules that diffuse in the environment, as happens during

paracrine or synaptic signalling. Long distance signalling, differently from the two we have just seen, is

performed by endocrine signalling. Autocrine and paracrine signalling is defined on the base of the target of

the signal. Local (systemic) signalling, then, does not rely on the circulation in the bloodstream. Long-distance

signalling does instead rely on the bloodstream to get a response via receptors in the target far away from

the signal source. Membrane receptor independent signalling means that the receptor is inside the cell and

that the signal can diffuse inside it (e.g. androgen receptors that bind steroid hormones). The receptor than

is able to enter the nucleus and perform the binding of DNA aimed to regulate transcription to produce

proteins.

Stages of signal transduction:

1. Reception of extracellular signal by cell;

2. Transduction mainly through signalling cascades,

from the outside to the inside of the cell. Note that

not-necessarily the ligand is transduced;

3. Cellular response.

If a cell doesn’t receive signals it will undergo apoptosis,

because signalling is essential to survival, growth,

proliferation and differentiation.

There are three main types of membrane receptors: G-

protein-linked, enzyme-linked and ion channel linked. 3+

G-proteins are interactors that bind with a receptor inside the membrane with a positive NH part on the

-

outside and the COO negative extremity in the inside. The G-protein can exchange GDP to GTP in response

to the interaction and activate other proteins to perform a cellular response. An example of this receptor is

acetylcholine signalling cascade or epinephrine signalling cascade. Commentato [EP2]: video

Enzyme linked receptors are like RTKs or S/TTKs that bind the ligand, undergo the dimerization (or

oligomerization) of different receptors that stimulates the intracellular activation through phosphorylation

of some residues in the internal tail. Every activation position can be recognised by another factor to start

the cellular response. The precise steps that allows their function are: ligand reception, dimerization, catalysis

of the phosphorylation, subsequent protein activation, further transduction and response.

Ion channel receptors are activated by a ligand that causes the opening of the channel, allowing the enter of

many ions. They regulate the essential ion concentration within the cell and cell compartments.

Cascades of molecular interactions signals go from one target to one other and at each step the signal is

converted into a different form, commonly a conformational change in a protein. Multistep pathways can

amplify a signal and provide more opportunities for coordination and regulation.

The reversibility of the phosphorylation, the main modification in signalling pathways, is fundamental. A

series of protein kinases add a phosphate to the next one in line, activating it. Due to the reversibility, this

modification helps the dynamic nature of cells, meaning that signals can’t last forever and for the

continuation of the response, more signal must be received.

Second messengers are small, non-protein, water-soluble molecules or ions. They include for instance cAMP,

2+

Ca , IP and DAG. cAMP is made out of AMP manipulated with adenylate cyclase activated by G-proteins.

3

They activate also phospholipase that cuts PIP in IP and DAG that remains bound to the membrane. IP acts

2 3 3

as a messenger that opens the calcium IP gated channels to trigger many cellular responses.

3

Cellular responses can be fast (second to minutes, based on direct activation of proteins) or slow (mins to

hours, needed to act on transcription inside the nucleus and translation). Cytoplasmatic (fast) response

8

induced by epinephrine involves a phosphorylation cascade that amplifies the signal of a 10 factor.

Epinephrine is involved in stress response but also in fight or flight response. Commentato [e3]: Video

Cell signalling is highly specific. Same ligand gives rise to different responses, cell differ regarding their protein

content, different proteins respond differently to the same environmental signals, different cells behave

differently because some, but not all proteins can differ between cell types and same receptors have different

relay based on the intracellular proteins.

Scaffold proteins work allowing the activation of many proteins on the same moment, with high efficiency

due to the maintenance in the same environment of targets and activator proteins. 25/09/2019

How do cells communicate? Name the different types of membrane bound receptors and what type is used

by adrenalin? What are second messengers and what do they do?

1.2. Pathways

Fight or flight response stimulates amygdala from environmental signal: signals are sent to hypothalamus

and in the pituitary gland, where adrenocorticotropic hormone is released into blood. It travels, with a nerve

pulse to adrenal gland to induce cells to release adrenalin into blood. ACTH binds MC-2 receptor on adrenal

cell, changing its shape and activating the binding of the G-protein that stimulates the action of adenylate

cyclase that produces cAMP. cAMP activates PKA, that releases its catalytic subunit which travels to the

mitochondria to switch on StAR protein, capable of importing cholesterol into the mitochondria. On the

inside of this organelle, cholesterol is converted into pregnenolone, transported to the ER and converted into

deoxycortisol. This last is sent back to the mitochondria and converted into cortisol, which diffuses through

membrane, travelling in the blood to increase its pressure, to increase the presence of sugar and to suppress

the immune system to ensure that all the attention in the organism is focused on the ongoing response.

Adrenaline travels also to different cell types. On liver cells it binds adrenergic receptor and causes its shape

to change. Inside, the G-protein gets activated and uncoupled, binds phospholipase C which produces IP3.

2+

This binds receptors on ER, resulting in Ca release that stimulates phosphorylase kinase to release glycogen

phosphorylase. This enzyme breaks glycogen into glucose subunits that go from the liver into the blood. In

the skin, adrenalin binds receptor on smooth muscle cell causing contraction. On sweat glands, adrenaline

binds adrenergic receptors causing gland contraction. In the lungs, adrenaline induces signalling cascade

relaxing muscles around bronchioles to increase respiration.