Lecture 14: Making Cells Different I - Partitioning, C.Elegans

C. elegan’s early development comes from…

its own genome

5 steps of fertilization

1) chemoattraction

2) binding

3) exocytosis

4) passage

5) fusion

Chemoattraction

• of the sperm to the egg by soluble molecules secreted by the egg 

Binding

• of the sperm to the extracellular matrix (jelly or zona pellucida) of the egg 

What surrounds the egg plasma membrane?

Jelly coat and vitelline envelope

Exocytosis

of the sperm acrosomal vesicle and the release of its enzymes

Passage

• of the sperm through the ECM to the egg cell membrane

• Components from the sperm acrosome degrade ECM

fusion

• of the egg and sperm cell membranes

The acrosome reaction (sea urchin) 

• A specilized exocytosis - acrosome membrane membrane fused with the sperm membrane 

• The acrosome is a cap-like, membrane-bound organelle derived from the Golgi apparatus located on the anterior half of the sperm head. 

  • It contains hydrolytic enzymes

  • Acrosomal enzymes digest the egg ECM (vitelline membrane in sea

  • urchin, zona pellucida in mammals)

• Actin polymerization pushes the front of the sperm outward, forming a finger-like projection 

• The finger-like projection is coated with bindin, a species-specific protein that allows the sperm to adhere firmly to the egg's vitelline envelope

blocks to polyspermy (fast)

• Change in membrane potential due to influx of Na⁺

• Sperm cannot fuse when the membrane potential is positive

• Occurs in sea urchins and potentially Xenopus, but not mammals

blocks to polyspermy (slow - cortical granules)

• Release of cortical granules from the egg via exocytosis

  • Cortical granules contain lots of glycosaminoglycans (GAGs)

• Contents modify the outer ECM layer so that sperm cannot fuse

• Includes a Ca²⁺ wave generated in the egg 

• Occurs in most species, including mammals and sea urchins

blocks to polyspermy (slow - Zn²⁺)

• ‘Zn spark’ - release of Zn²⁺ ions around the egg that strengthens the membrane to prevent sperm entry 

• Occurs in mammals and fish

C. elegans development

• ~15 hrs to hatch 

• Transparent 

• High brood sizes 

• Almost all organ systems are present (except the cytoskeleton)

• Asymmetric division  

• The 26-cell stage starts Gastrulation

• First complete genome to be sequenced

what cell stage starts gastrulation in C.elegans

26

Invariant lineage (C.elegans)

• A fertilized zygote (P0) always divides into 2 cells,

  • One is big (AB - anterior), one is small (P1-posterior)

• From AB:

  • Get ABa and ABp 

• From P1:

  • Get EMS and P2

• From EMS:

  •  Get MS and E 

• From P2: 

  • Get C and P3

• From P3 

  • Get D and P4 

Many ____ cell deaths occur a precursor cell divides asymmetrically

somatic, one daugther surives, and the other dies

assymmetric cleavage of blastomeres generates…

cell fate diversity and body axes

AB (big cell) gives rise to

anterior

small cell (P1) gives rise to

posterior

sperm entry point defines ____ in C. elegans

A-P axis

Wherever sperm enters is where the cell becomes….

P1 so posterior

Polarity proteins are

PAR proteins

Anterior PARs

• PAR-3, aPKC, PAR-6, CDC-42

posterior PARs

• PAR-2, PAR-1, LGL-1 

before fertilization, where are PARs

• Anterior PARs localized symmetrically around the cortex

• Posterior PARs in the cytoplasm

cortex

• thin, actin-rich network located directly underneath the plasma membrane

How does PAR function

• PAR-1 (kinase) recruits PAR-2 (ring protein) to the posterior and phosphorylates PAR-3 to kick it off posterior 

To put PAR-2 on membrane, need

tubulin

• sperm nucleus makes the MT, oocytes can organize MT a little

How is symetry broken?

• Anterior PARs are everywhere at the beginning, but sperm enters, and they start to attach to the MT

• Putting MT on the cortex displaces anterior PAR proteins on the membrane

• Posterior proteins bind immediately and expand  

  • Posterior proteins phosphorylate whatever is closest 

  • Posterior proteins also phosphorylate Air-1 (Aurora Kinase), which inhibits myosin

    • no contraction of actin in the posterior

• aPKC phosphorylates PAR-2 and LGL-1, removing them from the cortex 

• Active myosin shifts to the anterior 

  • Contraction of actin causes cortical flow of cytoplasm, which distributes things asymmetrically 

  • Myosin activity is what gives true asymmetry 

does actin contract in the posterior

no, because Air-1 is phosphorylated and inhibits myosin

P1 is ____ specified

autonomously, can laser AB and still get same fate

AB is ____ specified

inductively, if laser P1, don’t get pharynx in AB lineages

P granules

• key determinants partitioned into germline founder cells

• P granules are complexes of RNAs and proteins that function in specification of the germ cells 

• P-granules get pushed to the posterior in the one-cell zygote

PIE-1

• Key determinants are partitioned into germline founder cells 

• PIE-1 is a transcriptional repressor that keeps gene expression off in the germline founder cells early in embryonic development

• In the absence of PIE-1, germ cells adopt somatic fates

Autonomous development of P1

• Regulated by the partitioning of cell fate determinants 

• P-granules and Pie-1 in P1, P2, P3, P4 

• Pharyngeal determinant in P1, EMS, MSi

SKN-1

Pharyngeal determinant 

SKN-1 = Pharyngeal determinant 

• Posteriorly pushed

• Transcriptional factor

• High levels in P1 and it’s daughters and low levels in AB in its daughters-

SKN-1 Mutants

• EMS progeny not properly specified 

• Don't get pharynx, muscle, and intestine in mutants 

  • Skn-1 is essential for pharynx 

• Skn1 is also in P2, but if Pie-1 is there, then it is inhibited, so it’s only needed in EMS