Biologia dello sviluppo

Developmental biology

Luca Mennella

Date: September 11, 2024

University of Trento

Biomolecular Science and Technology

Via Sommarive 9, 38123 Povo (TN), Italy

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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.

Differentiation is possible only if the cells are moving in all directions. They are all coordinated, for example during gastrulation.

Mammalian development is initially very similar to other animals’ development. 4 main coordinate processes during development of multicellular organisms are needed:

• Cell proliferation
• Cell specialization
• Cell interaction
• Cell movement

This allows from one fertilized cell to obtain more than 200 different cell types with different functions in different body districts; this happens through cellular differentiation of a stem cell. All come from one cell, which needs to become more cells.

Stem cells

Cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. We have only 200 major types of cells, which derived from 1 single cell. Stem cells have the feature to become all of the cell type of our body. (POTENTIAL) they potentially divide in an asymmetric way. Asymmetric division is an important point: single cell can become different type of cell. They can be multipotent, totipotent, ecc. Therefore, they maintain this ability to transform only for a limited period of time, otherwise they become cancer cells. Another important point of stem cells is longevity: they can divide forever (in theory).

The main stem cell’s characteristics are:

• Longevity/self-renewing
• Quiescence
• Asymmetric cell division
• Potential to differentiate (toti-, pluri-, multi-, unipotency)
• Replenish other cells

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Longevity/self-renewing

Stem cells are not fully differentiated and are characterized by an unlimited capacity to divide (in culture). Every daughter cell can either stay as a stem cell or terminally differentiate. You can keep a stem cells culture forever (theoretically). In our bodies they can generate cancer.

Quiescence

Stem cells do not continuously divide, and they need activation to divide which also depends on apoptosis. They are not allowed to divide all the time. Normally in the body there are normal cells which divide in an equilibrium, and this is called homeostasis. If we increase the cell division or decrease the apoptosis, we form a tumor.

Asymmetric cell division

There are two types of asymmetric cell division:

• Environmental asymmetry: one of the 2 daughter cells receives signals from the environment, that is from surrounding cells, to differentiate; the other is in a different environment and does not receive that signal, therefore it does not differentiate. So surrounding cells influence daughter cells.
• Divisional asymmetry: proteins distribute asymmetrically during cell division and are therefore inherited by one cell, not by the other; again, one cell differentiates while the other remains staminal (either by keeping or losing proteins) depending on the protein subset. To summarize, molecules are not equally inherited by daughter cells. For example, in the animation we can see two nuclei but only one has inherited the proteins.

Potential to differentiate

Stem cells are not all equal, some can give rise to more, others to less cell types:

• Totipotency: give rise to all type of cells and embryonic annexa (the fertilized egg is totipotent).
• Pluripotency: give rise to cells from the three germ layers, not to embryonic annexa/extraembryonic tissue, those cells are more restricted, embryonic stem cells of the early 3 embryo (slightly restricted developmental potential). From ICM are pluripotent; those cells would be useful for experiments but cannot be used in all countries. These cells have a restriction in term of differential potential.
• Multipotency: have a restricted developmental potential, usually restricted to a certain tissues, adult stem cells in gut, skin, bone marrow...; those cells allow self-renewing of tissues and organs.
• Unipotency: have the potential to differentiate into a single cell type, like epidermis olfactory epithelium.

In blastocell there is a cavity called blastocoel and an inner cell mass which is composed of up to 256 cells which are considered pluripotent.

Progressing with development, cells lose their potential. For example, experiments of nuclear transplantation in frogs revealed that the earlier the nuclei from a donor were transferred, higher the chances of survival. This also proved that an adult nucleus is able to make a cell develop into an organism showing that it codifies for all the required “information”.

In 1962, during his PhD Gurdon applied nuclear transfer to X. laevis using albino frogs nuclei as donors, those nuclei reprogrammed to give rise to albino frogs, going back to pluripotency stage. This discovery led him to Nobel prize.

In 1996/97 nuclear somatic cell transfer (NSCT) was performed also in mammals generating Dolly. This sheep was born from an embryo enucleated and transferred with the nucleus of another sheep somatic cell. The embryo was implanted into a different breed surrogate mother. Dolly was born as a clone of the mother who gave the nucleus, it had a shorter and less healthy life compared to other sheep, but her “natural” daughter had a normal life, this difference is probably linked to epigenetic modifications in the nucleus during development which were not reversed with reprogramming.

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Replenish other cells

Different cell types have different life spans (intestine – few days, epithelia – 120 gg, muscle/heart/lens/neurons – life long...). Stem cells from hair follicle are multipotent. Stem cell niches are distinguished between those which contain several stem cells and those with one stem cell. Hair grows because they have stem cells in the bulge.

