Download Lab meeting 1.30.2015

Lab meeting 1/30/2015
Zebrafish have (inner) ears
http://www.bio.tamu.edu/USERS/briley/Research.html
Hammond, van Eeden, and Whitfield 2010, Development
Zebrafish ear development
• Otic placode  otic vesicle  inner ear
structures (semicircular canals, sensory patches,
and statoacoustic ganglion)
• The 5 sensory patches detect sound waves in
addition to vestibular functions.
• Placode appears at 14-16 hpf; vesicle at 18 hpf.
• Neuroblasts delaminate between 22 and 42 hpf.
• It is accepted that FGFR signaling is involved in
induction of otic placode
https://zfin.org/zf_info/anatomy/dict/ear/ear.html
FGFR signaling pathways
MAPK/ERK
AKT
PLCγ
Cell survival
Cell motility
(MEK)
(ERK)
Cell proliferation
Goetz and Mohammadi 2013
FGF2 promotes Pax2 via ERK pathway
• HH stage 8 embryos cultured 18 h with FGF pathway inhibitors
• FGFR inhibitor and MEK inhibitor decreased or abolished Pax2 expression
Yang et al. 2013
Atoh1 and Sox9 as candidate neurogenic
factors downstream of FGFs
• Expression of Sox9 and Atoh1 is dependent upon FGFR
signaling
• Sox9 and Atoh1 are implicated in otic neurogenesis
• Both are expressed in otic placode and vesicle of zebrafish
Millimaki et al. 2007, Development
Figure 1: FGFR and AKT signaling are
necessary for SAG development
D
P
A
V
HuC = neuron-specific RNA binding protein, used here as antigen to immunostain SAG
Heat-shock-inducible transgenic lines receive 40 minute exposure to 38.5°C
Heat shock applied at 12 hpf
hs:dn-fgfr1 inhibits function of WT FGFR1
hs:fgf8a drives expression of fgf8a
Figure 1: FGFR and AKT signaling are
necessary for SAG development
D
P
A
V
FGFRs
PI3K/AKT
ERK
PLC
Drug treatments applied at 12 hpf
SU5402
FGFRs
Wortmannin
PI3K/AKT
Figure 2: Sox9a and Atoh1a are independent
downstream targets of FGFR/AKT signaling
Heat shock applied at 10 hpf
Drug treatments applied during 10-12 hpf
Figure 2: Sox9a and Atoh1a are independent
downstream targets of FGFR/AKT signaling
Heat shock applied at 10 hpf
Drug treatments applied during 10-12 hpf
Figure 2: Sox9a and Atoh1a are independent
downstream targets of FGFR/AKT signaling
Morpholino knockdown of Atoh1a or Sox9a expression
Embryos injected at 1-2- cell stage with translation-blocking MOs
Expression of the other target assayed by qPCR
Figure 3: Sox9a and Atoh1a involved in otic
neurogenesis
Drug treatments applied during 10-14 hpf
Figure 3: Sox9a and Atoh1a involved in otic
neurogenesis
Drug treatments applied during 10-14 hpf
mRNA injections at 1-cell stage
ChIP assay
Cross-link protein to DNA
Add bead-attached antibodies
to IP the target protein
Shear DNA strands by sonicating
Unlink protein; purify DNA
Figure 4: Direct targets of Sox9a and
Atoh1a
Structural proteins
Transcription factors
Notch-related
Implicated in otic development
Inhibitor of FGF signaling
Neural development factor
Figure 4: Direct targets of Sox9a and
Atoh1a
Figure 5: Tlx2 and Eya2 are involved in otic
neurogenesis downstream of Sox9a and Atoh1a
Figure 6: FGFR-ERK signaling is involved in
otic sensorigenesis
Otic
region
Drug treatments applied during 10-12 hpf
Figure 6: FGFR-ERK signaling is involved in
otic sensorigenesis
Utricle
Drug treatments applied during 10-14 hpf in G-K
Model of FGFR signaling cascades leading
to otic neurogenesis and sensorigenesis