Week 9 - 10 morphogens

• Morphogenesis --> is a biological process of pattern formation that allows the organization of differentiated cells into specific and specialized structures - such as tissues or organs

  
• Morphogenesis is stimulated by a combination of chemical and mechanical factors that control the organized spatial distribution of cells during embryonic development. It can also occur in mature organisms for tissue homeostasis or stem cell regeneration after tissue damage

  
• Morphogens are a special signalling molecule (Chemicals/peptides/proteins) that play an important role in morphogenesis.

  
• morphogens act to define cell fate in a concentration-dependent manner, forming gradients across tissues or compartments --> a cell secretes morphogens, and things closer to that cell will have a higher concentration of that morphogen than things further. Depending on how much morphogen is taken in by the cell due to differences in concentration will determine the effects.

  
• Morphogens affect cellular processes that can stimulate cell movement or alter the transcriptional profile of the cell

  • influencing cell-cell adhesion
  • changing the composition of the extracellular matrix
  • alter the shape or size of a cell

  
• Morphogens are a type of paracrine/autocrine signal. it is also an analog type signal because of the different fates it causes depending on the distance from the morphogen producer

  
• the function of a morphogen: influence cell-cell adhesion

  • Cells can bind to one another using membrane-anchored extracellular proteins called cell adhesion molecules (CAMs)
  • Morphogens can influence the expression of different CAMS, affecting how cells attach.
  • high morphogen = a lot of cadherin
  • med morphogen = some cadherin some IgCAM
  • low morphogen = a lot of IgCAM

  
• Function. of morphogen: Influence the composition of the extracellular matrix(ECM)

  • The ECM is a dynamic network of macromolecules and minerals that provide structural and biochemical support to surrounding cells
  • Enzymes, polymers, glycoconjugates, carbohydrates
• Function of morphogens: influence the composition of the extracellular matrix (ECM)

  • The ECM can aid in the separation of cells
  • The ECM can create a system in which cells can move from one location to another

  
• Function of morphogens: Influence cell contraction

  • Morphogen can stimulate the production of proteins involved in cell contraction --> such as actin and myosin (proteins that move stuff around in the cell)

  
• Function of morphogens: influence cell contraction

  • Increased production of contractile proteins (actin and myosin) can change the shape of a cell, which can create a mechanical force on adjacent cells
  • this can lead to transcriptional expression differences or influence cell differentiation.
• morphogens specify the fate of cells, particularly during development. Morphogens are typically released from a specific cell type --> usually by paracrine or endocrine signalling.
• Morphogens often act via a gradient

  • the cell in which the morphogen is made secretes morphogens
  • Morphogens diffuse from the origin and establish a concentration gradient emanating from the site of secretion
  • Cells respond differently to different concentrations of the morphogen
• Morphogens can change tissue patterns by activating certain genetic transcription factors that regulate what genes are activated.
• The French flag model:

  • Genes A, B and C express different things in different cells because they are getting different amounts of morphogen from the source aka the gray cells.
  • There's a decrease in concentration of morphogen when you go further from the secreting cell.

  
• Neurulation occurs after the formation of the germ layers

  • Endoderm forms many structures such as endothelial GI tract, kidneys, lungs, livers, pancreas
  • Ectoderm forms the skin and nervous system
  • Mesoderm forms the muscular, skeletal, cardiovascular, excretory, and reproductive systems
• the notochord (NC) is a cylindrical rod of cells that spans the entire length of the developing embryo and directs the thickening of the ectodermal cells to form the neural plate

  
• Neural Tube formation:

  • The notochord stimulates the outward motion of the mesoderm, causing inward folding of the ectodermal neural plate. (plate between meso and ecto)
  • Neural plate invagination occurs, linking the two neural folds, which then form the neural tube and neural crest.
  • Neural tube gives rise to the central nervous system --> brain and spinal cord
  • Neural crest gives right to the peripheral nervous system --> ganglia

  
• Morphogens direct unique patterns of gene expression in the developing Drosophila embryo

  • FGF --> tail to head --> posterior to anterior
  • RA --> head to tail --> anterior to posterior
  • Shh --> front to back --> ventral to dorsal

  A decrease in concentration is shown from their start point to their endpoints

  
• The primitive streak runs around the mesoderm area which is what gives rise to the notochord

  
• • fibroblast growth factor (FGF) is secreted from the posterior
  • retinoic acid (RA) is secreted from the anterior
  • Sonic Hedgehog (Shh) is secreted from the ventral side
• Three key receptor signalling pathways important for development

  • Wnt
  • Hedgehog
  • Notch
• Wnt signalling

  • very localized action, creates steep gradients (requires high concentration to work)
  • Wnt ligands are covalently attached to a lipid tail at their N-terminus --> this way they can't go far (palmitoylated)
  • there are many different Wnts (ligands) and Frizzled (GPCR receptors)

  
• Planar cell polarity (PCP) is a polarity axis that organizes cells in the plane of the tissue. Morphogens act on this field allowing for different development. Morphogens either act on its apical, basal, proximal, or distal.

