Developmental Biology of Animals Quiz PDF

new Master\'s programme; oral exam at the beginning of January State exam ---------- Cell and Tissue Physiology, Pathobiology, Developmental Biology, Methods of Experimental Biology Syllabus -------- 1. Introduction to developmental biology, principles of morphogenesis and its regulation (M...

new Master\'s programme; oral exam at the beginning of January State exam ---------- Cell and Tissue Physiology, Pathobiology, Developmental Biology, Methods of Experimental Biology Syllabus -------- 1. Introduction to developmental biology, principles of morphogenesis and its regulation (MB) 2. Regulation of cell differentiation and preservation of cell differentiation status (TB) 3. The role of cell death in morphogenesis (MB) 4. Epithelial mesenchymal transformation in development (TB) 5. The role of extracellular matrix in development and adhesion (TB) 6. The role of senescence in development (MB) 7. Regulation of cell and organ size in development and examples of their necessity (TB) 8. The role of cell migration and polarity in development and examples of cell disorders (VB) 9. Role and regulation of epithelial branching in morphogenesis (MB) 10. ~~Influence of physical phenomena on development (TB)~~ 11. Regulation of symmetry/asymmetry in tissue formation (MB) 5/10/20 Lecture: Introduction to Developmental Biology (MB) =================================================== 1. Morphogenesis 2. Classification of morphogenetic processes 3. Morphogen definition, examples 4. Shaping 5. Main signaling pathways examples in development Morphogenesis ------------- ### Developmental biology includes: - Prenatal (embryology) and postnatal development - Growth - Differentiation - Metamorphosis - Tissue repair - Regeneration - EVO/DEVO = The process by which **a complex** multicellular organism is formed - Structural and functional cell reorganization - Creation of complex 3D structures - Cell group interactions - Regulation in space and time ### Basic nomenclature - **morphogenesis** = the main process by which a complex multicellular organism is formed - reorganization of the shape of groups of cells into tissues (3D) and further into organs - precise coordination - **reorganization of shapes** -- structural and functional, tissues from different progenitors and from different sites of the embryo (boundaries between certain types of tissues, e.g. an area where the regulation of intracellular signaling is not very well known and how cell movement is specified during embryogenesis???) - **Cell group** interactions - Spatio-temporal precision of development management! - e.g. cell cycle (BC), regulation of intracellular signaling, etc. - **processes involved in morphogenesis:** cell proliferation, differentiation, migration, growth and cell death - **morphogen** = secreted factor produced by cells that creates **a concentration gradient** in the tissue and thus gives **positional information** to other cells - How are complex 3D structures created? - e.g. cavitation, apoptosis, A-P polarization (development of glands, ducts) - How are tissues formed from cell populations? - e.g. rearrangement/fusion of progenitors (muscle formation) - How do organs consist of tissues? - proper interaction of different embryonic tissues - How are organs formed in specific places of the organism? - thanks to segmentation clocks in the formation of somites in early embryogenesis (organization of the collective behavior of differentiating cells) - interdigital spaces, tail, M -- e.g. cell death - How do organs and their cells grow? - e.g. development of limbs (symmetry coordination required -- see last lecture) - development of the intestine (necessary coordination of regeneration -- replacement/renewal of the epithelium and rate and polarity of mitoses) - How do organs acquire polarity? ### Processes involved in morphogenesis: - Cell proliferation - Cell differentiation - Cell migration - Growth - Cell death Classification of morphogenetic processes ----------------------------------------- During the entire process of morphogenesis, the following phenomena occur, depending on the ability of a particular tissue: - **condensation** (differentiation of bones, tendons, muscles) - **cavitation** -- blastocyst, gland lumens (lungs) - **MET** -- dispersed cells gain polarity and the ability to adhere, lose mobility (kidney development, somitogenesis) - **epibolism** -- a process where epithelial cells overgrow (during early embryogenesis), overgrow the future entoderm, which closes inwards - **involution** -- change of curvature - **invagination** -- protrusion (formation of germ sheets -- cell reorganization, formation of ectoderm derivatives such as nails, hair) - **EMT** -- loss of polarity, ability to migrate (disintegration of the secondary palate, neurulation) - **convergent extension** -- narrowing and elongation of structures along its axis (neurulation, elongation of the tail) - **branching** -- lungs, kidneys, salivary / mammary glands - How are these processes managed? - are controlled by morphogen gradients - The amount of morphogens is important - Target cell response ability - tissue specificity - Ability to trigger signaling and complete transcription Morphogens ---------- Morphogens are secreted factors that create a concentration gradient. This gives positional information to the cells. They induce a specific cell response depending on the gradient. **The fate of the cells is specified by the concentration of the growth factor**. Morphogens thus determine the control of proliferation of a specific area, define the polarity of cells, and thus coordinate the growth and modeling of the organ. - Depending on the purpose, morphogens can be used repeatedly, even with each other! - The main signaling molecules, referred to as morphogens, are: - **Shh, FGF, acid. Retinoic** ### **Intracellular** gradient of morphogens - **Bicoid** = gradient of transcription factors (TFs) within a single cell or in **a cell** (in **embryonic syncytium**) - indicates **the A-P** axis of Drosophilas (the first 13 divisions without cell division), more of it is in the anterior part of the embryo - first the RNA of Bicoid is formed, then the repression of the target genes - **VegT** = gradient of signaling molecules that are secreted and can pass between cells - in the future ectoderm - important for the early stages of Xenopus, it is on the vegetative pole and extends to the animal - gradient established before cellularization and persists into the blastula - **FGF8** gradient = gradient involved in somitogenesis (RNA forms a gradient) - in the most caudal region (in the tail), the ligands of FGF8 are the most - Moving to the head end accompanied by disintegration of the FGF8 transcript ### **Extracellular** gradient of morphogens - is more common **in embryogenesis** than intracellular gradient - It locally restricts transcription and produces a secretory protein that travels between cell populations, so it is a **diffusion** of morphogens **from a source** in the extracellular space - If a ligand **discovers its** receptor **along the way**, an **interaction occurs** and this affects the cellular response itself. The functions **of the receptor** are crucial. The receptor either stabilizes the ligand on its surface or targets the ligands for endocytosis/lysosome for degradation. **On the other hand, the ligand** does not necessarily respond only to receptors on the **cell surface**, but can also interact with **secreted** proteins or with the extracellular matrix (**ECM**), which then leads to an enhancement or weakening of extracellular movement. Ligands can also be **modified post-translationally**, e.g. by covalent lipid binding (plasma membrane linkage, PM). Both the biochemical and biophysical properties of ligands are important, as the latter affect the intensity of embryonic tissue transport. An example of the use of the extracellular gradient of morphogens can be **the formation of a neural tube**. There is a gradient of the Hh pathway in the inner part and the Bmp pathway on the dorsal side. Or **finger identity stinging**, where the Shh path in the 5th finger is most involved, and then it is anteriorly lowered to zero (the thumb is thus independent of Shh). #### **Movement** of morphogens in the extracellular space There are several models for the movement of morphogens in the extracellular space, including complex mathematical descriptions. We will introduce a model of a drunken sailor here. **The model of a drunken sailor** is immobile. The source of drunken sailors (morphogens) is a ship (cell). Sailors arrive at a port and randomly begin to move (random movement of each molecule through the target tissue). Each sailor takes a certain number of steps of a certain size, but they move in a random direction. There are the most sailors on the ship, and fewer and fewer on the ship. However, the random direction causes them to bump into each other, and each impact changes the direction of their movement (analogous to the diffusion of motion that leads to the formation of a gradient). ##### Simple **Diffusion** Model If sailors moved further and further away from the ship, their number would gradually decrease (over time, the morphogens in the tissue would be evenly distributed in the embryo and a gradient would not form). ###### Controlled **diffusion** model Therefore, there are several solutions -- various attractants and regulators cross their path -- pubs (clearance = attractant = negative regulator!), ladies (ECM), policemen (positive diffusion regulators, they bind to morphogens and control diffusion). #### How is the movement/transport of morphogens mediated? a\) Transport mediated by interaction with **HSPGs** **Heparan sulfate proteoglycans** affect signal transduction, morphogenesis stability, exocytosis and intracellular transport (Hh, Wnt, FGF pathways). These include: glypicans, syndecans, pearlecans). - **Dally** -- glypikan - **Dlp** -- glypican, both are used for movement of Hh in the disc of the wing of Drosophila (co-receptor Fzd from the Wnt pathway) b\) Lipid-mediated transport These lipids must be able to bind morphogens. It then forms a complex: **morphogen** + **lipid** + **lipoprotein**. They play an important role in the Hh and Wnt pathways, where they serve for posttranslational modifications **of palmitic acid** or **cholesterol**. It completely solves the problem of transport by simple diffusion (the need for a permeable form). Lipoprotein bodies (LPBs) are often deposited in vesicles, so-called extracellular vesicles (e.g. exosomes). These are formed from proteins from the Golgi complex (GK). After the formation of multivesicular bodies (MVBs) and xocytosis into tissues/organs, they serve as transporters = **lipoprotein particles**. It is therefore used to transport cholesterol and other lipids (and proteins) from the site of synthesis to elsewhere (to the liver, etc.). These particles are also involved in the formation and development of Drosophila wing discs (when lipoprotein levels are reduced, long-term signaling is lost, but the short-term response is not affected). LDL receptors **are also an essential part of the mentioned signaling pathways**. There is a whole large family of them. It forms an extracellular domain containing a ligand, a domain in the endosome for ligand release, and a cytoplasmic domain for signal transduction into the cell. One such LDL receptor is, for example, LRP6 in Wnt signaling (development of the forelegs and pelvic limbs). c\) Transport mediated by **binding** to **soluble** partners They can increase or decrease the activity of the ligand, and often have dual functions, e.g. cleavage. Such soluble factors are, for example: **Sog** (in the Drosophila embryo necessary to establish the boundary between the neuroectoderm and the epidermal ectoderm) and **Dpp** (in Drosophila it works only after cleavage by Tolloid, thus releasing the Dpp and allowing signaling towards the ventral cells). Model for transport **by transcytosis** On the border between intracellular and extracellular transport is the