Farin et al. Paper Investigation

• intestinal crypts are made of many cells constantly regenerating like intestinal epithelial, progenitor, stem, and paneth cells

  • progenitor cells -> descendent of stem cells, cannot indefinitely divide
  • paneth cells -> play roles in immunity and secrete Wnt signals to control stem cells, also secrete AMP, IgA and other factors to maintain sterile crypt environment
  • stem cells -> make most cell types, constantly dividing, they rejuvenate all cells within the IE cell layer

  
• differentiated cell types in the intestine

  • enterocytes -> responsible for nutrient uptake in intestinal wall, are absorptive
  • enteroendocrine -> secrete hormones associated with digestion
• cells in the intestinal crypt all end up being unique cell types by expressing different combinations of TF

  
• organoid cultures are used to study cellular interactions and organization in tissues

  • organoids are models of a regulated cellular assembly into a part of a tissue in culture media which recapitulates/summarizes
  • cell polarity
  • cell/cell contacts
  • influence of 1 cell type on another
  • organoid culture can be made from many different cell/tissue types as long as initial stem cell and growth factors are present

  
• organoids are used to study intestinal crypts by Farin et al. which contain stem cells and paneth cells

  
• to make the intestinal organoid culture in the lab

  • stem cells are isolated from an intestine -> cells become differentiated upon adding culture containing mitogens, morphogens, GF -> cells are grown in a supporting matrix to grow organoid

  
• Farin et al.

  • hypothesis -> Wnt3 (Wnt type known to function in intestinal stem cell maintenance) are secreted by paneth cells and act in a very localized manner at the bottom of intestinal crypts to maintain the balance between intestinal stem cell SELF RENEWAL and REGENERATION of DIFFERENTIATED intestinal cells like enterocytes
  • objectives
  • find location of Wnt3 in crypt/cell specifically
  • determine how Wnt3 travels from paneth to stem cells
  • determine how Wnt3 gradient is established
• Wnt3 is a 356 AA protein with few mechanisms to visualize it

  • couldn't tag the N terminal -> has signal peptide (toylation for transport to ER
  • couldn't tag the C terminal -> interacts with Frizzled and has to be available for that
  • thus made an internal HA tag which is downstream of the signal peptide but before the important coding region so Wnt3 could be monitored and visualized by western blot or immunofluorescence
  • Wnt3 HA/HA genetic mutation made in mice and isolated the stem cells to make organoids with this modified Wnt3
  • anti HA antibodies were used to detect Wnt3

  
• result: Wnt3 is exclusively found in the crypts of basolateral cell membranes

  
• result: Wnt3 is displayed on the surface of crypt cells

  • permeabilized cells -> detergent added, antibodies bind to Wnt3 displayed at cell surface or inside cells because it has to
  • non permeabilized cells -> no detergent, antibodies only bind to Wnt3 displayed on the cell surface, no possible way for it to enter the cell so it must be on the surface

  
• question: are Wnt3 signals diffusible from paneth to target cells or do they need physical contact?

  • made organoids from wild type stem cells and Wnt3 knockout stem cells and put them in the same dish without direct contact
  • found that the wild type that made Wnt3 couldn't rescue the knockouts because Wnt3 is not diffusible and needs direct contact to function
  • in each figure, there is a decrease in Wnt3 levels when the knockout is present

  
• question: since we know that Wnt3 is not diffusible, that means they must be in direct contact to sent Wnt3 signals to other cells

  • made organoid containing both knockout and wild type Wnt3 cells
  • found that the wild type paneth cells can rescue the knockout since they're in the same organoid and allowed differentiation to occur
  • therefore Wnt3 transfer needs direct cell contact

  
• wild type paneth cells can transit Wnt3 signals 1 to 2 cell distances away between wild type cells that make Wnt3 and knockout cells that can't make Wnt3

  
• Frizzled (Wnt3 receptor) is also needed for Wnt3 detection on surface of crypt cells

  • antibody that binds/blocks Frizzled receptor -> get less Wnt3 on cell surface thus Frizzled is needed for Wnt3 detection

  
• what determines which cells get Wnt3 on surface and how much? -> Wnt3 secreted from paneth cells binds to nearby cells and stays on those cells until they divide which dilutes the Wnt3 signal -> this causes differentiation towards enterocytes

  
• Wnt3 signal spreading requires cell division -> the Wnt3 gradient is formed by dilution during cell division

  • inhibited cell division in cells -> cells lose crypts, organoids aren't properly formed, Wnt3 signalling lost