Zebrafish have (inner) ears Hammond, van Eeden, and Whitfield
2010,
Developmenthttp://www.bio.tamu.edu/USERS/briley/Research.html
Slide 3
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
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
Slide 6
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
Slide 7
Slide 8
Figure 1: FGFR and AKT signaling are necessary for SAG
development HuC = neuron-specific RNA binding protein, used here as
antigen to immunostain SAG Heat-shock-inducible transgenic lines
receive 40 minute exposure to 38.5C Heat shock applied at 12 hpf
hs:dn-fgfr1 inhibits function of WT FGFR1 hs:fgf8a drives
expression of fgf8a D V P A
Slide 9
Figure 1: FGFR and AKT signaling are necessary for SAG
development Drug treatments applied at 12 hpf SU5402 FGFRs
Wortmannin PI3K/AKT D V P A FGFRs PI3K/AKT ERK PLC
Slide 10
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
Slide 11
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
Slide 12
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
Slide 13
Figure 3: Sox9a and Atoh1a involved in otic neurogenesis Drug
treatments applied during 10-14 hpf
Slide 14
Figure 3: Sox9a and Atoh1a involved in otic neurogenesis Drug
treatments applied during 10-14 hpf mRNA injections at 1-cell
stage
Slide 15
ChIP assay Y Cross-link protein to DNA Shear DNA strands by
sonicating Add bead-attached antibodies to IP the target protein
Unlink protein; purify DNA
Slide 16
Figure 4: Direct targets of Sox9a and Atoh1a Notch-related
Structural proteins Transcription factors Implicated in otic
development Inhibitor of FGF signaling Neural development
factor
Slide 17
Figure 4: Direct targets of Sox9a and Atoh1a
Slide 18
Figure 5: Tlx2 and Eya2 are involved in otic neurogenesis
downstream of Sox9a and Atoh1a
Slide 19
Figure 6: FGFR-ERK signaling is involved in otic sensorigenesis
Drug treatments applied during 10-12 hpf Otic region
Slide 20
Figure 6: FGFR-ERK signaling is involved in otic sensorigenesis
Drug treatments applied during 10-14 hpf in G-K Utricle
Slide 21
Model of FGFR signaling cascades leading to otic neurogenesis
and sensorigenesis
