Compensatory Regulation of the Size of the Inner Ear In Response to Excess Induction of Otic Progenitors by Fibroblast Growth Factor Signaling

Jian Zhang, Kevin D. Wright, Amanda A. Mahoney Rogers, Molly M. Barrett, and Katherine Shim

Summary: The Sprouty genes encode intracellular antagonists of receptor tyrosine kinase signaling, including FGF signaling. FGF signaling has in implicated, in multiple organisms and studies, to initiate otic development by inducing formation of the otic-epibranchial progenitor domain from the pre-placodal region. This paper focuses on using a Sprouty1 and Sprouty2 gene dosage series, by inactivating them combinatorially in mouse embryos. It was found that the otic placode was enlarged in a Sprouty gene dosage series at 8-11 somite stages (s), as the state at which cells are first committed to an otic fate. By 17-20s, size and morphogenesis of the otic cup in Spry1Het2Null embryos was restored to normal, thus a window of 12-15s was defined as the time during which the otic placode size may have been restored to normal size. In studying the cellular mechanisms behind the expansion and restoration, transcription factors Etv4, Etv5, and Dusp6 were used to suggest that FGF signaling was not elevated in Spry1Het2Null embryos at the stage of restoration otic cup size. Thus, normal levels of FGF signaling may contribute to the restoration, while elevated levels of FGF signaling, due to Spry1/2 knockout, in DKO embryos may explain why the otic placode was enlarged, and the morphogenesis of the otic cup and vesicle was abnormal.

Basic Methods: They used the Cre-Lox system to produce mouselines that were double heterozygote for Spry1 and Spry2, Spry1-/-;Spry2-/+, Spry1-/+;Spry2-/-, and double knockout. They used whole-mount in situ hybridization using the following labeled probes: Pax8, Dlx5, Hmx3 (otic placode markers), Foxi2 (marker for region surrounding otic placode), Spry1, Spry4, Etv 4/5 (ETS-domain containing transcription factors that are transcriptionally induced by FGF signaling in multiple organs), Dusp6, Dusp9, and Sef (negative feedback inhibitors of FGF signaling pathways). They also extracted RNA from the embryos to quantify gene expression using quantitative and real-time PCR.

Why it’s Important: This paper provides further motivation for our interests in the downstream pathways of FGF signaling in otic induction. This paper suggests that FGFs are necessary for OEPD and placode formation, but at some point must be limited in their effects, in order to maintain a crucial size of the regions preceding formation of the otic vesicle. But how does this translate to an in vitro system? Will we be able to make more hair cells if we knockout some of the molecules that inhibit FGF signaling in order to maximize FGF impact on our cells early on? Otic development relies on the effects of FGFs and by using knockout methods or molecules to involved in FGF negative feedback loops may help to maximize the effects of FGFs on our cells at the time points we have defined in our protocols.

Lab Meeting Paper

Lab Meeting Presentation 2.6.15


Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBL Targeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines

Cerbini T, Funahashi R, Luo Y, Liu C, Park K, Rao M, et al. (2015) Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBLTargeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines. PLoS ONE 10(1): e0116032. doi:10.1371/journal.pone.0116032

Summary: The focus of this paper was to investigate targeted gene insertion using TALENs as a means of creating an efficient, long-lasting reporter stem cell line. Stem cell transfection is typically challenging and results in low percentage efficiencies. Here, they seek to increase transfection efficiencies by using TALENs to target reporter genes to two “safe harbor” sites (AAVS1 and CLYBL), either separately or both at the same time. They compared efficiencies in transient reporter expression as well as long-term expression in both neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). They found that CLYBL-targeted transgenes resulted in higher transgene expression and resulted in clones that stably express the transgene through division and differentiation into cardiomyocytes and neurons. They also were able to knock-in transgenes at dual safe harbor sites. Finally, transgene expression in the CLYBL gene does not greatly disrupt normal local and global gene expression. These data offer support for a gene that can be targeted for safe harbor integration to maintain long-term expression of reporter genes in stem cell lines.

Basic Methods: They used TALENs to target transgenes expressing reporters (tdTomato, GFP, Nanoluc-Halotag) into either the CLYBL locus or the AAVS1 site, both considered to be safe harbor sites. After transfection of TALENs and reporter plasmids into NSCs or iPSCS using the 4D-Nucleofector X Unit, cells were assessed for transgene expression by staining with Oregon green and assessing Nanoluc expression or looking at expression levels of tdTomato and GFP. They allowed stem cells to differentiate into cardiomyocytes or neurons and again assessed reporter expression. They evaluated the location of transgene integration by using Southern blot analysis. Finally, they investigated local and global gene expression via microarray and RT-qPCR.

Why It’s Important: We are interested in methods to achieve stable expression of reporter genes in human embryonic stem cells that can be inserted into the inner ear, ultimately differentiating into hair cells. In conjunction with the Raphael lab, we have previously established a method that can insert non-stem cells into the inner ear with some success. The next step is insertion of stem cells, and using these cells (with successfully integrated reporter genes) would allow us to further investigate insertion, differentiation, and viability of cells after transplantation into the inner ear.

PDF of paper: Cerbini et al 2015

Presentation ppt: Cerbini et al lab meeting 1.23.15

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.


Lab meeting 1.30.2015

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


Hey lab!

This is the new lab blog site.  We will give this site a trial run over the 2015 calendar year.  Though it could evolve into something else, my intention is that it serves as a way to interact with the lab, provide some history for new folks to see what we’ve been up to, and provide a context for those away from the lab to see what’s going on.  Blog entries will be open for all lab members and will be (eventually) password protected so that only lab affiliates can access.

What to discuss?  I want to see some brief entries on papers of significant impact, including journal articles discussed at lab meetings.  Hosts for the lab meetings will be expected to make an entry before lab meeting, listing the basic why, how, what of the article and the relationship to our work.  If you read significant articles that you think would be good for lab meeting, subgroup meetings, or general information, it would be great to post here.  In addition, I would like to see the site used during scientific conferences, and possibly after you attend other seminars on campus.  Have other ideas?  Post.

Lastly, I would like to see how we might use this or another site as a WIKI for lab protocols, antibody use, etc.

No worries if this ends up failing.  As always, the goal is better communication and I want to try any tool that improves this.