Classification of Enteric Progenitors
in Zebrafish During Development
William Montagne
Eisen Lab
August 17, 2012
Enteric Nervous System (ENS)
Background
• ENS is the largest part of
the Peripheral Nervous
System
• Intrinsic innervation of
the intestinal tract
• Modulates gut function
• Functions autonomously
from Central nervous
system
• Composed of different
subtypes of neurons and
glial cells
zebrafish
Wallace et al. (2005)
ENS Cell Origin
• Entirely derived from one population of cells.
• Enteric progenitors migrate to the foregut.
• Continue to migrate and proliferate to
populate the entire digestive tract.
Enteric Progenitor Markers
• Progenitors express specific marker genes
(sox10, phox2b and ret)
• Key roles in migration, survival and
proliferation
• In mouse Ascl1 is present in enteric
progenitors but not analyzed in zebrafish
Aims
• Are there distinct subpopulations of enteric
progenitors?
• Is ascl1a an enteric progenitor marker in
zebrafish?
• Do the subpopulations change spatially and/or
temporally?
Methods
•
•
•
•
•
RNA probe synthesis
Tissue sectioning
Immunohistochemistry
Fluorescent in situ Hybridization
Fluorescent Microscopy
Tg (phox2b:GFP)
Aims
• Are there distinct subpopulations of enteric
progenitors?
• Is ascl1a an enteric progenitor marker in
zebrafish?
• Do subpopulations change spatially and/or
temporally?
1.
1. Section phox2b:GFP
2. Stain for marker/GFP
3. Focus on Progenitors
around the gut
2.
3.
phox2b Colocalization with GFP
phox2b Colocalization with GFP
100
phox2b
GFP
phox2b
GFP
90
80
Percent of Progenitors
70
60
50
40
30
20
10
0
phox2b+/GFP+
phox2b+/GFP-
phox2b-/GFP+
n=2
Distinct progenitor subpopulations
sox10 Colocalization with GFP
70
60
Percent of Progenitors
50
sox10 GFP
40
30
20
sox10 GFP
10
0
sox10+/GFP+
sox10+/GFP-
sox10-/GFP+
n=2
Distinct Progenitor Subpopulations
ret Colocalization with GFP
80
70
Percent of Progenitors
60
50
40
ret GFP
30
20
10
0
ret+/GFP+
ret+/GFP-
ret-/GFP+
n=2
ret GFP
Aims
• Are there distinct subpopulations of enteric
progenitors?
• Is ascl1a an enteric progenitor marker in
zebrafish?
• Do subpopulations change spatially and/or
temporally?
ascl1a Colocalization with GFP
ascl1a Colocalization with
GFP
70
60
ascl1a
GFP
Percent of Progenitors
50
ascl1a
40
30
20
10
0
ascl1a+/GFP+ ascl1a+/GFP- ascl1a-/GFP+
n=2
GFP
Colocalization Summary
100
90
80
Percent of Progenitors
70
60
50
40
30
20
10
n=2
0
phox2b+/GFP+
sox10+/GFP+
ret+/GFP+
ascl1a+/GFP+
Aims
• Is ascl1a an enteric progenitor marker in
zebrafish?
• Are there distinct subpopulations of enteric
progenitors?
• Do subpopulations change spatially and/or
temporally?
Spatial differences in marker gene
expression?
Mid-region
Rostral
Caudal
Spatial differences in marker gene
expression?
Colocalization with GFP
100
90
80
Percent of Progenitors
70
60
Rostral
50
Mid
Caudal
40
30
20
10
0
n=2
phox2b+/GFP+
ascl1a+/GFP+
sox10+/GFP+
ret+/GFP+
Summary
• ascl1a expressed in enteric progenitors in
zebrafish.
• Based on marker expression, distinct
subpopulations of ENS progenitors exist.
• Spatial differences for ascl1a+/GFP+ and
ret+/GFP+ subpopulations along the gut
Future Directions
• Double FISH with the different progenitor
markers
• FISH at different time points during ENS
development
• Lineage tracing to investigate functional
importance of subpopulations
ret+/phox2b+
Neurons
sox10+/phox2b+
Neurons
Thank You
Judith Eisen
Julia Ganz
Eisen Lab
SPUR Program
Peter O’Day
SPUR Interns
NIH
zebrafish
facility
Histology
Questions?