The Role of miR7 in the Zebrafish Pituitary

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The Role of miR-7 in the Zebrafish Pituitary

• Ima Paydar
• Honors Thesis
• April 2009

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Small RNAs and the Pituitary
Shariat N., Ryther R.C., Phillips J.A. 3rd,
Robinson I.C., Patton J.G. 2008. Rescue of
pituitary function in a mouse model of isolated
growth hormone deficiency type II by RNA
interference. Endocrinology. 149 580-6.
Thatcher E. J., Paydar I., Anderson K. K., Patton
J. G. 2008. Regulation of zebrafish fin
regeneration by microRNAs.PNAS. 10518384-9.
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miRNA Biogenesis
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The Zebrafish Pituitary
Pogoda Hammerschmidt, 2007
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miR-7
Wienholds et al. 2005.

• Forebrain, hypothalamus, pancreatic islets
  (Weinholds et al., 2005)
• Vasotocinergic neurons (Tessmar- Raible et al.,
  2007)
• Mammalian pituitary (Landgraf et al., 2007)
• Decreased expression in pituitary adenomas
  (Bottoni et al., 2007)
• Zebrafish pituitary?

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miR-7 Expression in Pituitary
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miR-7 Expression in Pituitary
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miR-7 cross-sections
10x 3dpf
10x 2dpf
40x 3dpf
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miR-7 Expression2dpf whole mount dbl in
situmiR-7 blue, pit1 red
63x
20x
10x
10x
10x
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miR-7 Expression2dpf whole mount dbl in
situmiR-7 blue, pit1 red
20x
40x
63x
20x
63x
40x
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miR-7 knockdownLNA in situ
miR-7MO
NIC
miR-7MO
NIC
3 dpf
2 dpf
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miR-7 Knockdown
miR-7MO
WT
WT
miR-7MO
gh
gh
prl
prl
miR-7MO
WT
WT
miR-7MO
pomc
pomc
pit1
pit1
miR-7MO
WT
tsh
tsh
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Pituitary Gene Expression
WT
miR-7MO
miR-7
pit1
WT
miR-7MO
pit1
WT
miR-7MO
gh
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Predicted Target of miR-7
mdh1b
AAAAA
miR-7
3 UTR
mdh1b
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MDH1bMalate Dehydrogenase
Citric Acid Cycle
Malate-Aspartate Shuttle
-MDH1 remains in the cytosol. It transports NADH
across the mitochondrial membrane and controls
the TCA cycle pool size. -MDH1 functions in
aerobic energy production, muscle contraction,
neuronal signal transmission, etc. -Highest
expression in heart, skeletal muscle, and brain.
http//www.javeriana.edu.co/Facultades/Ciencias/ n
eurobioquimica/libros/neurobioquimica/desarlanzade
ras_ archivos/malate-aspartate-shuttle.gif
http//www.femnutrition.com/images/ productphotos/
tca_cycle.jpg
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Does miR-7 target mdh1b?

• Make GFP reporter construct with mdh1b 3 UTR.

miR-7 3 UTR
5 3
3 5
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Does miR-7 Target mdh1b? (cont.)

• 2. Co-inject miR-7 and construct into zebrafish
  embryo at 1 cell stage.
• 3. Check GFP protein level with fluorescence and
  western blots at 1dpf.

miR-7
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Conclusions Future Directions

• miR-7 is expressed in the pituitary, both AH and
  NH
• miR-7 may regulate pit1 expression
• miR-7 may target mdh1b
• mdh1b is ubiquitously expressed
• In situ hybridization with NH markers
• Quantify pit1 RNA levels
• In vitro transcribe and inject GFPmdh1b 3 UTR
• Other miR-7 targets?

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Acknowledgements

• Advisor Dr. James G. Patton
• Research Mentor Elizabeth Thatcher