11/6 Development 2: Fertilization, patterning, & morphogensis

cell fate determination

• most of the time one directional, but can be reversible

• can be induced to de-differentiate from determined to totipotent/pluripotents state

adult cell to iPS cell

• cell fate determination is reversible

• start: fully differentiated adult cell (ex. fibroblast — connective tissue)

• 4 regulatory genes were identified by researchers (Oct3/4, Sox 2, Klf4, c-Myc)

• introduce genes via plasmid/vector

• dedifferentiation - new genes overtake current programming

• result: iPS cell, back into pluripotent state

fertilization

sperm and egg come together; sperm has genetic material from dad/egg has from mom

stages of fertilization

1. sperm penetration of follicle cell layer & zona pellucida

2. membrane fusion

3. egg activation — activates signal transduction pathway

what do membrane fusion & egg activation stimulate

PLCzeta signaling, Ca2+ wave 

PLCzeta

cleaves PIP2 to make DAG & IP3 (released (released from plasm membrane) 

fertilization pathway

• Trigger: A sperm fuses with the egg's plasma membrane, activating the egg.

• Enzyme Activation: This fusion activates the enzyme Phospholipase C zeta (PLCzeta).

• Cleavage: PLCzeta acts like molecular scissors, cutting a membrane lipid called PIP2 (phosphatidylinositol 4,5-bisphosphate).

• Products: PIP2 is split into two molecules:

  • DAG (diacylglycerol), which remains stuck in the plasma membrane.

  • IP3 (inositol trisphosphate), which is released into the cytoplasm to act as a second messenger.

• Calcium Release: IP3 travels to the Endoplasmic Reticulum (ER) and binds to IP3 receptors. This acts like a key opening a gate, causing the ER to dump a massive wave of stored Calcium (Ca2+) ions into the cytoplasm.

• Cellular Response: The spike in Calcium triggers cortical granules (tiny vesicles inside the egg) to fuse with the membrane and release their contents outside. This alters the egg's outer layer, creating a "slow block to polyspermy" that physically prevents any additional sperm from entering.

Taste Transduction Pathway

• Trigger: A tastant molecule (sweet, bitter, or umami) binds to a G-protein-coupled receptor (GPCR) on the outside of a taste bud cell.

• Enzyme Activation: The receptor activates a G-protein, which then activates Phospholipase C (specifically the PLC$\beta$2 version).

• The Shared Middle Steps: Exactly like fertilization, PLC cleaves PIP2 into DAG and IP3. IP3 then binds to the ER, triggering a release of Calcium (Ca2+).

• Cellular Response (Where they diverge): Instead of blocking sperm, the Calcium spike in the taste cell opens a specific ion channel (TrpM5) that lets Sodium (Na+) rush into the cell. This changes the cell's electrical charge (membrane potential) and causes the cell to release neurotransmitters (ATP via the Panx1 channel) to send a "taste" signal to your brain.

Why use amphibians?

The eggs develop outside the body, so it is easier to see the development 

Morphogenesis

• generation of ordered form & structure 

  • Body begins to develop recognizable structure 

  • Includes pattern formation (how does body pattern happen during body development) 

Gastrulation

cell and tissue movements during which blastula folds inward, generating 3 germ layers (ectoderm, endoderm, mesoderm) 

Layers within the embryo 

• Endoderm

• Ectoderm –

• Mesoderm  

In the early embryo

Pattern formation, Positional information leads to changes in signaling & gene activity 

Pattern formation

• use of positional information for body pattern 

  • We are 3D, can be on xyz axis 

  • Front (ventral) & back (dorsal) & (anterior) & (posterior)

what approaches have been the primary importance in determining the regions of the embryo that act in pattern formation?

Embryology

the Spemann-Mangold experiment 

• 1924 

• What part of the embryo plays an important role in that the dorsal side should develop into the dorsal side 

• What if you took dorsal tissue from an embryo and transplanted it into ventral → it would develop into a tadpole, but it has 2 dorsal sides & 1 ventral side 

• Indicates that there is something produced in dorsal tissue that instructs cells to take the fate of the dorsal structures 

the dorsal inducer noggin antagonizes the ventral inducer bone morphogenetic protein (BMP)

• Richard Harland – Noggin 

• Noggin – inhibitor of bone morphogenetic protein (BMP) 

  • By inhibiting BMP, cells in the dorsal region can develop into dorsal structures 

• What type of signaling scheme? Paracrine 

 which approaches have been of primary importance in identifying the genes & corresponding proteins that function in pattern formation?

Genetics in model organisms

• Note: just sequencing the genome will tell you all genes, but won’t tell you which genes are important 

Drosophila melanogaster

 model organism for studying A/P axis 

• Short lifespan 

• Develops quickly 

• Lots of progeny 

• Induce mutations → genetic cross (fly x fy) → egg → embryo → adult – look at altered body patterns 

Dropsophilia homeotic mutants

class of mutations that lead to the appearance of normal body parts in the wrong locations 

• Ex. mutant w/ 4 wings, mutant w/ legs as antennae 

• Are in homeobox (Hox) gene clusters 

Hox genes 

• control anterior/posterior pattern in animals, including in humans

• genes/proteins conserved in animals 

• Contain homeodomain that binds to DNA 

• Act as transcription factors 

• Differences in Hox gene sequence, position, or regulation result in striking differences in animal form 

  
  

Mutations in HOXA1

• disrupt human brain development 

• Rare: but some people have a disorder in the HOXA1 gene

• Why is it rare? Messing with these genes/expressions, won;’t be able to develop 

• 4 clusters – 1 could sub for the other, maybe there is some redundancy