1 Embryo, Cells and Genes

• Induction


  1 cell changes the fate of another
• Signalling Pathways (Induction-Paracrine)


  FgF, Activin/TGF-Beta, Wnt, Hedgehog (Hh)
• Activin/TGF-Beta Signalling

  • TGF-Beta ligand is a dimer - causes receptor dimerisation.
  • Can be modulated by extracellular antagonists.
  • Binds to specific SMADS (1,5 & 4).
  • Is a transcriptional regulator.
  • Cell growth, differentiation, apoptosis and cellular homeostasis.
• Wnt Signalling

  • Binds to a 7-pass transmembrane receptor (FRIZZLED).
  • Polarises the anterior-posterior axis.
  • Lead to activation of Rho GTPase.
  • Polarises anterior-posterior axis.
  • Role in somatic stem cell self-renewal.
• Hedgehog (Hh) Signalling

  • Required for proper cell differentiation.
  • Binds to PATCHED (receptor) on membrane.
  • Activates Ci - in turn activates gene expression of target genes.
• Paracrine Signalling


  Signalling to nearby cells.
• NOTCH Signalling

  • Delta (ligand on neighbouring cell) binds to NOTCH receptor via a modulator.
  • Initiates a conformational change.
  • Part of the receptor is cleaved.
  • Cleaved part is required for activation of transcription factor.
  • Involved in cell fate determination, differentiation, proliferation and apoptosis.
• Fate Map


  A picture of the normal destinations of cells in regions of the embryo.
• Differential Adhesion Hypothesis


  Cell types differ in their adhesion strength and will organise in a way that is thermodynamically stable.
• Selective Affinities


  Allow the cells to segregate into their "normal" embryonic structures after being aggregated together.
• Mesoderm Patterning


  Prospective mesoderm, even before it is internalised, is patterned by cells that will make the notochord.
• Drosophila Neurogenesis

  Specification of cell identity in the nervous system.

  A) One cell has more notch signalling.

  B) All cells could be neural.

  
• Signalling Pathway Conservation

  Signalling pathways are active in many different cell types. Some of the signalling pathways are used in different contexts. Therefore, the number of signalling pathways is very limited, and each pathway is re-used many times.
• How do we know about signalling pathways?

  • Mutations in Cancer.
  • Sensitised genetic screens.
• Screens for Enhancers/Suppressors of Phenotype

  • Mutations in a signalling pathway component that gives a viable, but obvious mutant phenotype.
  • Random mutagenesis in this strain.
  • Screen for mutations that suppress or enhance the phenotype.
  • Example: Rough eye in drosophila.
• Drosophila Early Embryo

  First ~ 180 mins of Drosophila development.

  A) Begins with a single nucleus deep within the egg/yolk.

  B) Nuclear divisions

  C) Migration

  D) Surface cortex

  E) Beginning formation of cells

  F) Occurs first at posterior

  
• Development of AP Axis in Drosophila

  • Gut-endoderm at the anterior.
  • 6 segments = Head.
  • 3 segments = Thorax.
  • 8+ segments = Abdomen.
  • Gut and pole cells at the posterior.
  • Mesoderm gastrulates to form the segments.
  • Serosa found at the dorsal side (temporary).
  • Staining can be used to plot the different regions of the blastoderm.
• Genetic Analysis of Drosophila AP Patterning

  Made mutations - scored embryo phenotypes to identify relevant genes.
• Maternal Effect

  Mutant mother gives abnormal embryo. Gene function is needed during oogenesis in maternal cells for later patterning of the anterior or posterior of the embryo.
• Zygotic Effect

  Mutant embryo is abnormal. Gene function is needed in cells of the embryo (zygote).
• Patterning in Oogenesis

  Patterning in oogenesis:

  A) Ovariole tubes - each tube works independently.

  B) Gradient of mature cells.

  C) Produce egg shell on the surface then die.

  D) Egg chamber

  
• Patterning the Oocyte

  Patterning the oocyte:

  A) Produce the signal.

  B) Nucleus signals to follicle cells above.

  C) Initiates formation of dorsal-ventral axis.

  D) Polyploid

  E) Nurse cells produce gene.

  F) Recpricol signalling

  G) Become aligned end-to-end.

  H) Forming and transporting yolk cells.

  I) Signal between posterior follicle cells and oocyte.

  
• Signalling in Oocyte Patterning (Drosophila)

  1. Bicoid (maternal nurse cell) is transcribed which binds dynein.
  2. Bicoid RNA localises in the oocyte.
  3. Gurken is transported to the dorsal-anterior region of the oocyte.
  4. Oskar RNA is transported to the posterior by kinesin.
  5. Oskar binds nanos RNA.
  6. Translation and protein diffusion in the syncitial cytoplasm.
  7. Transcription in response to gradient levels of bicoid.
  8. Patterened zygotic gene (requires large amount of bicoid) transcription.
  9. Segmentation.
• 'Morphogen' Concentration Gradient (Bicoid)


  Bicoid RNA - highest concentration is in the anterior of the embryo and lowest in the posterior forming a gradient.
• Patterning the Early Embryo (Posterior)


  Nanos protein has high concentration at the posterior end and lowest at the anterior creating a nanos gradient. The gradient binds maternal hunchback RNA. Hunchback is a transcription factor which leads to segmentation.
• Patterning - Gradients

  Atypical - Diffusion/Decay in the syncitial cytoplasm. Bicoid and a few others.

