2. Graded Expression: A gene being turned on in a high to low gradient.
Neocortical Arealization: The process of dividing the neocortex into
functional units.
Transcription Factors: Class of proteins that regulate the turning on
and off of specific genes.
Basic topics
3. Neocortex
• Mammalian
• Positions of areas are
conserved between
species
• This occurs during
formation of neocortex
(corticogenesis).
Yellow = auditory area
Red = motor area
Green = somatosensory area
Violet = visual area
4. adapted from Sahara, et al. (2007) Neuron 56:252.
high posterior-medial (P-M) to
low anterior-lateral (A-L)
Transcription Factors
Turn genes on and off
Work with other proteins to regulate transcription
Regulates arealization
5. Lhx9, from the Protein Data Bank.
Lhx2
406 amino acids, 44 kDa
A Lim homeodomain protein.
Involved in development of eye, olfactory cortex, spinal cord,
hippocampus, and neocortex.
6. From: The Atlas of Mouse Development
Mice are born 19 days
after conception.
7-day old (P7) mouse
Constitutive knock-
out Lhx2 mice die
at E15.5.
Specific areas
aren’t discernable
until 7 days after
birth.
Lhx2
7. V1 V2
Transcription factors Lhx2,
Sp8, Pax6, Coup-TFI and
Emx2 are expressed in
gradients.
These gradients determine
daughter cell fate.
Daughter cells migrate from
ventricular zone (VZ).
Cells migrate into the cortical
plate (CP) until they reach the
marginal zone (MZ).
Neocortical development
14. • When Lhx2 is not expressed in
the neocortex:
• The neocortex fails to grow
to the correct size.
• The piriform cortex grows
to compensate for the loss
of neocortex.
Lhx2 specifies regional fate in Emx1 lineage of telencephalic
progenitors generating cerebral cortex Nat Neurosci. 2009
Nov; 12(11): 1381–1389. Published online 2009 Oct 11.
Lhx2 Mutants
16. The Gal4 protein is a
transcription factor that
activates transcription of
yeast reporter genes.
Gal4 contains two
domains, each with
independent function.
By fusing a bait protein
with the Gal4 BD and
interacting it with a Prey-
Gal4 AD….
we can identify protein-
protein interactions.
Yeast Two-Hybrid
17. • pGBKT7 selects for SD/-Trp
• pGADT7 selects for SD/-Leu
• Contains both plasmids
selects for SD/-2(-Trp,-Leu)
Selects for protein-protein interactions
SD/-His
SD/-Ade
SD/A
SD/X (blue and white screening
Plasmids Used
18. Lane 1: pGBKT7-Lhx2 and pGADT7-Lbdv1
Lane 2: pGADT7-Lbdv1
Lane 3: pGADT7-T and pGBKT7-53.
Lane 4: Ladder
• Lane 1 shows pGBKT7-Lhx2 @ 64 kDa
• Lane 3 shows pGBKT7-53 @ 51 kDa
• Confirms Lhx2 produced as full length protein.
tRNA Percent used
in Mouse
Percent used
in Yeast
TTA (L) 6.5% 26.4%
TTG (L) 13.3% 27.1%
CTT (L) 13.2% 12.2%
CTC (L) 20.3% 5.4%
CTA (L) 8.0% 13.4%
CTG (L) 40.0% 10.4%
Confirmation of Lhx2 Production
19. y = 0.2391e0.2619x
R² = 0.9794
y = 0.089e0.3404x
R² = 0.9665
0.0625
0.125
0.25
0.5
1
2
0 1 2 3 4 5 6 7 8
Log2(OD600nm)
Hours
Control Lhx2
• Doubling time of control (pGBKT7-53) = 2.64 hours
• Doubling time of pGBKT7-Lhx2 = 2.32 hours
Lhx2 is not toxic to yeast cells
20. • Lhx2 and CLIM2 are known to
interact.
• Yeast Two-Hybrid with:
• GADT7-Lhx2
• GBKT7-CLIM2
• Confirms that Lhx2 is folded
properly.
Lhx2 Folds Properly
21. Prey Library yeast Bait (Lhx2) yeast
-Leucine -Tryptophan
-2 plates (-leu and –trp)
-4+A+X plates (-leu, –trp,
-ade, -his, aureobasidin
A, and X-gal)
Yeast Two-Hybrid Screen
22. • Streaked yeast onto SD/-leu plates.
• Extracted library plasmids from yeast.
• Transformed plasmids into E. coli.
• Isolated plasmids from E. coli:
A. Sequencing and BLAST searches were used to identify the proteins.
B. Library plasmids were cotransformed with pGBKT7-Lhx2 back into yeast to
confirm the interactions.
Preparing Screened Plasmids For Sequencing
24. Deadbox Ddx50
• Asp-Glu-Ala-Asp (DEAD)
• RNA helicases
• 38 human deadbox proteins
• Alters secondary structures of RNAs.
• Regulates embryogenesis, spermatogenesis,
and cellular growth and division.
Lhx2 Expression at E14.5
Visel A, (2004) Nucleic Acids Res. 1;32:D552-6
Ddx50 Expression at E14.5
Diez-Roux, G., et al. (2011) PLoS Biol. 9(1).
25. • Enhanced RNAi 3’ mRNA Exonuclease.
• Exonucleases are enzymes that cleave nucleotides one at a time
from the 3’ end of a polynucleotide chain by hydrolyzing
phosphodiester bonds.
• May degrade mRNA to repress translation.
