Cellautonomous mechanisms of fate determination

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Lecture 13

• Cell-autonomous mechanisms of fate determination
• Ascidians and nematodes

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Regulative vs. Mosaic development

• Cells made different by interaction with
  environment (other cells)
• Leads to regulative development

• Cells made different by inheritance of
  determinants that are segregated at a cell
  division
• Leads to mosaic development

Old view invertebrates mosaic, vertebrates
regulative Modern view shades of gray
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Ascidians (tunicates, sea squirts)

• Small eggs, transparent
• 24 h from egg to tadpole (2000 cells)
• position and fate of cells is the same from
  animal to animal--invariant

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Ascidian development Boltenia

• Movies of Ascidians from George von Dassow,
  Center for Cellular Dynamics, Friday Harbor, WA

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Ascidian development Corella

• Movies of Ascidians from George von Dassow,
  Center for Cellular Dynamics, Friday Harbor, WA

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reproducibility of development allows
construction of cell lineages

• describes ancestry of each cell
• made by direct observation or by cell marking

Fig 6.22
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Invariance of development does not tell us how
fates are determined

• Ascidian development is invariant
• so both the position and ancestry of any cell are
  invariant--which determines fate?
• do cells always inherit the same determinants?
  (cell autonomous)
• or do they always encounter the same
  environmental signals (nonautonomous)

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Evidence that cell fates might be determined
intrinsically

• Edwin Conklin (1905)
• 3-4 distinct regions of cytoplasm
• yellow cytoplasm--muscle
• light gray--notochord
• clear area--ectoderm

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Is muscle fate intrinsic?

• Chabry (1887)
• kill blastomeres, no regulation (but dead cells
  still present)
• Reverberi Minganti (1946)
• dissociate 8-cell stage into 4 symmetric pairs
• culture in permissive medium

only B4.1 cells can make muscle when isolated
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Other fates require interactions

• notochord
• normally formed by A4.1 AND B4.1
• isolated B4.1 still makes notochord
• isolated A4.1 does not.
• also neural tissue not made in isolation

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Evidence that myoplasm contains a determinant

• msotly correlates with muscle fate
• but some B4.1 cells do not become muscle
• and some muscle comes from non-B4.1 cells
• cytoplasmic transfers can sometimes convert
  other precursors to make muscle
• inject myoplasm into B4.2 blastomere switches it
  from ectoderm to making some muscle
• control transfer nuclei, no effect

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How to find the determinant?

• RNA or protein? must be localized by attachment
  to cytoskeleton--does not diffuse freely.
• Biochemical approach
• purify myoplasm, fractionate an activity
• Genetic approach
• look for mutants that lack muscle, clone ge
• Molecular approach
• check out homologs of genes involved in muscle
  differentiation in other animals

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How a determinant was found

• Nishida Sawada (2001)
• molecular screen for mRNAs differentially
  expressed between animal and vegetal blastomeres
  at 8-cell stage
• dissect 400 8-cell stage embryos
• extract cytoskeleton-associated mRNAs from animal
  and vegetal halves
• hybridize to remove messages present in both
• analyze remaining single stranded RNAs

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macho-1

• a transcription factor
• made maternally
• localized to myoplasm
• RNA interference depletion blocks muscle fates
• injection of macho-1 mRNA into A4.2 produces
  ectopic muscle

in situ hybridization to detect macho-1 mRNA in
embryos
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How is macho-1 mRNA localized?

• initially uniform in vegetal cortex of oocyte
• fertilization triggers cytoplasmic rearrangement
  (segregation)
• male pronucleus transiently moves to posterior
• mRNA must be attached to cytoskeleton
  (microtubules?)

