Zebrafishatoh1genes: classic proneural activity in the inner ear and regulation by Fgf and Notch

Abstract

Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. We have addressed these issues in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development.

Hedgehog Signaling Governs the Development of Otic Sensory Epithelium and Its Associated Innervation in Zebrafish

“…In the zebrafish ear, the prosensory function is thought to be mediated by atoh1a/b genes (Millimaki et al, 2007). We showed that the posterior domain of atoh1a expression in the zebrafish inner ear appears later than the anterior domain, and is selectively affected by reducing Hh signaling.…”

“…Tether cells and later-forming hair cells are specified in two distinct and successive phases. Their formation differentially requires the function of atoh1b and atoh1a respectively, two atonal homologs that have a proneural/prosensory function in zebrafish (Millimaki et al, 2007). Despite the common mechanisms leading to the formation of sensory cells in both maculae, anterior and posterior macula develops as two functionally specialized sensory epithelia.…”

“…It is assumed that the development of all otic neurosensory elements crucially depends on the function of three atonalrelated bHLH transcription factors: atoh1 for hair cell formation (Bermingham et al, 1999;Millimaki et al, 2007), ngn1 for sensory neuron determination (Ma et al, 1998;Andermann et al, 2002), and neuroD for sensory neuron differentiation and survival (Kim et al, 2001). …”

“…Whole-mount in situ hybridization was performed as described previously (Hauptmann and Gerster, 1994). Probes were as follows: atoh1a, atoh1b (Millimaki et al, 2007), ptc1 (Hammond et al, 2003), zath3/neuroD4 (Park et al, 2003), gata3 (Cheng et al, 2007), ngn1, and neuroD (Itoh and Chitnis, 2001).…”

Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line

“…Fluorescent in situ hybridization (FISH) was performed at 55°C as published previously (Julich et al, 2005), followed by myosin-VI immunostaining and counterstaining with 4Ј,6Ј-diamidino-2-phenylindole (DAPI) (Invitrogen). Plasmids were cut and transcribed with the following for generating antisense digoxigenin-labeled RNA probes: Pme1/T7 for atoh1a (a gift from Bruce Riley, Texas A&M University, College Station, TX) (Millimaki et al, 2007); SalI/T7 for notch3 (clone NAA32G11; Open Biosystems, Huntsville, AL); XbaI/T7 for notch1a (a gift from Jose CamposOrtega; University of Cologne, Cologne, Germany) (Bierkamp and Campos-Ortega, 1993); SmaI/T3 for notch1b [cDNA in pBS-SK plasmid (Addgene, Cambridge, MA)] (Kortschak et al, 2001); EcoRI/T7 for deltaA and deltaD (a gift from Bruce Appel, Vanderbilt University, Nashville, TN) (Haddon et al, 1998b); ApaI/Sp6 for jagged1a; EcoRV/Sp6 for jagged1b; and SpeI/T7 for jagged2 (all jagged plasmids were gifts from Natasha Tiso, University of Padua, Padua, Italy) (Zecchin et al, 2005).…”