process **of transcytosis**, where morphogens travel from one cell to another through endocytosis (intracellular transport), pass through the cell and finally go out through exocytosis (extracellular transport). This typically happens with the so-called S. **sticky = \"sticky\" proteins** (lipid-modified proteins). The process is such that the ligand binds to the receptor (extracellularly) first, the ligands are activated in the PM, form a complex, the cell is internalized and the entry occurs (this is a necessary process for the activation of cytoplasmic components and the start of subsequent transcription). Sticky protein transcytosis can be easily interrupted by enclosing in a lysosome, where it can be degraded. With Drosophila, the so-called Snobollah is taking place. **basolateral transport**, which is a type of transcytosis where ligands are secreted into the interstitial space and transferred further by passive diffusion (e.g. regulators transfer of Rab5 =GTPase, clathrins, dynamins, Rab11 to endosome recycling). **Planar tranytosis** is a process in which cycles of endo and exocytosis are repeated (e.g. in the wing discs, Dpp in the A-P region and Wingless in the D-V region). *Model for transport **through cytonomas*** **Cytonomas** are thin projections of PM based on **actin**, they are able to grow to morphogen-producing cells. After binding the ligand to the receptor, activation occurs and cytonomes act as second **messengers**, the cells do not retain morphogen, but only **attract it** in **different directions**. Cytonomas (protrusions attracting morphogen) therefore require cell contacts and precise coordination. However, this is a very **slow movement**, which is completely lost in the cytoma in the inactive state. Gradually, the signal strength decreases. Morphogens are transported through cytonemes, e.g. in the blastocoel of early embryos (the union of ICM (inner cell mass) and trophoectoderm), in the wing disc. ### **Interpretation** of the morphogen gradient by cells Interpretation means **the ability to distinguish the concentration of a given morphogen.** The interpretation of the gradient occurs in several steps, with the cell\'s response depending on the **number of receptors activated**. The relative activity of receptors is indicated by a catalyst, which is **ubiquitin-ligase**. It is the designation of a morphogen to degradation by a lysosome. The signalling can thus be terminated due to degradation. However, in vivo evidence is missing. Forming shapes in the embryo -------------------------------------------------- The formation of shapes in the embryo is referred to as **patterning**. It is the shaping of complex structures due to multiple morphogens (synergistic/antagonistic action). Two theories have been proposed for this process: 1. **Positional Information Theory** (FR Flag Model) - When a segment is transferred, the cells remember, but at the same time they listen 2. **Reaction diffusion model** (Turing model) - interaction of multiple morphogens, the first diffuses slowly (activator), the second acts as an inhibitor -- the interaction maintains spatial structures (tiger, zebra,\...) 3. **Salt and pepper function** (???) Signaling in the embryo ----------------------- Cells are constantly in communication during development. This is done by signaling pathways: - **FGF signaling -** limbs - from the receptor family of tyrosine kinases, serves for phosphorylation of residue Tyr - Course: ligand + receptor → dimerization → conformational change → signaling start - e.g. enchondral and intramembranous osophification - disorders → **achondroplasia** (activation of FGF3 stops the growth of long bones, loss of FGF4 blastocyst lethality or loss of FGF8 lethality in gastrulation) - **Hedgehog HH signaling -** limbs - course: modification of ligand → secretion into the extracellular space + Patch receptor on the cell surface → release of repression of the transmembrane molecule Smo → into the cilion → accumulation of activator proteins - e.g. limb patterning (ZPA -- a lot of Gly1, aneterior -- Gly3) - **BMP signaling -** skeletogenesis - 2 classes of receptors, homo/ heterodimers - Transforsforylation of the 1st receptor - inhibition by Noggin, Chordin - e.g. skeletogenesis, Darwin\'s finches (Bmp4 levels determine the size and thickness of the beak) - **Wnt signaling** -- body axis - Fzd receptor - canonical (stabilization of β-catenin in the presence of Wnt ligand) -- e.g. induction of the secondary axis of the embryo, important for the onset of gastrulation - Upregulation leads to duplication of the spine - non-canonical (activated by G protein) -- release of Ca inots, activated PCP signaling due to Wnt5a - Gradient: tail \> head - **Notch signaling** -- regulation of asymmetrical separation - process: binding of the transmembrane receptor to Notch → proteolysis → release of the intracellular domain → modification of target proteins in the nucleus - e.g. regulation of asymmetric division and polarity of the embryo, placentation, embryo implantation, angiogenesis - Disorders: Alagille\'s syndrome 12/10/20 lecture: Regulation of cell differentiation and preservation of cell differentiation status (TB) ================================================================================================ 1. Differentiation potential 2. Stem cells (basics and classification) 3. Differentiation mechanisms 4. Dedifferentiation 5. How to change cell fate (Reprogramming & Transdifferentiation) How do cells get their own destiny? How can this fate change then? Differentiation potential ------------------------- Stem cells (SCs) ---------------- The stem cell retains the **ability to divide** and re-regenerate **(self-renewal)** while also having the ability to generate offspring capable of specializing in a more differentiated cell type. They therefore have two main tasks: **to proliferate** and **to differentiate**. ### Classification of stem cells The best way to classify SCs is by their **potency**, i.e. the ability to differentiate. In other cases, they are divided into **embryonic stem cells** and **tissue/organ-specific stem** cells (adult SCs). ### Embryonic SCs They come from **inner cell mass** = **embryoblast** of pre-implantation embryos (see video). During the cleavage of the zygote, the body axes are already specified. Several consecutive grooves give rise to morule, in which blastocoel is formed from morula is blastula. An envelope is formed all around = **eucida**. The embryo itself arises from the **epiblast**, which gradually generates all cell types. **The trophectoderm** and **primitive endoderm** give rise to extraembryonic structures such as the embryonic side of the **placenta**, **the chorion** and **the yolk sac**. If we isolate the embryoblast and put it in a suitable environment, embryonic SCs capable of self-renewal and differentiation can be isolated. Pluripotency is then maintained by transcription factories Growth factory in the medium, **cell contact** and **ECM are needed**! By changing this policy, we allow cells to differentiate into specific types (by the presence of other GFs, ECM or co-culture with other cell types). ### Adult SCs They are normally responsible for maintaining **homeostasis**, **keeping the tissue** **cell populations** (**morphogenesis**), various structures important for embryogenesis or related to adaptation to a certain environment must be removed. E.g. **Elimination of the tail in amphibians during metamorphosis**. Apoptosis induction occurs in the area of the spinal cord - **spinal ganglia** - motor neurons die and eventually the muscle cords around the spinal cord degrade. Muscle fibers are separated from the EC mass and are eliminated. In addition to amphibians, we can also see the surrounding structures in other vertebrates -- including humans -- reduction by apoptosis during the embryo period, if it fails, the tail structure remains -- surgical removal. Another typical example: degeneration of internal organs, e.g. during the development of excretory and genital tracts, such as pronephrosis and mezonephrosis in amphibians and in some amniotes. Furthermore, **regression of the umbilical cord** after birth. The umbilical cord is eliminated in two ways -- high levels of proteoglycan in the umbilical cord through blood vessels -- apoptosis of blood vessels and ligaments. Only a thin ligament remains from the cord. Another example is the creation of **interdigital spaces**. Shaping of tissues and organs ----------------------------- It used to be assumed that apoptosis was only used to remove structures. However, analyses now show that they also play a role in **the modulation of** autopodium or the shaping of folds in interdigital spaces -- genes helping tissue remodelling: metalloproteinases, FGF18, Hmgn1, etc. **However, there are no changes in the expression of chondrogenesis genes** (Sox9 or alcian blue). ### Interdigital spaces -- apoptosis and tissue remodeling -- What is the mechanism controlling cell death synchronicity? The interdigital region is used as a model for controlling **collective cell death** -- apoptosis is induced simultaneously over a large area. Apoptosis is induced by external factors. However, the cells **do not respond simultaneously**, but can produce a number of **death factors on their own**, which can then control the entire cohort of cells and induce synchronized cell death. Apoptosis controls the expression of genes that help \"carve\" the contours of the fingers (tissue remodeling genes **Hmgn1** and **FGF18** at digit-interdigit junction). ### Involvement **of apoptosis** in morphogenetic processes -- Drosophila vs. vertebrates Apoptosis promotes tissue movement and shape change. In Drosophila, it is involved, for example, in the **dorsal closure of the embryo.** Active process - the movement of the lateral ectoderm up in the dorsal direction, thus closing the ectoderm, during which the extraembryonic tissues such as **amnioserosa shrink.** In addition to shrinking, it partially undergoes **apoptosis** and partially moves **inside the** embryo. Changes in amniosesis are quite complex. They are also affected by changes in the surrounding lateral ectoderm, which put pressure on amniosesis Amnioserosis also affects **the dynamics of** dorsal closure. If we inhibit **apoptosis**, it leads to **a delay in closing the dorsal end of the Drosophila embryo**, ectopic induction, on the other hand, accelerates this closure. *(Mechanism: several changes -- induction of apo signals such as **caspases** and **reapers** at the base of cells, changes in cell shape and formation of apical constriction, the upper part narrows and the lower part thickens and this area gradually constricts. Rearrangement of the cytoskeleton. Changes in **mitochondria**, their morphology changes from **tubular** to **fragmented** mitochondria, mechanical stress activates **caspases** and thus cells are **expelled** from the **original layer).*** Apoptosis is also involved in the morphogenesis of Drosophila **during the development of male gonads.** When it allows **the gonads** to rotate **360°.** A typical example of apoptosis in vertebrate morphogenesis is the closure **of the neural tube** from **the neural plate**. During the insertion of neural folds and in later stages, a specific induction of apoptosis occurs, not only during **the fusion itself**, but also during **the formation of dorsal structures**. When closing, the apo cells move along the axis of the body, i.e. in the same direction as the neural plate gradually closes in the cranial and caudal directions. ### Apoptosis promotes **tissue movement** and **shape change** Several models have been created to study the significance of apoptosis: Mouse model with **chemical inhibitors of apoptosis** and model with **caspase-3** or **APAF1 deficiency** -- **anterior and posterior brain closure defect**. Life imaging has been used to show **the occurrence of two types of apoptotic cells** with different tissue distribution **in one region**: - [**C-type** Apoptotic cells] are in NP = nerve plate, BC = border cells, but also in SE = superficial ectoderm. They typically have **shrunk** cytoplasm and occur in the tissue **Short** Time. They express only **Casp3**. - [**D-type** apoptotic cells] are in NP = nerve plate and in BC = border cells. D cells do not shrink, they are still **round** and persist for **a long time** in the tissue, they are **able to migrate**. They express both **Casp3** and **Casp7**. In **an APAF mutant, there are neither C nor D cells.