Lab meeting 1/30/2015 – Signaling pathways downstream of FGFs in zebrafish otic development

Wang, J., Y. Wu, F. Zhao, Y. Wu, W. Dong, J. Zhao, Z. Zhu, and D. Liu. 2015. “Fgf-Signaling-Dependent Sox9a and Atoh1a Regulate Otic Neural Development in Zebrafish.” Journal of Neuroscience 35 (1): 234–44. doi:10.1523/JNEUROSCI.3353-14.2015. PMID: 25568117

Summary: This paper focuses on signaling pathways downstream of FGF receptors in neural and sensory cell development in the zebrafish inner ear.  It shows that FGF signaling is necessary for development of the statoacoustic ganglion (SAG, equivalent to spiral ganglion).  FGF receptors can signal through multiple parallel downstream pathways, including ERK and AKT pathways.  In this paper, AKT is implicated in mediating the effect of FGFs on SAG development.  FGF signaling is also necessary for expression of Sox9a and Atoh1a; in this paper, these transcription factors are shown to be involved in otic neurogenesis.  A possible mechanism for this is suggested: Sox9a and Atoh1a promote expression of eya2 and tlx2, which also are necessary for normal SAG development.  Finally, blocking the ERK pathway leads to reduced Atoh1a and 1b expression–the effect on 1b is more dramatic–but has no effect on Sox9a.  Since Atoh1 is known to mediate hair cell development, the paper suggests a model in which FGFR-ERK signaling mediates otic sensorineurogenesis whereas FGFR-AKT signaling (leading to Sox9a, Atoh1a, Eya2, and Tlx2) mediates otic neurogenesis.

Basic methods: Transgenic zebrafish lines were engineered to express heat shock-inducible genes to allow modulation of FGF signaling at specific timepoints in development.  Morpholinos and pharmacological inhibitors of FGFRs (SU5402) and downstream pathways AKT (wortmannin), ERK (PD98059), and PLC (U73122) were also used to reduce expression or activity.  HuC was used as an antigen for immunostaining the SAG to measure its area.  In situ hybridization was used to visualize Neurod expression to assess neurogenesis.  Targets of Sox9a and Atoh1a transcription factors were identified via chromatin immunoprecipitation.  Utricular hair cells were quantified via MyoVI immunostaining.

Why it’s important: We are interested in signaling mechanisms downstream of FGFRs in otic induction and how these can be manipulated in context of our stem cell cultures.  This paper suggests that we could apply FGFs, favor ERK signaling by blocking AKT, and influence our cells to differentiate toward a hair cell fate rather than a neuronal fate.  Alternatively, we could block ERK and influence them to become neuronal.  Yang et al. 2013 also suggests the role of ERK in otic induction in chicken.  This paper lends further support to that idea and strengthens the rationale behind our idea to use pharmacological inhibitors to favor the specific downstream pathway (ERK, we think) that promotes hair cell fate.

Model

Lab meeting 1.30.2015

Wang et al. – 2015 – Fgf-Signaling-Dependent Sox9a and Atoh1a Regulate

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