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.