** The rotation and flexion of the neural folds is reduced, especially in the area of the midbrain and posterior, the closure of the neural tube, the position of the cells during the zipping process is violated. Recently, another process has been described in which apo cells are involved during the closure of the neural tube. Apo is also involved in **the removal of specific cell populations**. Red crosses = cells undergoing apo, produces **FGF8**, only **dorsally** located cells remain, ventral cells are removed by apoptosis. In **KO**, there is a persistence of cell populations and **accumulation of FGF8** and thus an **increase in proliferation**. This leads to defects such as multiplication of nerve cells, overgrowth of ectopic folds. Api is not only involved in **cell number control**, but **also** modulates signaling **and participates in** the modulation of morphogen gradient**.** Another apo -- the formation of folds on the **limbs Drosophilas** -- apoptosis localized in the area of the emerging folds. ### Apoptosis aids in **the formation of folds** Change of apicobasal forces in the epithelium -- it forms myosin bundles, **actino-myosin rings are formed here** and thus the constriction is affected, this filament is first formed in the apo cells and then transferred to the surrounding cells -- change of forces on the cell surface -- formation of folds -- rapid process. **Ectopic apo is a sufficient stimulus for the induction of crease formation** -- even in flat tissues. If there is a dissipation of apoptosis, folds do not form. ### Apoptosis **induces cell fusion** E.g.: in muscle tissues -- myoblast into myofibrils during **the formation of muscle fibers**, here apoptosis occurs in a part of myoblast cells, dying cells never fuse with healthy cells, they remain in their close proximity -- if we block myoblasts with pancaspase inhibitors, the induction of myoblast fusion is suppressed. **Defect in myogenesis of Bai1-deficient mice.** In addition, it supports the transmembr protein **Bai1 = phosphatidil serine receptor**, which can bind to the apo cell body phosphatidylserine -- activation of Rak signaling -- when Bai1 expression increases, myoblast fusion is formed and increased, on the contrary, myoblast fusion is inhibited when lost of function is inhibited. So are mutants -- reduced myoblast fusion. Bai deficient mouse -- reduction of myofibrils, reduction of their number. Not only do myoblast fusion disorders occur during development, but this protein is also important in muscle repair. ### Apoptotic cell signaling Historically, the sig. has been studied in ***C. elegans*** -- due to the precisely defined number of cells that undergo apoptosis (131 cells out of 1090) -- an advantageous model for experiments. If these key genes are lost, 131 cells survive, but it does not have a significant effect on the overall survival of the animals :-O The system is more complex in ***Drosophila***, the whole signaling is more complex, cell fate and number of cells are not precisely determined, they also depend on extracellular signals and factors. However, it is advantageous to study it in the context of cellular and tissue plasticity and homeostasis. Inhibition of apo here does not lead to the extinction of some cell populations that need **nurturing cells** -- they are not affected -- not only apo is involved in cell elimination. The last model -- **mouse embryo** -- increasing complexity -- complexity of apo signaling, the surprise is that some kt genes encode key components, they lead to only small changes in delelation -- proven functional redundancy between apo proteins, but some cells are also reduced by other zp than apo. The cells have a distinct phenocytosis during death\... Nematode -- binding of Egl1 to Ced9 -- inhibited, is an analogue of Bcl2 -- this binding causes the release of Ced4 (apaf1) -- this allows cells to eliminate Ced3 (= execution caspase). Drosophila -- the so-called **Jap complex** = inhibitor of apo proteins, this complex must be inactivated in order to activate apoptosis, this process occurs thanks to antagonists: **Hid, Reaper, Grim,** these cause degradation of **the Diap 1** protein and release of effector caspases (**Ice**, **Dcp1**) -- this allows the interaction **of Dronco** = initiating caspase Casp 9, apaf homologum -- key for the formation of the apoptosome and activation of effector caspases. An important molecule is also **p35** -- a specific inhibitor of these caspases ### Apoptosis-induced proliferation (AIP) Not only is internal signalling activated, but these cells can also secrete a number of factors, AIV = **apoptosis can induce proliferation** -- apoptosis can induce proliferation or AIA = **apoptosis induces apoptosis**. It is important to activate **Junk signaling** and thus to secrete various factors. If AIP is activated, the cell is able to survive in and after some time, then it produces signals to its surroundings, these cells respond to these factors by increased proliferation -- **replacing the cell population with proliferating cells** and tissue repair. In Drosophila, two processes can occur -- on the one hand, in the opposite p35 tense there is inhibition of execution caspases and the cell is kept in an undead state, p53 is activated via Dronc and Arc -- activation of Junk signaling and production of DPP and Wingless -- cell hyperplasia is induced. In Drosophila, the apo is independent of p35, the apo takes place through activation through executor caspases. **Imaginary discs** in Drosophila -- **future wing + basis for the eye**. After stimiluation **of Hid** -- through the enhancer GMR -- (specif for the eye) -- induction of apo in 2 waves. During compensatory proliferation, and in the area of the cells by semi-proliferation, the cells expand and differentiate later, which leads to the reduction of the eye, because in the head part the area of the dif cells is enlarged. Another example: proliferation of inductive apo during **regeneration of the apore** -- after cutting off the head area, here induces a lot of apo cells -- they produce mainly **Wnt3** and these subsequently induce **compensatory proliferation** in neighboring cells. If we blocked the caspase activity, the Wnt synthesis would stop and there would be no compensation. prolif. The opposite is the ectopic formation of two head parts in the case of the marshmallow. AIP also occurs in **regeneration of limb processes after their amputation/tail**. It has been observed in **reptiles** and **amphibians**. Specifically, induction of apo occurs at the site of tail amputation -- associated with induction of **casp 9** and **apaf1** and **Wnt secretion**. However, when experim is performed, inhibition of Kasp 9 and 3 -- inhibition of proliferation and non-inhibition of tissue regeneration. A similar process occurs after cutting the **Dania osacus** -- proliferation is induced at the edge of the wound and the caudal fins grow back. Associated with Ros induction -- induction of caspase activity, Junk signaling + other factors (Wnt, Fgf20 are associated with proliferation, but lack of evidence that they are directly produced by apo cells). AIP can also occur in mammals -- liver damage/ removal of lobes -- induction of Ros production and secretion of IL11 hepatocytes -- drying of compensatory AIP proliferation. Wound healing depends on casp 7 and 3, which are facilitated by proteolysis of calcin-independent phospholipase 2 and prostaglandin F3 proliferation??? It causes prolique of liver tissue. ### Apoptosis-induced apoptosis (AIA) Apoptotic cells can produce **factors** (**TNF**, **Eiger**) affecting other cells around. This creates large areas subject to apoptosis. AIA occurs, for example, in ***Drosophila* discs.** First, **Eiger** is produced (he is an orthographer of mammalian TNFs), -- binding to surrounding cell receptors -- activation **of Junk signaling**: activation **of Dronc** and **AIS** and **DCP1**. In addition to acting on surrounding receptors, Eiger can also act on distant populations of cells. If it is **inhibited** at the site of the first dying cell or if Junk signaling is inhibited in the target cells, **AIA is suppressed**. AIA is also known in **vertebrates** -- during various stages of hair/srtsi exchange. There is a specific induction of apoptosis in **the hair follicles**, these are cyclical changes. First it is in the resting stage -- **telogen,** then in the growth phase -- **anagene,** finally in **catagene**, when regression occurs -- huge elimination of cells -- the lower 2/3 of the follicle disappears. During catagene, **the TNF** molecules in the lower parts -- the primary cells, which then send signals to the higher cells -- are first induced, sending a wave of apoptosis that spreads further to the dorsal regions of the follicle. 26/10/20 Lecture: Epithelio-mesenchymal transformations in development (TB) ==================================================================  1. EMT and MET in general 2. Gadgetry 3. EMT and MET in development 4. Bonus: The Role of EMTs in Metastasis [From the previous lecture]: ***Are cells kept in a permanently differentiated state?*** **NO** -- the original concept that terminally differentiated cells only perform their function and are \"static\" is no longer valid! Indeed, cells in **terminally differentiated epithelium** **can change their phenotype by activating the EMT program**, which allows **transdifferentiation**, leading to the transfer of epithelial cells to mesenchymal derivatives during development and adulthood. EMT and MET in general ---------------------- ### Epitetio -- mesenchymal transition (EMT) = a series of events where epithelial cells are transformed into mesenchymal cells. During embryogenesis, these events are responsible for the formation of new tissues. Epithelial cells have both **basal** and **apical** parts -- the basal part is attached to the **basal lamina** by integrin. Through the EMT process, these **polarized stationary** cells become mesenchymal cells with high migration potential. Mesenchymal cells then have the ability to penetrate tissues. ### Epithelial cell characteristics: - Polygonal/columnar cell shape - Apico-basolateral polarization - Strong cell-cell interaction due to **adherens junction** (APC complex and E-cadherin) - Limited migration potential - Markers (expressed genes): **E-cadherin, Cytokeratins, Occludin, Claudin** ### Mesenchymal-epithelial transition (MET) = a series of events when mesenchymal cells are transformed into epithelial cells. ### Properties of mesenchymal cells: - Spindle-shaped cells (e.g. fibroblasts) - Anterior-posterior polarization - Focal cell-cell interaction - Strong migration potential -- thanks to paracrine signal - Markers (expressed genes): **N-cadherin, Vimentin, Fibronectin + Snail and Snug** Changing EMT in MET and vice versa is a very dynamic process, it changes as needed. Transformation of mesenchymal fibroblasts into induced epithelial pluripotent SCs can be induced by four factors: Oct4, Sox2, Klf4, c-Myc. **Epithelial cells** typically perform some tissue-specific function, while **mesenchymal cells play a more supportive role**. EMT mechanism ------------- During the development or healing of wounds, epithelial cells begin to lose intercellular contacts, **enzymes** necessary for dissolution **of the basement** membrane begin to be expressed , the cells become migrating mesenchymal cells and go to the site where they subsequently form **a scar**. - So at the beginning, there is **a decrease in** the expression of **Cadherins**, especially E-cadherin. The cytoskeletal actin is reorganized. Enzymes are secreted that disrupt and degrade the basal lamina (**metalloproteinases**) and cell proliferation **increases** (it doesn\'t have to, but it can). There is no \"master regulator\" EMT or MET! Which is a great pity, if it existed, we could also discover the Achilles\' heel of secondary dissemination of cancer, because it is because of EMT that metastases develop. The main signaling pathways involved in EMT are mainly **TGF-β, FGF** and **Wnt**. The others, of course, are also involved, but less so. We do not have to know the molecular mechanism exactly. It is only necessary to know what plays the most important role -- triggering the expression of genes important for mesenchymal cells. ### Where EMT can take place: a. in inflammation and fibrosis -- in the case of an inflammatory reaction, TGF-β or metalloproteinases are produced, which causes EMT and the cell travels to the site of inflammation and forms a scar. Fibroblasts are taken in fibrosis thanks to EMTs, but the scar can also be formed only by resident finbroblasts or cells that travel all the way from the bone marrow. b. in embryonic development c. in cancer (or metastases) ### EMT in embryonic development During development, some epithelial cells are \"plastic\" -- they are dynamically able to cross the epithelium\mesenchyma through EMT and MET processes. This happens in processes such as: embryo implantation, embryogenesis, organ development or regeneration and homeostasis. **The key role of EMT in development -- without it, development would not be possible at all!!