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Two components

• Determination by cell-autonomous mechanisms
  requires two separate yet equally important
  components
• a determinant (e.g. macho-1)--the red boxes
• a cytokinesis that segregates the determinant to
  only one daughter

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Asymmetric cell division

• if two daughter cells differ in size, mother cell
  must have divided asymmetrically
• mother cell must have been polarized
• examples from single-celled organisms

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Bacillus subtilis
unstarved
starved
symmetric division
spore inside mother cell
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How is the plane of cytokinesis determined?
unstarved
starved
random?
localization of FtsZ protein (bacterial
equivalent of tubulin also found in chloroplasts)
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How are mother and spore made different?
--transcription factors (sigma factors) become
differentially activated in mother and
spore --exactly how remains unclear
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Asymmetry in budding yeast (Saccharomyces
cerevisiae)

• bakers yeast, brewers yeast (lager yeast,
  S. carlsbergensis, is a variety)

mother
daughter
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Mating type switching
a

• Two mating types (sexes) a and a
• Mother cells can switch, daughter cells cannot
• switching does not depend on cell size
• only mothers express endonuclease HO

a
a ? a
a ? a
a
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Why is HO only expressed in mother cells?

• Ash1, a repressor of HO, is segregated to
  daughter cell
• Ash1 mRNA transported along actin cables (myosin
  motor)
• block Ash1 function--both cells switch
• block Ash1 segregation--mother does not switch

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What determines where the bud forms?

• Often adjacent to previous bud site (old moms
  have lots of bud site scars)
• but can also be random
• cellular machinery can initiate a bud anywhere.

actin in red, bud scars in green
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Conclusions from bacteria, yeast

• useful to study polarity in single cells
• asymmetry of cytokinesis
• segregation of determinants (e.g. Ash1)
• Bacillus switching between symmetric and
  asymmetric division
• yeast always polarized due to inherent
  asymmetry of budding
• polarity generated and maintained autonomously

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Caenorhabditis elegans
nervous system
pharynx (feeding)
dorsal
anterior
posterior
ventral
gut
gonad with eggs
--ubiquitous soil nematode --invariant
development --transparent, 1 mm long
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Questions

• how are body axes set up?
• AP, DV and left-right
• how are tissues specified?
• the pharynx (feeding organ, mesendoderm)

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C. elegans development
timelapse microscopy
John Sulstons drawings of nuclear positions (6
May 1980)
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Cell lineage
959 somatic cells (adult hermaphrodite)
(John Sulston, Bob Horvitz, Judith Kimble
1977-1983)
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Early development

• oocytes appear spherically symmetrical
• fertilization
• activation
• complete meiosis II
• sperm entry side becomes posterior
• sperm contributes haploid nucleus centrosome

oocyte
sperm
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the first division

• pronuclei fuse
• centrosomes duplicate and organize MTs
• zygotic nucleus and one aster shift posteriorly
• first cleavage is unequal
• AB cell big--mostly ectoderm
• P1 cell small--mesoderm, endoderm (roughly)

AB cell (anterior)
P1 cell (posterior)
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origin of germ layers in C. elegans

• germ line and gut arise clonally
• everything else mixed ancestry, e.g. pharynx
• Differences between AB and P1
• AB divides equally
• P1 divides unequally

Anterior Pharynx
Posterior Pharynx
segregation of P granules
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P granules and germline

• swept to posterior of zygote by cytoplasmic
  streaming
• only inherited by P1
• segregated in subsequent divisions to germline
  (P4)
• Similar ribonucleoprotein granules found in
  germline precursors (primordial germ cells, PGCs)
  of many animals

Fig 6.3
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The DV axis

• P1 spindle parallel to first division
• AB spindle starts at 90 to previous division
• steric constraints mean that one daughter is
  forced posterior (ABp) and one anterior (ABa)
• ABp defines dorsal, EMS defines ventral

AB spindle pushed obliquely
Fig 6.4
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Reversal of DV axis

• Are AB daughters different prior to skewing of
  spindle?
• Jim Priess wait until spindle starts to skew,
  then push it back
• result worm is normal
• conclusion ABa and ABp are initially equivalent
  
• DV axis is labile, determined by relative
  positions of cells at 4-cell stage

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left-right asymmetry

• first visible at 6-cell stage
• ABa/p divisions skewed so that left daughters
  more anterior
• in adult gonad, nervous system have left-right
  differences