Eri3
26. Lane 9-11: pGADT7-Ddx50
Lane 12-14: pGBKT7-Ddx50
Lane 15: Ladder
Lane 3-5: pGADT7-Eri3
Lane 6-8: pGBKT7-Eri3
Lane 15: Ladder
Eri3: .5 kB
Ddx50 3 KB
pGBKT7: 7.3 KB
pGADT7: 8.0 KB
• Lane 4 shows Eri3 and pGADT7
• Lane 7 shows Eri3 and pGBKT7
• Lane 10, 11 shows Ddx50 and pGADT7
• Lane 12-14 shows Ddx50 and pGBKT7
- 8 - 8
- 3 - 3
- .5 - .5
Cloning Full Length Open Reading Frames for Eri3 and Ddx50
31. LIM Domain Homeodomain
LIM Domain
No Homeodomain
No LIM Domain
Homeodomain
0
Amino
Acid
180
Amino
Acid
255
Amino
Acid
406
Amino
Acid
Eri3-Lhx2 No Lim domain
32. LIM Domain Homeodomain
LIM Domain
No Homeodomain
No LIM Domain
Homeodomain
0
Amino
Acid
180
Amino
Acid
255
Amino
Acid
406
Amino
Acid
Eri3-Lhx2 No Homeodomain
33. LIM Domain Homeodomain
LIM Domain
No Homeodomain
No LIM Domain
Homeodomain
0
Amino
Acid
180
Amino
Acid
255
Amino
Acid
406
Amino
Acid
This suggests that the LIM
domain of Lhx2 is
sufficient and necessary
for binding to Eri3.
Eri3-Lhx2 Lim domain
35. Transcription:
• Binding of Ddx50 or Eri3 to Lhx2 could
modulate which genes Lhx2 turns on and off.
Possible transcriptional functions for the Lhx2-
Ddx50 and Lhx2-Eri3 interactions
36. Possible Posttranscriptional
Lhx2-Ddx50 Function
• Lhx2 could recruit RNA to Ddx50:
• Remove proteins from RNA
• Allows the mRNA to be translated
• Allows the mRNA to be degraded
• Allow for proper folding of mRNA to
allow transcription
Lhx2 binds mRNA
Lhx2 recruits Ddx50 to mRNA
Ddx50 unfolds mRNA
37. Possible posttranscriptional
Lhx2-Eri3 function
Post transcription:
• Lhx2 could inhibit the Eri3 for
degrading mRNA
• Lhx2 could recruit RNA to Eri3:
• Degradation of mRNA
Lhx2 interacts with mRNA
Lhx2 recruits Eri3
Eri3 degrades mRNA
38. Potential Pathway
• Lhx2 may interact with both Eri3 and
Ddx50.
• Lhx2 may recruit mRNA.
• Ddx50 unfolds mRNA and allows Eri3 to
degrade the protein.
• The mRNA that are destroyed could be
transcription factors that repress
formation of the visual area.
• Determining the expression pattern of
Eri3 will help to refine these models.
Lhx2 interacts with folded mRNA Ddx50 and Eri3 get recruited by
Lhx2
Ddx50 unfolds mRNA
Eri3 is able to degrade the mRNA now that the mRNA is
unfolded
39. • Need to clone the full length Ddx50, to
confirm that it interacts with Lhx2.
• Compare expression of Ddx50 and Eri3 to
Lhx2 in neocortex using in situ hybridization.
• Yeast 3 hybrid
• RNA experiments:
• Determine whether Lhx2 interacts with
mRNA.
• Functional assay will detect whether Lhx2
affects the Eri3-dependent degradation of
mRNA.
Future Research
40. • Dr. Kroll
• Thesis Committee
• Dr. Carin Thomas
• Dr. Holly Pinkart
• Dr. Kroll’s research group
• Family And Friends
• CWU
Acknowledgements
41. • Montagnini, A.; Treves, A. The evolution of mammalian cortex, from lamination to arealization. Brain Res Bull
2003, 60 (4), 387-393.
• O'Leary, D. D.; Chou, S. J.; Sahara, S. Area patterning of the mammalian cortex. Neuron 2007, 252-269.
• Tétreault, N.; Champagne, M. P.; Bernier, G. The LIM homeobox transcription factor Lhx2 is required to specify
the retina field and synergistically cooperates with Pax6 for Six6 trans-activation. Dev Biol 2009, 541-550.
• Matchmaker® Gold Yeast Two-Hybrid System User Manual; ClonTech Laboratories, Inc: Mountain View, CA.
• ERI1 Exoribonuclease Family Member 3.
http://www.genecards.org/cgibin/carddisp.pl?gene=ERI3&search=e268f81180ef17c582e4d36fc56ae1db
(accessed Dec 12, 2014).
• Dubnau, J.; Struhl, G. RNA recognition and translational regulation by a homeodomain protein. Nature 1996, 379
(1), 694-699.
• Takashi, K.; Masaya, O.; Takahiko, U.; Shoko, K.; Kohji, O.; Toshihiko, E.; Fumihiro, F.; Yasufumi, M. Dbp9p, a
Member of the DEAD Box Protein Family, Exhibits DNA Helicase Activity. The Journal of Biological Chemistry
2004, 279 (20), 20692-20698.
• B, S.; Hari, P.; Huilgol, D.; Tole, S. Dual role for LIM-homeodomain gene Lhx2 in the formation of the lateral
olfactory tract. The Journal of Neuroscience 2007, 27 (9), 2290-2297.
• Susa, T.; Sato, T.; Ono, T.; Kato, T.; Kato, Y. Cofactor CLIM2 promotes the repressive action of LIM
homeodomain transcription factor Lhx2 in the expression of porcine pituitary glycoprotein hormone alpha subunit
gene. Biochimica et Biophysica Acta 2006, 1759 (8), 403-409.
Citations