** - [4 examples of the necessary presence of EMTs in development:] 1. **Embryo implantation -- the** mother\'s epithelium must be parted and EMT must pass through to allow implantation of the fertilized egg. 2. **Gastrulation and mesoderm formation** - EMT occurs already during **the development of the blastoper** and the cells migrate inwards, forming the mesoderm and developing blastoporus. The basal lamina is in the inner part of the blastuli and the apical pole is in the ring. If a cell receives **an EMT signal**, it breaks away and migrates inward in the **blastopore** area. In the **blastula** stage (14th day, epiblast, hypoblast), when **gastrulation** gradually occurs (16th day), **a primitive band must be formed** and then a projection **furrow**. This determines the polarity of cranial and caudal and at the same time right-to-left symmetry. The principle of gastrulation is that the cells of the epiblast proliferate and migrate through this primitive strip and furrow outward, replacing the **cells of the hypoblast**, giving rise to **endoderm**. The initiator of gastrulation is **the Hansen knot.** In the end, 3 germ layers are formed -- they are distinguished from each other only by the different composition of initiation factors, but it is not the case that one layer is formed, then the second and the third, it is continuous. EMT in gastrulation is governed by canonical Wnt -- **Wnt3**, **TGF-β** (Nodal, Vg1) and **FGF**. The fertilized egg has the highest gradient Wnt3 at the apical pole, even after asymmetrical division occurs. Still until the stage of gastrulation, Wnt3 is maintained in the apical part. The FGF track often cooperates with the Wnt track. If FGF1 receptor is used, no mesoderm will be established in the embryo. 3. **Formation of the neural crest** - \"Fourth germ layer\" - of ectodermal origin, transient - not in the adult, only temporarily; it arises from **the neural tube** (from the neural tube also peripheral nervous system such as neurons and glia, adrenal medulla, pigment cells, connective tissues and teeth) through EMT and cells migrate along the anterior-posterior axis and differentiate due to various growth factors and physical barriers (change of the environment of the A-P axis leads to the formation of different cell types); Neural crest cells lose their adhesive connections and separate from the epithelium -- this process is called **delamination** (in the video this can be seen as the stage when pigment cells appear on the embryo). ***How is it possible that so many cell types arise from the neural crest?*** ***Lineage tracing** = a method where we can monitor the individual movement of cells in development. This is done by cloning a cassette of a specific assembly of fluorescent proteins into mice. Among the genes for these fluorescent proteins there are sites for Cre-recombinase, and if we start expressing Cre-recombinase during the development of the embryo (day 9 = before migration of neural tube cells to the neural crest), for example, we start the expression of Cre-recombination with a promoter, the marker stains recombine and we see on the 10th day that cell X is yellow and all the offspring of the cell in the neural crest will be only yellow during development. So we can see how far the cell travels and what it creates.* To put it simply: The entire delamination is controlled by the Wnt gradient and **BMP signaling**. In the upper part, the BMP is the most, which increases the expression of Snail2 and thus disrupts Cadherin-6B. Its disruption leads to EMT = delamination phase. At that point, the cells begin to migrate from top to bottom along the AP axis, forming the tissues mentioned above. Sox2 prevents the expression of Snail2 (BMP, on the contrary, increases its expression). 4. **Formation of vertebrae from somites** - Somites are epithelial blocks (clusters) of cells that are located near the neural tube, migrating thanks to EMT to a specific location; **They determine the articulation of the body -- future vertebrae**. The BMP gradient determines the placement of body segments. - Sclerotoma - Myotoma - dermomyottoma - ### 3) MET in embryonic development -- **formation of somites** However, **the somites** themselves are formed **by the opposite** process, namely **MET,** so the **pre-somitic mesoderm** is formed **by mesenchymal** cells!! (but the architecture of the Somites consists of **epithelial** blocks). Therefore, mesenchymal cells must transform into epithelial cells =\> MET!! This process is controlled by the **Mesp** gene (Mesodermal posterior). It is expressed in the epithelial part of the entire somite and causes it to be further expressed **by Ephrin-E4**, which thus increases the expression of its binding partner **Ephrin-B2**, whose essential role is to ensure that transition (through another cascade) of the epithelium in the mesenchymal along the AP axis. This leads to the establishment of new somites and new and new links and lengthening of the body. ### Bonus 1: The role of EMT in **metastasis** Normal epithelium -- mutation -- tumour -- EMT -- metastatic cells escape through the bloodstream -- metastases are formed. In addition to various growth and transcription factors, vesicles such as exosomes also leak from the tumor into the bloodstream. These can then spread further along with the tumor cells into the body, and if they are coated with platelets, the immune system does not recognize them as harmful and therefore does not degrade them. ### Bonus 2: Embryonic Origin of the **Adenohypophysis** The adenohypophysis is an endocrine gland that synchronizes long-term processes of vertebrates such as growth, metabolism, reproduction, puberty or the menstrual cycle. In adulthood, it can be found on the underside of the vertebrate brain, but it arises from the **plaoda** (i.e. the area of \"thickening\" of the ectoderm, from which the \"new head\" of vertebrates arises), which migrates relatively far from the front of the brain to the bottom of the future head. For a long time it was thought to be **of ectodermal** origin (the entire upper part of the head does not have endoderm, only ecto and mesoderm), through **lineage tracing** it was discovered that **part of the adenohypophysis** is of **endodermal** origin. P. Fabiál et al. found out by staining the endoderm red and finding that **the dorsal part of adenohypothesis** is formed by **endoderm**. Simply put, he showed that in the adenohypophysis, the structure of the so-called \"new\" vertebrate head, it is possible to detect an \"old\" endodermal contribution. However, cells of endodermal origin do not form a separate cell population, but are **randomly scattered** throughout the adenohypophysis. **The evolutionary origin of the adenohypophysis** can therefore be found in the ancient **pharyngeal region of the so-called Hypophysis**. **preoral intestine**, which in the course of evolution was involved in the development of the structures of our new vertebrate head. This discovery will probably rewrite textbooks. Peter Fabian, Kuo-Chang Tseng, Joanna Smeeton, Joseph J. Lancman, P. Duc Si Dong, Robert Černý, J. Gage Crump**, Lineage analysis reveals an endodermal contribution to the vertebrate pituitary**, Science 23 Oct 2020: Vol. 370, Issue 6515, pp. 463-467, DOI: 10.1126/science.aba4767 9/11/20 Lecture: The role of ECM in development and cell adhesion (TB) ============================================================== 1. **Extracellular matrix** - Function, Role in development, Composition, Components, Remodelling, Receptors, Anoikis 2. **Cell adhesion/cohesion** - Sorting-out, Cadheriny Extracellular matrix (ECM) -------------------------- It is not just a network of insoluble proteins and sugars in which the cell just sits and does nothing. But it\'s a **physical barrier** that the cell can attach to, but it also tells the cell what to do. **Similar** environment, but **inside** the cell is **the cell cytoskeleton**. And it is connected by **integrins** to the ECM -- integration of the signal from the outside to the inside -- into the nucleus -- the initiation of expression. Thus, the ECM is a three-dimensional insoluble network of extracellular macromolecules such as collagen, enzymes, and glycoproteins that provide structural and biochemical support to surrounding cells. It mediates cell adhesion, cell-to-cell communication, and differentiation. It instructs cells \"when to grow, when to divide, when to produce different molecules, when to die.\" It consists of about 300 proteins, mostly: **collagen** (which accounts for up to 30% of all proteins in mammals), **proteoglycans**, **elastin**, **glycoproteins** (fibronectin and laminin). Each component has certain physical and biochemical properties. ### ECM function 1. attaching the cell to the ECM + inducing its polarity 2. acts as a migration barrier -- the ECM paves the way for the cell to migrate 3. it works as a reservoir of signaling molecules -- it forms a concentration gradient of morphogens/GFs, which is not only created by being produced/degraded somewhere, but that these morphogens bind to the ECM in **the concentration gradient** 4. ECMs, as co-receptors embedded in the membrane, can provide juxtacellular signaling and may even provide perception of the stiffness of the surrounding product outside the cell ECM components -------------- The classification of matrix components can be taken from several perspectives, the most common being biochemical, i.e. what the ECM components consist of: Proteoglycans (heparan sulfate and chondroitin sulfate), glycoproteins (laminin, fibronectins), proteins (collagens), Polysaccharides (hyaluronic acid). But it can also be looked at from the point of view of where these components are, either in the epithelium - basal laminae: collagen IV, laminins and HSPGs, entactin, etc. Or scattered around the matrix, which are then other collagens, fibronectin, PGs, GAGs, Tenascin C and Elastin. #### ECM Components - Proteoglycans Key role in **the transport of paracrine signals**. Large molecules that consist of a central protein and a covalently bonded polysaccharide chain (each proteoglycan has specific binding sites for different amounts of carbohydrates). They have the ability to bind GFs, so they act as a reservoir and can also bind them, thus inhibiting the triggering of potential pathways. The proteoglycan complex is made up of **protein** + **carbohydrate** + **polysaccharide**. Examples of polysaccharides include: **Heparan Sulfate**, **Chondroitin Sulfate**. ###### Heparan sulfate (HS) It is a linear polysaccharide that occurs in the form of proteoglycan (protein with **covalently bound HS chains**) -- in almost all animal tissues. There are two types of HS: a. loose, membrane-free: agrin, perlecan b. membrane-based: glypians, syndecanes -- act as co-receptors or mediate paracrine signals It plays a key role in **the binding of ligands to receptors** - \> **without it, binding does not have to take place at all!