Advanced developmental biology 1

• What is juxtacrine signaling?

  Signaling of surface proteins of cells that are in contact with each other.
• Is notch signalling paracrine, autocrine or juxtacrine?

  Juxtacrine
• What is paracrine/autocrine signalling?

  Signals that are released and encounter receptors by diffusion
• What is the difference between instructive and permissive signals?

  Instructive = initiated new program

  Permissive = provides a favourable environment for a specific program
• The gastrula gives rise to 3 germ layers, what are they?

  Ectoderm (external layer)

  Mesoderm (middle layer)

  Endoderm (internal layer)
• Pattern formation


  Process by which cells are organised in space and time to produce a well-ordered structure within an embryo.
• Morphogenesis


  Cell and tissue movements and changes in cell behaviour that give the developing embryo or organ its 3D shape.
• What is involved in morphogenesis?

  Cell adhesion, cell death, cell migration and cell shape
• What type of signalling are Shh, Wnt, TGFbeta, BMP and FGF an example of?

  Paracrine/autocrine
• cell differentiation


  Process where cells become different from each other and acquire specialised functional property whilst losing pluripotency.
• What are the steps involved in differentiation?

  1. Stem cell/ progenitor cell

  2. Specification

  3. Determination

  4. Differentiation

  5. Maturation
• Growth


  Increase in mass or size
• What 3 things can growth result from?

  Cell proliferation, cell enlargement or ECM production.
• What methods can be used to establish where and when a gene is expressed?


  - In situ hybridisation

  - Reporter lines

  - High throughput analysis (e.g. RNA-sequencing)
• What methods investigate the distribution of proteins i.e. is it expressed in the same location and at the same time as the gene?

  - Immunodetection (immunofluorescence/ immunohistochemistry)

  - Fusion protein construct
• What methods can be done to determine if a gene/ protein is essential for development?

  - loss of function (forward and reverse genetics)

  - gain of function (transgenesis)
• What methods demonstrate how the gene is regulated or the inductive functions of one tissue on another?

  - embryology: tissue manipulation (graft, transplantation, ablation)

  - manipulating signalling pathways: drugs, transfection/electroporation, bead/cell implantation
• What is fate mapping: lineage tracing used for?

  To understand what structure a single cell or group of cells in an early embryo contribute to in a later-stage embryo.
• Is a morphogen instructive or permissive?

  Instructive
• What tests can be done to ensure a morphogen is instructive?

  1) provide a second source of the initial signal

  2) provide a source of initial signal at uniform concentration
• What tests show a morphogen has direct action rather than bucket brigade?

  1) using genetic engineering to make the proposed morphogen juxtacrine (e.g. adding a transmembrane domain)

  2) making a genetic mosaic that lacks the receptor for the initial signal in one of the cells
• What must a molecule do in order to be a morphogen?

  1) induce different outputs at different concentrations

  2) act directly at a distance
• How does restricted diffusion establish a morphogen gradient?

  They generate a steep gradient by binding to molecules in ECM with high concentrations of receptor. Rapid degradation of signals in ECM can also generate a steep gradient.
• How does planar transcytosis establish a morphogen gradient?

  Certain morphogens travel through cells in a tissue by repeated cycles of endocytosis and desecration (cell membrane engulfs morphogen in a vesicle).
• How do HSPGs regulate morphogen diffusion?

  - sequestration or slowing diffusion (BMP- TGFbeta)

  - facilitating diffusion (Hedgehog)
• Provide an overview of Smad signalling?

  1. Reception: Ligand-receptor complex assembled then r2 phosphorylate multiple serines/threonines in GS domain of r1 to activate it.

  2. Transduction: r1 phosphorylate Smad proteins. They act as secondary messengers.

  3. Response: tissue-specific TFs modulate Smad binding to target genes.
• How do RTKs signal downstream?

  1. FGF binds HSPG to form oligomers that bind RTKs in order to dimerise.

  2. Dimers on cells in close proximity to one another cross-phosphorylate, creating docking sites for intracellular proteins.

  3. SH2 of GBR2 binds FGF receptors while SH3 domains bind Sos.

  4. Sos is complexed with FGF and GBR2. This complex binds Ras to promote GDP release.

  5. GTP-bound Ras binds Raf activating Mek, which phosphorylates ERK/MAP.

  6. ERK/MAPK phosphorylates TF to regulate target genes.
• Why does FGFR activation by paracrine FGFs require HSPGs?

  HSPGs are chains of sugars that can modified in many different ways, such as sulphation. These modifications can result in a code that creates binding sites for specific proteins, e.g. FGF2. Paracrine FGGs have a high affinity for HSPGs so are retained and act locally but endocrine FGFs have low affinity for HSPGs so they diffuse into the bloodstream.
• FGF signalling can trigger different cell behaviours through distinct pathways. What result comes from 1) Mapk 2)Akt 3)Calcineurin

  1) cell proliferation

  2) cell survival

  3) cell motility
• What are the steps involved in the Hedgehog signalling pathway?

  1. Hh binds Patched outside the cell, Patched is degraded.

  2. This allows Smoothened to move to the PM through vesicle fusion.

  3. Kinase binds/phosphorylates Smoothened. Cos-2 and Fused proteins are also phosphorylated leading to the disassociation of Ci and SUFU complex.

  4. Ci can move from the cytoplasm to the nucleus to bind CBP, acting as a transcriptional activator of Hh target genes.
• What are the steps involved in the Wingless signalling pathway?

  1. Wnt binds LRP and Frizzled.

  2. Frizzled recruits Dishevelled.

  3. Destruction complex moves to PM, where it's phosphorylated by GSK3 and CK1. It's then degraded.

  4. Beta-catenin accumulates in the cytoplasm then moves to the nucleus to bind LEF/TCF. It then displaces Groucho (repressor) to activate WNt target genes.
• What are the negative regulators of the hedgehog pathway?

  Suppressor of Fused (SuFu), protein kinase A (causes formation of CiR), GSK3 and CK1 (kinases).
• What are the negative regulators of the wingless pathway?

  Destruction complex (APC, Axin, CK1 and GSK3), which degrades beta-catenin.
• What is the role of hedgehog in development?

  Many functions.

  A-P patterning of limbs, wings and gut.

  Neural tube differentiation.

  Lung branching.

  Muscle differentiation.

  Etc.
• What is the role of wingless in development?

  Segmentation in early development.

  D-V boundary of Drosophila wing, required for patterning and outgrowth.

  Regulation of neuronal fate in C.elegans.

  Important for upkeep of stem cells in gut, where there's lots of cell turnover.
• What would the expected outcome of a mutated Gli3 mutation be?

  Low level of Hedgehog signalling everywhere
• What cancers are formed as a direct result of defects in Wnt or Hh signalling?

  Colon cancer Wnt active; Rhabdomyosarcamo Hh active.
• What is spermatogenesis?


  production of sperm.

  In drosophila, it involves hub cells secreting factors (e.g. unpaired), stem cells being adjacent to hub cells and the cells further away differentiating.
• What is oogenesis?


  Egg production. 4x incomplete 'cytoblast' mitotic divisions, where an oocyte is in the centre of 4 nurse cells.
• What is the hierarchy of genes?

  1. Maternal genes

  2. Gap genes

  3. Pair rule genes

  4. Segment polarity genes