Sea Urchin Embryos and Larvae as Biosensors

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Sea Urchin Embryos and Larvae as Biosensors for
Teratogenesis by Pre-nervous Neurotransmitter
and Retinoic Acid Receptor Ligands Gennady A.
Buznikov Lyuda G. Nikitina Robert E.
Peterson Jean M. Lauder University of North
Carolina School of Medicine Chapel Hill, NC
27599-7090
The perfect DNT screen?
Supported by CAAT grant 2005-03 to JML
unclau_at_med.unc.edu
Lytechinus variegatus year round residents of
the Lauder Lab
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Neurotransmitters act as Developmental Signals
across Species

• Serotonin
• Acetylcholine
• Dopamine
• Norepinephrine
• Opiates
• Excitatory Amino Acids
• GABA
• Peptides

Sea urchins
 In the early 1960s, the Russian developmental
biologist Gennady Buznikov began the field of
Neurotransmitters as developmental signals by
providing the first evidence that substances like
serotonin (5-HT), catecholamines and
acetylcholine regulate early phases of sea urchin
development (cleavage and gastrulation)
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Table 1. Perturb and rescue by retinoic acid
(RA) and neurotransmitter ligands
High Through-put Screen
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Teratogenesis by retinoic acid and rescue by
pan-RAR antagonist, BMS
50 mm
Control (ASWvehicle)
RA 5 mM BMS 0.01 mM
BMS 0.1 mM
All substances were given at the 2-cell stage (1
h 50 min after fertilization) and embryos imaged
at the early gastrula stage. RA blocks
gastrulation and a low concentration of BMS (0.01
mM ), but not higher concentration (0.1 mM)
rescues, even when added at later stages (mid- or
late blastula).
Lytechinus variegatus
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Teratogenesis by retinoic acid and rescue by
5-HTR ligands or forskolin
Lytechinus variegatus
RA 6 mM AA-5-HT BW723C86
forskolin Control
40 mM 12
mM 20 mM
All substances were given at the 2-cell stage (1h
50min after fertilization) and embryos imaged at
the early gastrula stage. RA blocks
gastrulation AA-5-HT and forskolin rescue.
5-HT2B R agonist, BW723C86 only partially rescues.
Mechanism Opposing regulation of
morphogenesis by 5-HTR and RA mediated by
cAMP signal transduction
pathways
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Reserpine teratogenesis and rescue by 5-HTR
ligands
RE 5-HT
AA-5-HT
Control
40 mM 40 mM
All substances were given at early blastula stage
(5 hours post fertilization), and embryos imaged
at early gastrula. VMAT inhibitor, RE blocks
blastula-gastrula transition AA-5-HT, but not
5-HT, rescues almost completely. No rescue by
hydrophilic 5-HTQ.
Mechanism Intracellular VMAT sites regulate
morphogenesis
Lytechinus variegatus
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Teratogenesis by pan-5-HT2 R antagonist,
ritanserin and rescue by lipophilic AA-5-HT
50 mm
RIT
AA-5-HT 5-HTQ
Control 12 mM
40 mM
40 mM All substances were given at mid-blastula
2 stage, when there was maximal sensitivity to
ritanserin, and embryos imaged at early
gastrula. Ritanserin blocks gastrulation
AA-5-HT, but not 5-HTQ, rescues almost
completely.
Mechanism Intracellular 5-HT2R regulate
morphogenesis
Strongylocentrotus droebachiensis
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Effects of pan-5-HT2 receptor antagonist,
ritanserin, on patterning of primary mesenchyme
cells (PMC) during blastula stage
PMC progenitors of the larval skeleton
Ritanserin decreases the number of PMC, and
disturbs patterning
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Conclusions

• A pre-nervous neurotransmitter system,
• acting together with other growth-regulatory
  signals,
• regulates early development of sea urchin
  embryos.
• and probably early vertebrate
  embryos as well.
• RA, mediated by RAR acts together with
  intracellular 5-HT2 receptors to exert opposing
  influences on development of the
• larval skeleton
• An intracellular vesicular monoamine transporter
  (VMAT) regulates blastula-gastrula transition.
• Developmental dynamics of pre-nervous
  regulation changes immediately before
  gastrulation, at the mid-blastula transition.
• The sea urchin model can provide a DNT screen
  for prenatal exposures,
• and may provide a window into the evolutionary
  history of the vertebrate nervous system.