** Mutations that block the synthesis of Heparan sulfate cause various **defects** in cell **migration**, **morphogenesis** and **differentiation**, often these mutations mimic mutations in signaling pathways!! (FGF1 is required in gastrulation, the HS mutation has the same phenotype as the FGF1 mutation). Different organs and tissues produce different HS compositions during development. HS also changes during development =\> is therefore **important for proper development**. These findings led to **the \"sugar code\" hypothesis** -- specific HS modifications control individual events during development through **interactions with signaling pathways** = a change in the environment determines whether a cell will migrate or differentiate. Many organs, including the hematopoietic system, skeletal systems, and liver, lungs, and kidneys, do not form properly if HS is absent. - General structure: Proteins have a different number of carbohydrate binding sites, some may also contain chondroitin sulfate - Core proteins have a different number of carbohydrate binding sites - HS is synthesized in the Golgi apparatus - As **receptors**nutrients. After IGF binds to the receptor, bifurcation occurs, with part leading through **Erk1/2**, **PI3K** and **AKT.** The second part, also activated by the IGF, runs through Raf, Mek1/2 and MAPK signaling. However, all signals via IGF are pro-survival (they increase proliferation, reduce apoptosis, increase protein synthesis, increase glucose metabolism). b\) Size -- signalling pathways - Ras/Raf/MAP - Also activated via IGF - This part is common with the receptor tyrosine kinases of the EGF/FGF/VEGF pathway - Signaling is the same for everyone, ligands have the same result (por-survival) c\) Size -- signal pathways -- TOR Like IGF, it also regulates growth based on the presence of nutrients, energy, oxygen. A classic example of the preservation of amino acids from the environment. It is preserved from yeast to humans. **IGF** and **TOR** are the main players involved in **transmitting information** about the **presence of nutrients** in order **to coordinate growth**. TOR = target of rapamycin. No other pathway has been discovered yet! d\) Size -- signaling pathways -- Hippo A relatively newly discovered pathway, conserved from Drosophila to mammals. This pathway is responsible for the actual size of the cells and the number of cells. - Drosophila vs. Mammals - Hippo = Mst1/2 - Salvador = Sav1 - Warts = Lats1/2 - Mats = Mob1A/B e\) Size -- signalling pathways -- JNK - Stress pathway, regulates cell death, tissue regeneration, wound healing - A key path for negative growth regulation f\) Size -- signalling pathways - Other - Signaling pathways that control patterning - They control the shape and arrangement of the organ - changes in cell growth and proliferation - Hh, Wnt, TGF-β -- indirectly regulate the size/patterning of tissue or organ 3\) Size control aspects The signaling pathways mentioned above regulate growth and proliferation, but this does not explain the nature of size control. **The size of the tissue/organ/individual determines the growth rate and the duration of growth**. A. **GROWTH RATE** -- is controlled by signaling pathways that regulate cell proliferation and growth B. **GROWTH TIME**/ **DURATION** -- is regulated by systemic **hormonal** signals that coordinate growth arrest throughout the body, as well as **organ-autonomous processes** that ensure that organs stop growing once they reach their final size. Size Control Aspects -- Drosofila - **Imaginal discs** -- model system -- The size of the disc determines the size of the organ. - These discs are established in the larval stage and the only thing that happens during metamorphosis is that the cells in the imaginary disc divide very quickly (legs, eye, wings grow from it). - Drosophila has 15 disks, which are first divided into A-P and D-V parts (4 parts) - the most common model is the Drosophila wing - e.g. GAL4 is used as an Engrailed promoter (determines the posterior part of the wing). This way, GAL4 subscribes to express itself only where it is Engrailed, so it then mounts the UAS sequences and triggers the expression of the desired gene (e.g. GFP), the posterior part of the offspring wing then shines under a fluorescence microscope. Or we can use another promoter, such as spez (for the dorsal side of the wing) and again put GAL4 behind the promoter. We cross such a fly with another fly with a different promoter, such as UAS, and instead of GAL4, it expresses GFP. The offspring then have such a locus that GAL4 mounts the UAS and GFP is expressed. A. Size control considerations -- Growth rate Regulated by cell growth and proliferation =\> more larger cells/time = larger organ/individual. Growth and proliferation are regulated by different pathways. Growth: 75% of the cell is made up of water, ions and small molecules; **18%** is made up **of proteins** =\> **growth depends on protein synthesis**. - S6K (S6 kinase) is a ribosomal 40S protein =\> controls ribosomal protein synthesis (TOR signaling) - **S6K deficiency** (I-1/I-1) -- delayed development, smaller individual, but the same number of cells, only smaller. - *FSC = forward scatter flow cytometer -- shows the distribution of cell population size, the distribution is normal for WT / shifted to the left towards smaller cells in mutants* *\ * - S6K is a ribosomal 40S protein =\> controls protein synthesis. S6K deficiency (I-1/I-1) -- delayed development, smaller individual, smaller organs, but the same number of cells -- cell cycle is not damaged/disrupted - S6K is a ribosomal 40S protein =\> controls the synthesis of ribosomal proteins. dS6K (**overexpression**) -- larger cells, twisted wings (one layer overgrown over the other) - This study was carried out only on the wings *Insulin/Insulin-like growth factor (IGF)* - 2 parts of signaling - **FOXO** regulates organ size by controlling the number of cells - **Higher** expression of **FOXO** (is the last target of this pathway), **fewer** cells -- smaller eye, regulates proliferation, but if we express FOXO before the eye is found, the eye will not develop at all - **Rescue** phenotype via **dAkt overexpression** (growth inhibitor inhibition) - **[=\> The size of the cells and the number of cells are regulated independently. ]** *Growth rate -- cell cycle* - the most important BC regulators are CDKs + cyclins - **Oncoprotein** - promotes the expression of multiple growth regulators - Positively regulates ribosome biogenesis and hence global protein synthesis Growth rate -- cell cyclus -- MYC de la Cova, Claire, Abril, M, Bellosta, P, Gallant, P, and Johnston, L A. \"Drosophila myc regulates organ size by inducing cell competition.\" *Cell* 117.1 (2004): 107-16 Overexpression of Myc in wing cells: During development, cells with overexpressed Myc (in Fig. Gal4), as they grow faster, enter the wing disc and lose Myc through mitotic recombination (we are talking here about clones of cells marked Fig. Sibling). Sibling is therefore without Myc. Myc from Gal4 somehow represses clones of Sibling cells. Cells that are in close contact with cells with high amounts of Myc have some growth disadvantage, but it is not known why. Cells in close contact with the dMyc clone have a growth disadvantage and, conversely, cells with a growth advantage (Myc overexpression) induce apoptosis of surrounding cells -- cell competition -- this leads to uniform cell size. If we suppress apoptosis, we lose control of cell size and thus lose cell uniformity. **Size control is induced by apoptosis!** - **MYC Summary** - Myc **overexpression** = more larger cells, faster proliferation, larger individual, loss of size control (**loss of control of growth correction, control means apoptosis**) - Myc **deficiency** = smaller cells, smaller individual - The size of the organ (wing) is controlled by **Wnt** (Wg) -- if we remove the Wnt signaling, the cells begin to express c-Myc B. Size control considerations --- Growth duration Regulating the growth rate is not enough to fully control growth. Differences between individuals (and also between species) are caused by different size and number of cells - \> also regulated by growth time. Many signaling pathways and molecular mechanisms that regulate growth rates are also involved in regulating growth time. - Drosophila -- 3 larval stages - - - - *Growth duration - IGF* However, the decision to pupate is at a much earlier stage (beginning of the 3rd instar) and is associated with the acquisition of a certain size (*critical size*), which is accompanied by the synthesis of the hormone **ecdysone.** - **Ecdysone** is synthesized in increasing **pulses** and **each pulse is associated with a specific event in development (metamorphosis**). - Critical size is missing in humans -- Drosofila is therefore not very suitable\... - Dwarfism in Pericentrin Mutation (Incorrect Mitosis) - IGF regulates growth rate during TGP - If it is deficient, we will affect the length of growth -- the time for Critical Size is extended! Nothing more! Everything is smaller\... but different phenotypes can be saved by doing so - **Increasing the growth time** leads to the \"rescue\" of the phenotype **of the cycE hypomorphic mutation** -- we give the individual enough time to set the correct number of cells (in this case, the cells of the eye) - **We can save certain phenotypes by extending the duration, but the phenomena are separate from each other!** 4\) Aspects of size control -- Growth duration -- Drosofila x human **Drosophila**: The exact mechanism by which the larvae register their *critical size* is not clear\... but\... \....it has been found that the size of tissues is determined by how these tissues are supplied with oxygen -- with the growth of the larva, its weight increases, the volume of the trachea does not increase (it increases sharply for a given stage), as soon as the volume of the larva increases, there is a low oxygen saturation and this is the impulse that monitors the size of the body -- so metamorphosis occurs in the next stage \... in addition, signaling pathways regulating Ecdysone synthesis are known and thus respond to critical *size* -- **IIS, TOR, RAS/RAF/MAPK** **Human**: Growth arrest is associated with the end of puberty - \> **the timing of puberty is an important factor in size regulation**. Hormonal changes at puberty are known, BUT the **mechanisms** that control **when** these hormonal changes are initiated are much less well understood. - Higher BMI -- formerly puberty - This suggests that the timing of puberty and growth arrest in humans, as in Drosophila, are regulated by the nutritional status and body size of juveniles. *Article:* ***LIN28B* the first genetic determinant regulating the timing of human pubertal growth** The allele of this specific gene is associated with an earlier onset of puberty. \"This allele was also associated with earlier breast development in girls; earlier voice breaking and more advanced pubic hair development in boys; **[a faster tempo of height growth in girls and boys; and shorter adult height in women and men in keeping with earlier growth cessation.