  
• Frizzled/Wnt in planar cell polarity and morphogenesis:

  • Frizzled and Dishevelled on one cell form a complex with Vang/Fmi on the next cell
  • This complex represses response to Wnt, so that each cell "accepts" Wnt signal from ONE side but not the other. --> polarization

  
• Polarization causes cells to have a leading edge that are always receptive to Wnt signalling and the other isn't . Frizzled will always be engaged in a protein/protein interaction which either activates (Wnt) or outcompetes for Wnt (Vang).

  
• Planar cell polarity is the ability of cells in a monolayer to exhibit polarity perpendicular to the apical-basolateral polarity. Frizzled and Wnt are important for establishing planar cell polarity. Frizzled on one cell can bind to Vang/Fmi on another (nearby) cell, which impairs normal activation by Wnt, and establishes planar cell polarity.

  
• Hedgehog signalling:

  • Hedgehog ligands are covalently attached to cholesterol (on the C-terminus), as well as a fatty acid chain (palmitate on the N-terminus)
  • Hedgehog receptor is called Patched, with iHog as a co-receptor
  • Inside cells, the main target of hedgehog signalling is called Cubitis interruptis (Ci)

  
• Notch is a receptor that is synthesized and trafficked to the plasma membrane.

  • upon binding to its ligand (Delta, which is also a transmembrane protein on a different cell).
  • notch undergoes cleavage to remove a large portion of the extracellular domain
  • The smaller notch membrane-associated fragments undergo endocytosis
  • In endosomes, the smaller Notch membrane fragment undergoes further cleavage, releasing a small fragment
  • This last fragment is also a transcription factor/regulator, leading to the activation of notch-specific genes

  
• Wnt, Hedgehog and Notch are all morphogens involved in development.

  • Characteristics of morphogens:
  • Act in a very short-range manner (paracrine, juxtacrine)
  • Form gradients that specify different fates at different concentrations of ligand
  • Morphogens often act in gradients, with cells distinctly responding to different concentrations of morphogens
  • Different thresholds must be met for activation of different characteristics

  
• The intestinal crypts are made up of many cells that are constantly regenerating:

  • Enterocytes: nutrient update in intestinal wall
  • enteroendocrine: secrete hormones associated with digestion
  • Paneth cells: roles in immunity, also secrete Wnt signals to control stem cells
  • Stem cells: can make most other cell types, and are constantly dividing.

  
• stem cells differentiate into different types of cells depending on the transcription factors that are activated within the cells. Paneth cells have control over Wnt which holds control of TF expression. The proximity of the cells will determine what TF will be activated in stem cells for it to differentiate

  
• Organoids: are model organs that is a regulated cellular assembly of a part of a tissue in culture.

  • it recapitulates:
  • cell polarity
  • Cell-cell contacts
  • influence of one cell type on another

  It's pretty close to an organ without having to explant tissues from a live subject.

  • it is also able to to made from many different cell, tissue types

  
• Making intestinal organoid cultures in the lab

  1. Stem cells are isolated from in-vivo (in organism) intestine
  2. the intestinal stem cells are then differentiated into different cells and controlled in-vitro (outside organism)
  3. cells grown in supporting matrix to form intestinal organoids

  
• The hypothesis of the Farin et al paper was:

  • Wnt3 (a Wnt that is known to function in intestinal stem cell maintenance) is secreted by Paneth cells and acts in a very localized manner in the bottom of the intestinal crypt to maintain a balance between intestinal stem cell self-renewal and generation of differentiated intestinal cells.

  
• The Farin et al. experiment starts off by generating a mouse model that can be used to look at the tissue distribution of Wnt ligands in the intestinal crypt.

  Then, use organoid cultures from different mice background to study the behaviour of Wnt signals in detail

  
• Wnt is hard to tag because we can't just tag the N-terminus or C-terminus because they get cut off in the process. So in order to tag Wnt we need to make an epitope tag. We have to find a spot somewhere inside the Wnt that had no interactions and allows us to monitor the function.

  • an HA epitope tag was added downstream of the signal peptide but before any important sites.
  • A bunch of antibodies were then used in order to visualize Wnt3

  
• Wnt3 HA/HA was found exclusively in the basolateral (bottom) cell membranes of the crypt cells

  
• Wnt3 is displayed on the cell surface of crypt cells --> to check this they went and used permeabilized cells that allow antibodies to bind to Wnt inside or on the cell surface.

  • Wnt was only found on the cell surface --> this meant that signalling w/ Wnt must happen outside the cell

  
• Separated wild-type organoids DO NOT rescue knockout cell defects for making organoids

  • what they did here was make a knockout Wnt3 cell organoid and make a WT-cell organoid
  • they put both in the same dish BUT there was NO direct contact between the two. What they end up seeing was that Wnt3 from WT did not reach the Wnt3 knockout organoid
  • this meant that Wnt3 is not freely diffusable (can't be shot out).