Stem cells from gut epithelium are multipotent and their activity is continuous, replacing dead cells. Those cells divide slowly and move upwards from the crypt, where are located, towards the surface or the villi, undergoing differentiation and passing through progenitor stage. This could be uncovered thanks to the use of transgenic models which allow to follow the cells expressing a particular marker. It has been discovered that APC, a Wnt pathway inhibitor if deregulated allows continuous proliferation and therefore its mutations are associated, in particular to colon and gut cancer.

The replenishment needs to be quick, and it has to happen in dangerous situation.

Developmental biology does not happen without stem cells. You choose the animal model in order to answer specific questions. Mouse are used for animal questions.

Crypt in the intestine: embodiment in which differentiated cells are originated by stem cells. Colon cancer develops from cells that originate as stem cells in the gut. Tumor suppressor gene APC: this is one of the gene involved in signaling pathway. So, if we have mutation in this gene, the proliferation of cells increases and develop tumor cells.

Stem cells from olfactory system are unipotent with a 1-month life span, the continuous renewal of olfactory neurons starts from basal cells. The division is asymmetric. Basal (stem) cells are unipotent. Mettere immagine

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Blood cells have to be generated constantly, in particular after blood loss. Stem cells of the blood are called hematopoietic stem cells (HSCs) and are multipotent, divide slowly and generate quickly dividing transient amplifying cells. HSCs are located in the bone marrow (1 out of 100000 cells). HSCs are multipotent and give rise to many cell types. The cells exit from quiescence induced by factors; it is a multistep process to ensure regulation and control at each step through division frequency, cell death, number of committed progenitors division before differentiation, differentiated cells lifetime. This stem cells are quiescent but can be activated in the back when we lose blood. They are multipotent. They divide very slowly and become committed transit amplifying cell in order to increase the pool of stem cells. The intermediate steps are important because in each step is possible a control of the process and the alteration probability decrease significantly.

We need controls at every level. The multistep mechanism of blood cell development ensures optimal control.

Stem cells and cancer

Stem cells are involved in cancer due to deregulation of proliferative mechanisms, in particular a reduced number of apoptotic cells combined with normal cell division or increased cell division associated with normal apoptosis. Due to their continuous proliferation stem cells are prone to accumulate mutation, if that occurs inside important gene/promoters/enhancers, the risk of malignancy increases, and those cells will keep generating malignant cells (e.g. B-lymphocyte leukemia), making the tumor continuously growing. If they have too many cells division and proliferation we have tumors, if the apoptosis is altered, we have tumors.

Mutations in stem cells or transit amplifying cells can influence the proliferation rate.

Stem cells: accumulation of mutations due to life long proliferation (DNA replication).

Transient amplifying cells: mutations can confer stem cells properties, e.g. proliferation, self-renewal (B-lymphocyte leukemia).

This transit amplifying cells will transfer the mutations inherited by the stem cells to the daughters. Mutation can influence the proliferation rate. The accumulation of mutation led to too long proliferation and in determinate cases tumors.

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Cell death

There is also cell death not only proliferation. There are two forms of cell death:

• Controlled death, the apoptosis. Controlled cell suicide often occurs by apoptosis. Dying cells shrink, chromatin breaks up, cytoskeleton collapses and cell surface becomes chemically altered. Macrophages engulf them without triggering an inflammatory response. Many purposes:
  • Removal of irreversibly damaged cells.
  • Removal of cells no longer needed (hands, feet, tail, lymphocytes after infection is cleared).
  • Removal of potentially dangerous, abnormal or misplaced cells (non functional T and B lymphocytes).
• Not controlled death, necrosis. The cell just died in an uncontrolled way. It is a not controlled cell death due to for instance an acute insult (trauma, lack of blood supply). Cells swell and burst resulting in an inflammatory response.

Apoptosis is needed during the development of frog. Initially all of us has “pinnate” hands, but through the development we lose some the cells in order to have normal fingers.

Simplified apoptosis mechanism

Apoptosis is mediated by FAS receptor expressed on apoptotic cells binds to Fas ligand on macrophages forming DISC complex that leads to caspase activation. Mutations of genes associated with apoptosis are often lethal before born. An example is the role of microglia engulfing apoptotic neurons.

Only cell that want to be killed are making this receptor that are presented to the outside and will be bound by caspase. The apoptotic cell needs to be removed because it can originate inflammatory response.

For example, microglia are called the guardians of the brain because they remove apoptotic neurons in the brain.