\"]** Size Control Considerations -- Growth Duration -- LIN28 -- How Does It Work? LIN28 is important in microRNA processing (inhibits Let-7 miRNA). It controls growth and metabolism. It binds to Let-7 and so the miRNA cannot pass into the cytoplasm, when it passes, LIN28 binds to it again -- degradation. Back to the flies\... - **Lin-28** *deficiency* leads to earlier roundness, smaller and lighter individuals. On the contrary, **overexpression of** lin-28 leads to larger individuals and problems during hatching (problems during metamorphosis). - In **Let-7 deficiency,** cells are unable to \"step out\" of the cell cycle. Flight-7 overexpression leads to cessation of development in L1, L2 and high lethality =\> overexpression of miRNAs in vestigial (vg) -- wing-specific -- they have larger wings. Negative growth controls - apoptosis -- inhibition of apoptosis - Drosophil discs are not as uniform as in WT, individuals are then larger - see. previous lecture with MB 5\) Regulation of organ size - Organs \"know\" what size they should be. And they also \"know\" when to stop growing =\> **autonomy** in terms of organ size. The concept was introduced back in the 70s. - Compensatory role of the remaining kidney in transplantation -- hypertrophy - The same applies to the fruit fly, as well as the autonomous signal of what the wing should look like\... - Mammals have a similar body plan, but the size of the organs varies dramatically! - The size is not random, but the result of thorough regulation - The size of the organ must also be adapted to physiological needs (when the kidney is removed, the other one hypertrophies) - Coordination of proliferation and cell death is crucial for the correct size of the organ. - **Each organ has an autonomous size control** (but is also partially subject to systemic control - hypertrophy) - Growth duration/timing seems to be key! - Each organ has autonomous size control - Link to the previous lecture -- myostatin normally negatively controls muscle mass, but when mutation occurs -- hypertrophy occurs -- systemic regulation **Coordination of proliferation** and **cell death** is crucial for the correct size of the organ\.....**Hyppo pathway!!** Size control considerations -- Organ size -- Hippo The hippo pathway integrates proliferation and cell death. - **YkiS168A** -- if it is not phosphorylated, it gets into the nucleus, but if we mutate it (serine for alanine), it still gets into the nucleus, the organ/individual is then larger. - **YAPS127A** -- analogue in mammals -- the mutation also leads to hypertrophy and the cells are larger, and when we put the human analogue into Drosophila, we get the same phenotype :-O With increased expression of YAP, our liver enlarges. Systemic regulation through blood circulation is equally essential as autonomous organ regulation! - Hippo and cell death -- induces both positively and negatively; YAP is important for the induction of apoptosis - Hippo and cancer -- long-term YAP expression induces pathological cell division and cancer development; YAP is now a therapeutic target (by inhibiting YAP phosphorylation -- it has only one phosphorylation site) Size Control Considerations -- Organ Size -- Mechanical feedback Cells at the edge of the imaginary disk have completely different forces (much smaller) than in the middle of the disk, where much greater pressure is exerted on the cells. We\'ll talk next time. Conclusion: - Growth rate vs Growth duration control + examples (not details) - Organ growth/size control (Hippo) - Know that there is also negative regulation. 24/11/20 Replacement 8. 1. Asymmetric segregation of determinants in the ovule/zygote 2. Planar Cell Polarity 3. Cellular processes and principles in collective migration -- neural crest 4. \"Migration\" of parts of cells 5. Common sub-cellular basis of polarity and migration determination 1\) Segregation of cytoplasmic determinants in the egg Revision: How is gene expression regulated? Genes turn on transcription factors (TFs). TF is a protein produced and activated in the cytoplasm and transported to the nucleus. There, TF binds to specific regulatory sequences of the gene = promoters/enhancers. RF pulls the cell machinery there in the center with RNA polymerase II. It will start to produce mRNA. Before that, the introns are spliced. Each transcription of the mRNA then takes place on the ribosome and after this a protein is formed. The process can stop at any time -- e.g. by stabilizing the mRNA by moving it to some part of the cell/ adding the mRNA to the complexes and be protected from degradation or there may be factors preventing transcription. All these factors are used by the fertilization of the egg. Formation of asymmetry -- C. elegans - Model: First cell divisions in a nematode model (Caenorhabditis elegans) - **Segregation of P granules and the formation of asymmetry** Phase separation principle = liquid-liquid face separation (LLFS) We can think of it as oil droplets in water. In the beginning, there are two liquids, one of which spontaneously forms droplets, but they are not enveloped by a membrane!! It is not yet fully understood. What is known is that the formation of these granules is due to sequence motifs in proteins and mRNA. These granules can then attach to cytoskeleton molecules and transfer to one part of the cell. P granules are just that example. The mRNA is marked in blue in the figure, then the PGL proteins (P granules) -- they have the property of separating from the cytoplasm and thus allowing themselves to be moved elsewhere in the cell. Notch and the emergence of asymmetry Asymmetry is determined by the mother -- the egg, it differs in different types, such as the place where the sperm enters, etc. In C. elegans, each cell has its own name. P1 divides asymmetrically and P granules go further and further up to the germ line. However, cell AB divides symmetrically. The P2 cell has ligands of the Notch pathway that can only signal by contact with one of the daughter cells, not both. In this way, the Notch signal indicates the A-P cell (which receives different signals despite the fact that it originally divided symmetrically). Symmetrical division leads to the formation of asymmetric daughter cells (Notch Role). *Embryonic development of Drosophila melanogaster* Drosophila has a very special early embryogenesis. It has a developmental stage where one cytoplasm contains a lot of nuclei = syncitium, which is initially non-cellular and later these nuclei multiply and begin to form membranes of separate cells. These separate cells must already carry a different mRNA because dramatic specification is already taking place at that stage. How can this be achieved? Individual mRNA coding proteins are differently distributed in cells and, after membrane formation, distributed differently in embryonic cells. - egg cell - Syncytial blastoderm - Cell blastoderm - Gastrulation and segmentation Genes involved: - g\. maternal effect - g\. Governing articulation of the body - g\. responsible for the identity of articles -- homeotic genes - g\. directing the creation of complete organs Maternal genes The most well-known genes include maternal genes, whose mRNA is present from the egg stage. Transcription in the ovary, translation to fertilization. a. Determining anterior-posterior polarity (antero-posterior): e.g. nanos, **bicoid** (anterior), **oskar** (posterior), **gurken** b. Determining dorso-ventral polarity: dorsal, toll Reasons for mRNA localization -- why is mRNA not localized later? (EXP) **The localization of mRNA prevents undesirable expression elsewhere than at the point of need.** But it can also allow skipping the mRNA production step in some rapid embryonic stages, no need for proper TFs. - e.g. cytoplasmic determinants in the embryo of D. melanogaster -- oskar, nanos; Change in localization leads to developmental abnormalities - Faster response to local protein requirements -- especially important for large, polarized cells (e.g. neurons) Mechanisms of mRNA localization -- they are interconnected 1. **site-specific synthesis** (rare) - mRNA is transcribed only from the nucleus, where it is present from the beginning, e.g. gurken mRNA in Drosophila melanogaster 2. **local protection against degradation** - degradation of incorrectly localized transcripts (what should not be there, is thrown away); requires two different cis-elements in the 3 ́UTR region (degradation and protection); evolutionarily conserved 3. **diffusion and anchoring** - passive diffusion of mRNA through cytoplasm until binding is captured. protein nanos, cyclin B, gcl -- posterior of Drosophila oocyte 4. **active transport** -- the most common is controlled cytoplasmic transport with the participation of the cytoskeleton (myosins, dyneins, kinesins + organelles and other structures) -- shifts RNA protein complexes (liquid droplets) Sequential elements for mRNA localization - Ex. *bicoid* mRNA - \>600 nucleotides - 3 ́UTR = untranscribed part of mRNA - mRNA can be used in one place and then moved and reused later *Formation of mRNA bodies (**phase separation**)* - 30 mRNA molecules - P-granules = RNP complexes = mRNA bodies - it is always a complex/clumping of RNA + bound proteins that allow the complex to separate from its surroundings and materialize into transport particles - oskar mRNA -- travels on other oskar mRNA molecules - bicoid mRNA -- dimer formation Translation regulation In addition to regulating how mRNA is degraded or moved, it also regulates when it should finally undergo translation into a protein. In addition, there are proteins that bind to regulatory sequences, which can then enhance/block translation. - mRNA is not translated until it reaches **its destination** - Proteins interacting with [5 ́-UTRs]: mRNA stabilizers and translation inhibitors, translation enhancers - Proteins interacting with [3 ́-UTRs]: stability and localization of mRNA; importance of mRNA polyadenylation -\> higher stability of mRNA 2\) Non-canonical Wnt pathway (planar cell polarity pathway -- PCP) - Participates in: - establishment of proximo-distal polarity in the simple epithelium (out of the body axis, fingertips are the most distal ones) - convergent extension - regulation of morphogenesis -- formation of shapes - Wnt signaling pathway: includes ligands (proteins secreted from the cell), a receptor complex headed by Fzd and a number of proteins - non-canonical signaling pathway NEVER stabilizes beta-catenin Planar cell polarity in the fruit fly\'s wing **There are not many possibilities that evolution has invented -- how to organize the cell so that it is stably organized as polarized, so that some complexes are on one side and the other on the other side of the cell. And so the PCP signaling pathway is used again and again by different cell types in different connections, in various relatively small modifications. However, the core remains the same.