Download Identifying Wnt Target Genes Involved in Tracheal Patterning

IDENTIFYING WNT TARGET GENES
INVOLVED IN TRACHEAL PATTERNING
Lauren Leesman,
John Snowball, Manoj
Ambalavanan and Debora
Sinner
Honors Program
INTRODUCTION – TRACHEAL PATTERNING
Patterning of Trachea
 Patterned on the dorsal-ventral axis.
 C-shaped cartilaginous rings on ventral side, smooth muscle on the dorsal side
 Enables protection, stability, and flexibility necessary
gene expression - Tomato ShhCre mice
INTRODUCTION – TRACHEOMALACIA
Tracheomalacia
 Congenital disorder characterized by the underdevelopment of the
trachea
 Cartilaginous rings which are located on the ventral side are either
flaccid or absent
Those diagnosed with tracheomalacia can experience
decreased air flow and have trouble breathing
Can lead to trachea collapsing
Other Abnormalities in patterning
 Tracheal sleeve
 Cartilaginous rings extending around the entire trachea
INTRODUCTION – GOAL
We know a fair amount about the patterning of the trachea
Little is known about how the patterning is established
Previous studies show significance of the Wntless gene (Wls)
 its associated pathways help in understanding the embryonic development of tracheal patterning.
Aim to identify the genes that are expressed in the developing trachea and mediate
tracheal patterning.
HOW ARE WNT LIGANDS PRODUCED AND HOW DO THEY REACH THEIR
TARGET CELLS?
HSPG
Lipoprotein particles
Wnt
Wnt
Wls
Wnt
Porcnp
Canonical
Pathway:
SCD
Wnt producing cell
Cell fate
determination
NonCanonical
Pathway:
Tissue
polarity, cell
movement
Wnt
responsive/receiving
cell
PREVIOUS STUDIES
Deleting Wls from the tracheal epithelium
10
8
Gene expression in E13.5 tracheal
tissue
6
4
2
0
-2
-4
-6
-8
-10
FC ([M] vs…
SOME GENES OF INTEREST SHOW EXPRESSION IN
. THE RESPIRATORY TRACT AS WELL AS NEURAL
Lgi3
Sost
Cer1
Lrrc7
WLS TARGET GENES ARE EXPRESSED IN DISCRETE
COMPARTMENTS OF THE TRACHEA.
Whole mound showed respiratory tract expression- hard to tell dorsal/ventral
patterning
RNA Scope allows to see more detailed expression pattern
Studying the expression of Notum in tracheal tissue in the frog
Notum
Wnt 5a
Wls
Lgi3 – dorsal expression
GENES MODULATED BY EPITHELIAL WLS
SIGNALING ARE POTENTIAL MODULATORS OF
WNT/ B-CATENIN SIGNALING
Top Flash Promoter in Luciferase Assays determined if target genes were intracellular
modulators of Wnt/B-catenin within the canonical Wnt signaling pathway.
Target Genes Top Flash
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
No stimuli
B-catenin
B-cat Fop
flash
Lgi3
Lrrc7
Fgf14
Sost
Also looking at extracellular by using the addition of Wnt3a.
WNT TARGET GENES ARE POTENTIAL MODULATORS
OF SOX9
Use of Sox9 Luciferase construct showed if genes were modulators of Sox9
Sox 9 Luciferase Assay 0.2 kb
Sox 9 Luciferase Assay 2.5 kb
50
8
7
Luminescence in relative units
(Luciferase/Renilla activity)
Luminescence in relative units
(Luciferase/Renilla activity)
45
40
35
30
25
20
15
10
5
6
5
4
3
2
1
0
No
stimuli
Lgi3
Lrrc7
Fgf14
Sost
empty
0
No
stimuli
Lgi3
Lrrc7
Fgf14
Sost
empty
SOX9OX
How cartilage is effected in over expression of Sox9
Overexpressed in epithelium using Sox9ox/ShhCre
Overexpressed in mesenchyme using Sox9ox/Dermo1Cre
Control
Length (mm)
Crown-Rump
Measurements
12
10
8
6
4
2
0
Control (Sox9OX)
Mutant
(Sox9OX/ShhCre)
Sox9ox/ShhCre
Sox9ox/ShhCre
Sox9ox/Dermo1Cre
CONCLUSIONS
Identified candidate genes never described before in respiratory tract that may play
roles in tracheal patterning
Looking at target genes expression with RNA Scope
How target genes contribute to muscle and cartilage development in developing
trachea
PERSONAL REFLECTION
What I have learned




New techniques
Patience
Time management
How to interpret challenging literature