Altered programmed cell death is linked with human diseases. Too many cells die:

• Heart attacks: mainly necrosis due to ischemia
• Strokes: mainly necrosis due to ischemia

Too few cells die:

• Autoimmune diseases: mutation in Fas ligand or receptor causes lymphocyte survival.
• 7 Lymphocyte cancer: overactivation of the Bcl2 gene prolongs cell survival.
• Cancer in general: tumor suppressor gene p53 is mutated in 50% of human cancers.

Regeneration

Regeneration is limited to some organs e.g. liver, muscle, bones, but not CNS (spinal cord injury) and limbs (amputation). To find genes involved in regeneration multiple model organisms have been exploited: hydra, planaria, salamander, Zebrafish. Some of those genes might have been lost or assumed a different function in humans. Hydra is a lot far from us in terms of evolution.

Hydra

Hydra is characterized by radial symmetry and is able to regenerate from aggregates of cells; in situ hybridization ISH of Wnt, mRNA was found during regeneration of head, meaning that Wnt pathway is involved in regeneration. In hydra, you can displace the organism in single cells and each of them are able to re-applicate and form another organism of hydra.

Some of the signals involved the Wnt mRNA. In situ hybridization: we can visualize the messenger RNA for Wnt gene. Only we can knock out and identify the loss of function, we can understand the function of the gene.

Planaria

Planaria has 2 photosensitive cells, is cheap, if chopping a part, that will regenerate giving rise to another planaria in 14 days because 20% of its cells are multipotent, which are important for the regeneration of cells and tissues. You cannot destroy planaria because the replication abilities are incredible.

Extracting mRNA from regenerating planarias allow to detect genes expressed during that moment (expression profile) through candidate gene knock out/down. You always need a control for a comparative analysis of the gene that are expressed.

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Salamander

The limbs of a salamander can regenerate between 1-3 month.

Zebrafish

The tail fin regenerates in 3 weeks and is completely functional, meaning that cells must have memory of what they had been.

4 types of regeneration

In this case, stem cells mediated regeneration give rise to a particular type of cells in our body. It is a highly controlled process.

• Stem cell-mediated regeneration that occurs in hair, blood; multipotent SC get signal to give rise to different cells.
• Epimorphosis: differentiated cells get signals to dedifferentiate e.g. pancreatic cells, salamander limbs.
• Morphallaxis (transdifferentiation) is the direct conversion from one cell type to another, e.g. hydra.
• Compensatory regeneration: differentiated cells can regenerate e.g. liver.

NB transdifferentiation is not known in human, only induced transdifferentiation. In fact, there is an induced transdifferentiation of pancreatic α-cells into β-cells.

Stem cells grow in medium containing growing factors allows differentiation into cells of interest.

Glia cells support neurons: fixation of neurons in place, supply nutrients and oxygen, separation of neurons, removal of useless neurons. You can use retinoic acid to obtain differentiation in neural stem cells.

Stem cells can be exploited in therapy like for ALS (Amyotrophic Lateral Sclerosis) in mouse, however induced SC (iPSC) are not as good as embryonic SC which cannot be used in all the countries.

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We take somatic cells and use them to reprogramme in iPS cell and programme them to differentiate in a specific type.

The advantages of using stem cells are:

• Therapies

The contras are:

• Ethical discussions: for example, on the level pluripotent stem cells. So prelevate stem cells from fetal. In Germany you can use stem cells that were generate before 2007.
• Religion

Cell interaction

Cells communicate thanks to ligand-receptor interactions and transduce signals to determine changes in the expression pattern or proteome to induce a response to a given “message”. Cells development is also subjected to the interactions due to neighboring cells, tissue identity, position in the body and each cell also gives signals to the ones close by. An important signaling pathway involved in development is Wnt signaling. Others are p53, SRY gene in chromosomal Y, hedgehog (HH), RTK signaling (FGF, TGFb), Notch/Delta pathway.

Cells develop in the context of their environment, including:

• Their immediate cellular neighborhood
• Their tissue identity
• Their position in the body

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

Cell-cell signaling is divided into 3 categories:

• Cytoplasmic connections between cells (free diffusion)
• Cell-to-cell contact mediated signaling
• Cell contact-independent signaling

• Local signaling
• Long distance signaling (hormones, etc.)

Reception means Molecule-to-Molecule Contact.

Signaling with cell-cell contact

Small molecules like Ca²⁺ and cAMP and other ions can diffuse through gap junctions, marking those molecules with fluorescent dyes allows to discover those mechanisms. Cells can react to signals quickly and coordinated. Through ligand–receptor interactions, transduction occurs thanks to receptors’ cytoplasmic domain. An example is Notch signaling.

Gap junctions are important for the transport of small molecules. The size of the molecule is an important factor. To analyze the dimension of the molecules, we can isolate the vesicles and label them with fluorescent markers.

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Signaling without cell-cell contact

There are signaling