** The PCP trajectory is studied on the fruit fly\'s wing because it goes very well. It has long been discovered that some fruit fly mutants do not have properly arranged wing hairs. Normally, they point distally -- out of the wing -- the cells must have polarity! The fact that it directs them distal is due to the actin cytoskeleton and the localization of actin. The same pathway is also used to polarize omatidia in the compound eye. In the P-D direction, the proteins of the PCP pathway are asymmetrically localized. **The main participants in this signaling pathway are:** - Distal: Dsh -- Dishevelled (**DVL 1,2,3**)/ Fz -- Frizzled (**FZD1-10**) - Proximal: Stbm (Strabismus) -- **VANGL 1,2**/ Pk -- **Prickle 1,2,3,4** - - Stabilizing: Fmi (Flamingo) -- **CELSR 1,2,3** = 7 trans-membrane protein (as a receptor roasted with G proteins) coupled to a large ECM domain that looks like a cadherin domain. It\'s such a hybrid. On the apical part (the opposite of the basal part) we have these polarized proteins. Molecular mechanism of PCP alignment Polarization is ensured by molecular interactions (positive affinity) both extracellular between FZ and VANGL, and intracellular (negative affinity), which ensure movement to opposite poles of the cell. This is achieved in several ways, including local degradation in a specific part of the cell. In this way, a domino effect can occur -- once one cell is properly polarized, it attracts the extracellular part of VANGL and CELSR. Polarity is propagated by the entire embryo. PCP in the epithelium in mammals: inner ear In mammals, there are not so many epithelia that would be similarly polarized. One of the few examples, however, is the orientation of the hair cell stereocilia in the inner ear. Actin is organized here in a very similar way to that in the fruit fly\'s wing. This hair organization is 100% dependent on the PCP pathway. *Disturbances in the non-canonical Wnt signaling pathway in mammals* Disorders lead to a \"disheveled\" mammalian fur coat. Mutants do not have properly polarized hairs. These mutations are used in the breeding of some species (e.g. guinea pigs). The hair follicle is tilted due to PCP. Wnt/PCP pathway and **convergent extension** From the point of view of embryology, convergent extension is much more fundamental than polarization. All genes that have ever been linked to PCP in fruit flies and found by their orthologists in mammals show a clear phenotype. The orthology of the \"key\" genes involved in PCP signaling shows typical phenotypes related to the elongation of the body axis in vertebrates. Morpholine causes a mutation in Xenop (turning off the gene for Prickle) and there is no elongation of the body axis. This is used to form an elongated bilaterally symmetrical body originating from a round egg. *PCP path in convergent extension* **Convergence** means aiming the cells towards the centre of the future embryo. And **extension** , on the other hand, that the whole embryo begins to lengthen. This is an essential and universal process that is again completely under the control of the PCP pathway. DVL and Rac1, Pkcδ is at the edges (media-lateral direction) and Prickle is in the middle. By simply rearranging cells, cells converge and extend the entire structure. *Cell principle of convergent extension* - Convergent extension -- the migration of cells towards the centre of the body -- leads to the elongation of the body axis - **CE Study Options - Keller\'s Explants (Xenopus)** - a piece of tissue with the largest number of cells is taken from the clawed embryo, which performs CE and put it on a dish -- then it grows on its own - in the case of KO (morpholine blocks Lrp5) -- CE does not occur - after adding mRNA for Lrp5, a so-called \"rescue phenotype\" occurs - Cell shape changes at CE **And how does it happen?** Where there is a lot of Prickle, which is near the membrane, it can activate myosin, pulling it towards itself, thus stretching the entire embryo. **PCP proteins thus activate the actino-myosin cytoskeleton during CE.** *Consequences of impaired convergent extension (CE)* CE also overlaps in time with neurulation, which is the process by which a neural tube is formed. If the width of the neuroepithelium does not correspond to what they are programmed for, i.e. that CE is not optimal, it ultimately prevents the neural ramparts from fusing together. Typical phenotypes are various exocephaly and spina bifida (mutants VANGL and Prickle). Wnt/PCP pathway in morphogenesis However, it does not end there, PCP is also involved in other processes during later development. The most important players are: **Wnt5a** (ligand), **ROR1** and **ROR2** (receptors), **DVL** (dishevelled) -- cytoplasmic protein (converter) To explain the importance of these molecules, a very educational example is a mouse with a lack of essential Wnt5a. **Wnt5a knockout embryo** (1999) - defects in the morphogenesis of structures protruding from the body (outgrowth) such as limbs, tail, head structures or genitals - completely absent elongation!! At the same time, it has all the structures, only their extension is disrupted. Mutant cells are not capable of organized or oriented division. They lack a signal that would direct them proximally-distally. **Wnt/PCP affects morphogenesis by controlling cell orientation and asymmetric division.** Mutations in the receptor for Wnt5a - **ROR2** (as well as WNT5A, DVL2 and 3) cause Robinow\'s syndrome. **Robinow\'s syndrome dwarfism** -- a set of symptoms of autosomal dominant hereditary dyschondroplasia. Dwarfism is disproportionate, brachymelia, protruding forehead with hypertelorism, hypoplasia of the lower jaw with numerous anomalies of teeth, scrotal and penile hypoplasia, cryptorchidism are present. Fertility and mental development are normal. A mutation in DVL2 similar to that observed in MS patients determines a specific phenotype of bulldogs and bulldogs. 3\) Neural crest cell migration A cellular view of this process is summarized in the review. figure. The neural crest runs in several areas and in different ways. It is a process that leads to the formation of many organs in distant locations of the body. Overview of the main migration streams NC -- anteroposterior view -- here the cells have a slightly different cell fate. **The neural crest goes in two directions:** a. **ventro-medial** = from below to the middle b. **dorso-laterally** = top and side Dorsolateral and ventromedial migration of NC cells is essentially limited only by the somite and the neural tube. The main **molecular regulators** can be seen in the figure. Green are the neural crest cells and yellow are the factors that attract migration. Such soluble factors are, for example, those that control angiogenesis (VGF, FGF -- they are also strong chemoattractants, Sdf1 -- chemokine guiding cells in IS). These factors tell the cell where it has an ancestor, the so-called **leading edge**. Other molecules are such molecular repellents that are expressed in various places where the cells should no longer migrate (on somites, neural tube). These are ECM components, semaphorins, ephrins, etc. Migrating cells, on the other hand, have receptors for these semaphorins such as neuropilin, receptors for ephrins such as F-molecules, receptors for VGF or for chemokines (cxcr4). All of them must perceive the factors regulating migration. *Collective mesenchymal migration* In the neural crest, we can observe the phenomenon **of collective mesenchymal migration**, when the epithelium does not shift (as in healing), but the cells migrate together, but each separately. The cells are not interconnected, but rather form transient contacts -- as if in a herd, they determine the direction and stick together. Basic principles controlling the migration of (not only) neural crest cells a\) Route restriction (confinement) Collective cell migration is regulated by spatial **limitation** (e.g. due to ECM molecules such as proteoglycan **versican**), which acts as a physicochemical barrier and promotes the emergence of organized migration in separate streams. They support ephrine, semaphorine signaling, etc. They generally have a repulsive effect. b\) Contact inhibition of locomotion Contact inhibition of locomotion (**CIL**) is a process in which cells that come into physical contact with each other -- establish a mutual connection via N-cadherins (a marker of mesenchymal cells) etc. as shown in the picture, stop migrating in the original direction. After contact, they **repolarize** and **migrate** in **the opposite** direction. This has various consequences -- for example, cells migrate to somewhere where there are no other cells. However, repolarization can also be incomplete, only one cell out of two colliding cells can react, which then leads to ? c\) Autocrine chemotaxis (co-attraction) Autocrine chemotaxis (co-attraction) between mass-migrating cells is a process in which cells of the same type secrete **a chemoattractant** (e.g., part of the C3a complement in neural crest cells) that stimulates other cells in **the group** that have receptors for this chemoattractant. Cells respond to stimulation by moving towards each other and thus maintain a high cell density. They keep themselves in the \"herd\". The most important in this process are complement molecules, specifically C3a (re-use nature). When the cells sense it, they migrate together. Collective chemotaxis Collective chemotaxis is a process that takes place only if contact inhibition and locomotion are observed. Cells are able to perform more effective chemotaxis in collective chemotaxis if they are in a group/joint contact. One cell rotates (it has the right cell fate), which causes the other cells in contact to rotate, so that cell takes them like a train machine. Such a grouping is then much more sensitive to the perception of chemokine gradients (because the gradient is normally relatively small). The longer the train, the easier it is for the cells to navigate in this way. A typical example in NC cells (best described) is chemotaxis towards CXCL12 (SDF1). 4\) Cellular processes and principles in collective migration Axon guidance This is an example where the whole cell does not migrate, but only a part of it -- the axon -- the projection of a neuron. Axon guidance -- the process(s) by which the emerging axon is guided to target neurons, with which synaptic connections are then established. Axons are very long. And they work quite similarly to migrating cells, except that the axon on the neuron body works partially autonomously. Also, the connection works over much longer distances than the cellular ones. At the end of the axon, there is a special structure, called the Axon. **growth cone**. It answers almost the same as **the leading edge** for migrating cells. It responds to repulsive signals, from which it deviates and, on the contrary, is guided by signals that are chemotactic for it. It then extends in that direction as it detects these signals. *Molecules regulating axon guidance* - Slit/Robo - ephrins (**the Eph receptor** is on the growth cone and tells the axon *to stop and turn around*) - semaphorins - classical morphogens (Wnt, BMPs, FGFs\...) - **Eph/ephrin complex** - ephrin -- **are membrane-bound ligands** (similar to **ligands of the Notch** pathway in the sense that they respond only to contact with the cell) = receptor kinases - Both the receptor and the ligand are membrane-bound - ephrins **A** -- fixed to the membrane by means of a so-called **GPI anchors** - ephrins **B** -- transmembrane ligands that are themselves able to signal to the cell - The eph/ephrin system is mainly involved in the \"**navigation**\" of cells (e.g. blood vessel cells) or their parts (e.g. axon guidance in the nervous system -- they make a kind of lining), and in \"**contact-mediated cell sorting**\" in the developing embryo. It is a general mechanism regulating cell migration. Inwards, they signal to the cytoskeleton and activate small GTPases. - A unique property of ephrins: **reverse signalling** -- i.e. it signals not only the receptor, but also a ligand!!! - if we inhibit GTPase signaling, there is no retraction -- the signal is interpreted at the cytoskeleton level - **Semaphorins** - Semaphorin/plexin system - Their receptors are: plexin, neuropilin - semaphorin (Sema4D) is a membrane-bound ligand that binds to its receptor (plexin or neuropilin, Nrp1) - activation leads to activation of small GTPases (e.g. RhoA), remodeling of the cytoskeleton and a change in the direction of guidance *Neurogenesis -- axon ingrowth into the limb bud* It is regulated by the semaphorin family. They ensure that specific types of neurons that grow out of the neural tube find the right places and innervate the parts they are supposed to innervate. - SC-sympathetic ganglion - LMC-Lateral Motor Column - (m-medial; l-lateral) *Migration Study Options -- attractant/repellent assays* We can differentiate an explant from neural SCs and observe how our axons rotate relative to the chemoattractant. 