Fig 6.5. Views from ventral side
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reversal of L/R asymmetry

• Bill Wood skew spindles of ABa/p so that left
  daughters are posterior
• result healthy, fertile mirror image worms

conclusion left-right differences made by
cell-cell interactions at the 6-cell stage or
later.
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specifying an organ the pharynx
bacteria in this end
ground-up bacteria out this end
peristalsis, filtering, grinding

• combination mouth/esophagus
• about 90 cells muscles, neurons, glands..
• anterior part from AB, posterior from EMS (P1)

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how are pharyngeal precursors specified?
X
X
X

• kill AB or EMS precursors at 28-cell stage, no
  regulation
• therefore cell fates are specified by then

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blastomere isolation/recombination
isolated P1
isolated AB
no pharynx
some pharynx
P2
EMS
no pharynx
pharynx
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Conclusions and questions

• Ability of P1 (EMS) to make pharynx is cell
  autonomous
• Ability of AB to make pharynx is non-autonomous,
  requires a signal from EMS
• What are the determinants, signals?
• How do they become localized?
• genetic approach look for mutants lacking
  pharynx or part of it.

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skn-1 is required for pharynx formation

• skn-1 (skinhead)
• maternal-effect lethal mutations
• mutants have no pharynx excess skin
• EMS is transformed into sister P2 (makes
  epidermis but not pharynx)

ABp
ABp
P2
P2
ABa
ABa
P2
EMS
embryo from skn-1 mom
wild type
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SKN-1 localization

• SKN-1 mRNA in all cells (maternally deposited)
• SKN-1 protein only in EMS, P2
• functional only in EMS (inhibited in P2 by
  another protein, PIE-1)
• nuclear localized, transcription factor
• why is SKN-1 mRNA only translated in
  EMS/P2?--other factors

EMS
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MEX-1 localizes SKN-1

• In mex-1 mutants, SKN-1 translated everywhere
• Isolate AB from a mex-1 mutant, it can make
  pharynx
• ectopic SKN-1 sufficient to direct pharyngeal
  fates?

WT
mex-1
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glp-1 is required for induction of anterior
pharynx
EMS

• GLP-1 protein is a receptor on surfaces of ABa,
  ABp
• receptor for (unknown) inductive signal from EMS?

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why are AB daughters different?

• ABa and ABp both contact EMS, so why dont both
  get induced?
• P2 expresses APX-1, another GLP-1 ligand, that
  inhibits ABp from responding
• regulation of competence

ABp
P2
ABa
EMS
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how do proteins like GLP-1 and SKN-1 get
localized?

• mRNAs ubiquitous regulation of translation by
  proteins already differentially localized in
  zygote
• requires 3 UTR sequences of glp-1 mRNA
• Ken Kemphues par (partitioning) mutants
• maternal-effect lethals
• gt6 genes
• mutants lack polarity at 1-cell stage
• defects in localization of P granules, SKN-1, etc

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The par genes
AB
P1

• wild type
• par-2 mutant P1 develops like AB
• par-3 mutant AB develops like P1

AB
AB
P1
P1
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Polarity of the zygote
AB
P1
P1
P1

• First cleavage in wild type versus par-6

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PAR proteins are localized

• define distinct domains in cell cortex
• PAR-3/6 anterior cortex, PAR-1/2 posterior cortex
• whos on first?

PAR-2GFP
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PAR-2 and PAR-3 exclude each other from cortical
domains
PAR-3 protein PAR-2 protein
par-2 mutant
par-3 mutant
wild type
what sets this up?
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model--the early worm
PAR-3 PAR-2
sperm centrosome/MTs polarizes cortical cytoskelet
on (actin?) (intensively studied, we have
glossed over the details)
Zygote becomes polarized --cytoplasmic
streaming --posterior movement of nucleus,
centrosomes --localized translation of GLP-1
(anterior) SKN-1 (posterior)
PAR domains set up in cortex, exclude one another