  Typical - Diffusion/Decay in the extracellular matrix. Hedgehog, Wnt, Fgf, BMP etc. Animal intracellular signalling pathways.
• Patterning Conclusions

  1. Development of the embryo starts in oogenesis.
  2. Patterning depends on spatial interaction.
  3. Development of differences between cells occurs by control of gene transcription.
  4. Details will vary between organisms.
• Amphibian Embryo

  • Traditional system to analyse developmental mechanisms.
  • Maternal gene products are moved way from the site of sperm entry as the cortical cytoplasm rotates (before division starts).
  • Gene products are moved towards dorsal side.
  • Dorsal mesoderm folds in during gastrulation and forms a blastopore.
  • Ventral mesoderm also tucks in later.
• Blastula Fate Map (Xenopus laevis)

  Labeled fate map:

  A) Ectoderm

  B) Epidermis

  C) Nervous System

  D) Notochord

  E) Head Mesoderm

  F) Blood, Kidney

  G) Endoderm

  H) Head Endoderm

  I) Mesoderm

  J) Ventral

  K) Dorsal

  L) Animal

  M) Vegetal

  N) Somites

  
• Spemann & Mangold 1920s Experiment

  • Experiment on xenopus laevis.
  • Transplanted dorsal cells to ventral side of different embryo.
  • Formed a mirrored organism.
• Identifying Maternal (Vegetal) Determinants

  • Find maternal determinants via differential expression.
  • Vg1 (part of the TGF-beta superfamily).
  • Vg1 RNA - persists in vegetal cells of embryo until mid blastula. Veg T is a transcription factor that works with Vg1.
  • Vg1- showed reduced mesoderm, no notochord and very reduced nervous system.
• Establishing Dorsal (Xenopus laevis)

  1. Sperm entry at the ventral side - microtubules extend from the centriole.
  2. Initial rotation of the cortical cytoplasm.
  3. Alignment of extending microtubules.
  4. Transport of maternal gene products (dishevelled, wnt 11 RNA) along the microtubules.
  5. Beta-catenin stabilised due to dishevelled protein on the dorsal side - Wnt signalling pathway.
• Zygotic Vegetal Signal - Nodal (Xenopus laevis)

  • Zygotic nodal transcription in early-mid blastula.
  • Nodal transcription gradient - lowest at ventral and highest at dorsal.
  • Maternal products VegT and Vg1 (activates) control nodal transcription on the vegetal side.
  • Beta-catenin upregulates nodal's transcription on the dorsal side.
• Mesoderm Induction & Patterning (Xenopus laevis)

  Nodal signalling & Beta-catenin turns on Siamois (transcription factor) at the dorsal side which leads to inhibition of BMP signalling and activates BMP antagonists Chordin and Noggin. Therefore, BMP4 patterning is formed as a gradient from highest at the ventral side to lowest at the dorsal side.
• BMP4 Gradient Function (Xenopus laevis)

  Graph of BMP and Noggin/Chordin gradient:

  A) Chordin & Noggin

  B) BMP4

  C) Block

  D) Active

  E) Gradient

  F) Signalling

  G) Ventral

  H) Dorsal

  I) Neural crest

  
• Amphibian

  Amphibian Embryo:

  A) Forebrain

  B) Hindbrain

  C) Spinal cord

  D) Notochord

  E) Neural crest

  F) Limb

  G) Limb

  H) Somites

  I) Endoderm

  J) Lateral plate, Blood

  K) Epidermis

  
• Amphibian Patterning Conclusions

  1. Development of pattern starts at oogenesis.
  2. Patterning proceeds by spatial interaction.
  3. Development of differences between cells occurs by control of gene expression.
  4. Details vary between organisms.
• Why study plant development?

  • Plants and animals are thought not to share common multicellular ancestors.
  • Evolved multicellularity independently.
  • Comparison of plant and animal development important.
  • No cell migration or contraction in plants.
  • Genome sequence and CRISPR have made many plants accessible to analysis (Evo-devo).
• Immobility (Plants)


  Immobility of plants means they are a sitting duck in a changing environment however, plants have pockets of flexible stem cells which are easy to clone.
• Plant Organs

  Plant organs initiate at apical meristems.

  A) Leaf organ primordium

  B) Shoot apical meristem (SAM)

  C) Axillary meristem

  D) Ground tissue

  E) Vascular tissue

  F) Epidermis