5\) Common sub-cellular basis for polarity and migration  Synthesis and reminders All the processes discussed in today\'s lecture have a common denominator at the cellular level: Dynamic processes at the cytoskeleton level -- let\'s recall. ***Movement of the cell on the substrate*** - From the cytoskeleton point of view: - philopodium = the ancestor of lamelipodium, the explorer, is in the direction of the desired movement of the cell - The actin network in **the lamelipodium** -- the key role of **Arp2/3** (branches of actin filaments) -- stabilizes the actin (polymerizes there, pushing the membrane forward and thus moving it). In the case where repulsive signals are not present, there is attachment and eventually attraction\... I climb the rock -- lamelipodium is my hand that I grope for the rock. In case there is something to hold on to = attechment and protrusion phase. Finally, the traction phase = pull-up is one step higher. Quiescent cells = resting cells. Overexpression of proteins/fumbrine produces filopodia. Or they form lamelipodia or form stress fibers attached to the substrate, between them myosin works for contractility -- all these phases take place in the movement of the cell on the substrate. - Movement of the cell on the substrate -- key role **of integrins** = it knows that it is not possible to catch back (contact inhibition / locomotion), allows attachment and connection to the leading edge (shift due to myosins = motors) - in the front/in the back completely different contractile function of the cytoskeleton -- regulation due to **small GTPases**\... - Small GTPases from the **Rho family** are key regulators of the cytoskeleton -- each GTPase determines a different \"appearance\"/phase of the cytoskeleton - These small GTPases must be regulated spatially separately -- **Rac** must be expressed in the anterior part of the cell (promoting networking) and inhibit myosin activity (the one that makes up stress fibers). On the contrary, a small GTPase **Rho** promotes the formation of stress fibers and myosin activity -- cells will have rubusty fibers for myosin and the whole cell. - All this, how the small GTPases -- cytoskeleton -- cell organize themselves are determined by the **attractive**/ **repulsive signals** (in the case of Sbf1, VGEF -- activation of own receptors, coupled to G proteins/ receptor-tyrosine cyanase -- this activates Rak -- polymerization forward and due to the antagonism of Rak and Rho, there is a movement towards the chemoattractant. 30/11/20 9th lecture: The role and regulation of epithelial branching in morphogenesis (MB) During development, a number of organs, such as the salivary gland, mammary gland, lungs or kidneys, undergo the process of branching during morphogenesis. 1. Formation of new branches -- budding 2. Formation of new branches -- oriented cell division 3. Formation of new branches -- formation of grooves/slits = \"clefting\" 4. Process for creating new branches 5. Salivary glands 6. Regulation of branch extensions and their maturation 7. Examples of organs with branching epithelial tissues 8. Branching disorders Morphology of branching organs Several morphogenetic processes are involved in organ branching, such as migration, proliferation, cell rearrangement, shape deformation or ECM dynamics. During embryonic development, buds are formed first, which gradually enlarge over time. This process is important for the secretion or absorption of epithelial cells in the alveoli, acins or tubules. These branching structures occur not only in animals, but also in plants. In animals, they are found in Trachea Drosophila, blood vessels, lungs or nuclei. Morphology varies greatly -- length and diameter of branches. The morphology of branching organs also changes during development. For example, the bud of a mouse kidney is lobular in earlier stages with rounded tips and short branches, but later these branches become very elongated into tubular branches. [Branching can be divided into two basic types: ] **a) stereotypical** (the pattern of branches is genetically strictly controlled -- mammalian lungs, Drosophila tracheas, kidneys) **b) stochastic** branching (no branching pattern is given in advance -- there are no spatial limits in these structures -- mammary, salivary glands, blood vessels). 1\) Budding = creating new branches Morphogenesis of branching organs includes both the formation of new branches and the reorganization of old ones. New branches are formed **by budding** or by creating grooves by \"**clefting**\". Budding is *the de novo* formation of new branches from the superficial primordial bud and from the lateral sides from pre-existing branches, while furrow formation divides the existing branch top into 2 to 3 new vertices. Spatially, each bud creates a branch tip, and each slit formation creates at least 2 new vertices, but at the same time the old tip perishes. At the cellular level, collective cell migration, the formation of shapes through cell proliferation, as well as coordinated cell deformations mostly related to the cytoskeleton and cell rearrangement are involved in the formation of branching. a\) Invasive collective cell migration It takes place in **Drosophila traches** and in **blood vessels,** e.g. **mouse retina**. The leader cells migrate forward and the other group of cells rearrange behind them into a stem, where a new branch is then formed. The identity of the leading cells is then specified by RTK signaling in both systems. In the case of Drosophila tracheal it is FGF and in the case of mouse retina VEGF. Furthermore, the Delta/Notch is used in these systems, which is used for lateral inhibition (see the previous lecture). This serves to prevent the subsidiary cells from becoming leaders. It happens in both systems. The difference, however, is in the response of the stop sign cells to the RTK signaling. In Drosophilia trachea, FGF/Bni signaling is used, important only for the leading cells at the branch tip. The other cells of the peduncle respond only passively due to the stretching force exerted by the leading cells. When it comes to blood vessel branching, both tip and peduncle cells respond to VEGF signaling, which regulates the migration of apex cells and the proliferation of peduncle cells. b\) Non-invasive collective cell migration It has been described in the budding of the mammary gland, when multi-layered terminal buds are formed at the end. The leading cells have no invasive projections and there is no penetration of the basement membrane. In the terminal buds, there may even be changes in the position of the cells -- they can be dynamically exchanged for basal cells or leave the secondary position and get to the center and vice versa. If it is the lungs and ureter bud, here a simple or pseudostratified epithelium is formed at the tip of the bud. The cells remain at the tip and are pushed forward by cell division and rearrangement of the cell stalk. So it\'s different than with the mammary gland. 2\) Formation of new branches -- oriented cell division Oriented cell division also participates in the formation of new branches. This is mainly involved in the formation of the so-called **crevices**. It is most described in the formation **of grooves of lung buds**. In the lungs, at the beginning of development in the terminal parts, there are no differences in the distribution of proliferating cells inside the peak before its division. However, the cells in the center of the bud divide preferentially longitudinally according to the axis of flattening. This helps the bud to expand in the same direction and thus prepare for the formation of a slit. The exact regulation of this process is not yet known, it is the subject of current studies. Only the effect of PCP signaling, which is involved in the regulation of oriented cell proliferation in different bud regions, has been described. There is also a great influence of the extreme contraction of smooth muscle cells that differentiate around the peduncle and in the place of the future slit. Also, during the formation of the ureter bud of the slit, the homogeneous distribution of proliferating cells at the tips of the branches before the actual branching is first observed. First, the bud is expanded, then the foundation part is flattened, and finally grooves are formed, dividing the bud into two bases. Cell division is similar to that in the lungs. Here it is characteristic that the premetatic cells first delaminate from the simple epithelium, enter the central part of the bud, complete cell division there and then reinsert into the epithelium in specific places, deforming the shape of the bud. 3\) Formation of new branches -- creation of grooves Thus, regionalized proliferation and oriented cell division occur, which then contributes to the formation of new branches depending on the type of organ. At the same time, there is a rearrangement of the basal membrane and, in particular, accumulation of some basal proteins of this membrane. On the contrary, on the tips of the branches, the slits in the BM are broken. When the slitches of the salivary gland are formed, a layered bud is first formed, the outer layer of cells is made of tall columnar cells, very well arranged. Unlike the inner cells, which are arranged chaotically. In addition, the outer cells move much faster than the inner cells. During the formation of grooves, the ECM also invades the outer layer of the epithelium, widens and stabilizes. This process is also accompanied by the formation of microscopic perforations of the basal membrane and the accumulation of BM components in locations outside the forming grooves. 4\) Processes of creating new branches In addition to oriented cell division, different cell motility of individual cell populations, ECM remodeling (in the area of form grooves and in the tops of buds), external physical forces influencing the shape of the bud and then also cell rearrangement due to actino-myosin contractility are applied in the formation of new branches. Budding is independent of this contractility, but the formation of slits is already dependent. The completion of the organ architecture takes place as the final stage of the entire branching process. This is due to the lengthening of branches and the patturation of the final parts. The final morphology of the branching organ depends not only on the formation of new branches, but also on the ratio of branching peduncles to tips. Various cellular mechanisms can control peduncle elongation and tip maturation. Peduncle elongation occurs both by cell rearrangement by intercalation or convergent extension, and also by proliferation or simple elongation of cells (e.g. tracheal branches of drosophils -- occurs without cell division, peduncle cells elongate significantly and cell intercalation occurs). During convergent extension, rosettes can also form. In this way, the renal tubules of the mouse and Xenopus are lengthened. This convergent extension is also dependent on PCP signaling, which regulates the orientation of cell division. It is also important for general stimulation of tubule elongation. Oriented cell division is also involved in the regulation of branch shape in mouse lungs, where this division takes place longitudinally, probably due to delays in certain areas or due to the production of active extracellular kinases. Furthermore, the differentiation of the end parts of the branches also participates in the formation of the final shape. In the case of the salivary gland, the epithelial cells in the terminal part arrange themselves in tall shapes, which then surround the lumen of the acines. Some of these cells retain proliferative activity and serve for the gradual renewal of acina cells. There is no contribution from the stem cells. As far as the differentiation of alveoli in the alveoli is concerned, there is a differentiation into two cell types -- flat pneoumocytes type I = **AT1** cells (used for gas exchange, lining the alveoli themselves) or -- cubic cells = **AT2** (producing surfactant). Some of these cells always retain proliferative activity. The inner part contributes to the renewal of the epithelium in cycles. 5\) Salivary glands They are established as solid cell buds growing from the lining of the primitive oral cavity. The salivary glands can be of ectodermal origin (glandula parotins) or endodermal (glandula mandibularis and sublingualis). The bud-shaped bases of the salivary glands gradually lengthen into a cord during the process of ingrowth, which then branches into the bases of the glandular lobules. The bud of the salivary gland, unlike the base of the lungs, is made up of layered epithelium. Gradually, the outer cells are rearranged into columnar cells. They are generally more organized than internal cells. Morphogenesis However, during branching, the cells are not static, there are intensive movements between the outer and inner layers of the cells. Especially on the periphery of branching buds. The study -- using the stra