Fgf and Hh signalling act on a symmetrical pre-pattern to specify anterior and posterior identity in the zebrafish otic placode and vesicle

Abstract

SUMMARYSpecification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20 somite (early otic vesicle) stages. We now show that Fgf signalling is both necessary and sufficient for anterior otic specification during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia -/-(fgf3 -/-) mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear.

Shaping sound in space: the regulation of inner ear patterning

“…One obvious possibility is that FGF signaling in the anterior region of the otocyst actively inhibits Hh signaling, restricting its influence to the posterior domain. However, Hh signaling, as revealed by Patched gene expression, still localizes to the ventromedial domain in the absence of FGF signaling; similarly, FGF signaling, as revealed by pea3 expression, remains localized to the anterior domain of the otocyst in the absence of Hh signaling (Hammond and Whitfield, 2011). Loss of both FGF and Hh signaling in zebrafish leads to a grossly abnormal inner ear that lacks most sensory cells and exhibits vestigial semicircular canals, and to a loss of most markers of anterior and posterior identity (Hammond and Whitfield, 2011).…”

“…For example, mutations in the zinc-finger transcription factor mafb gene cause a loss of rhombomeres 5 and 6 and an expansion of rhombomere 4 markers such as fgf3. These mutants have an expansion of anterior otic markers, whereas the fgf3 mutant limabsent (lia) displays a partial loss of anterior otic markers (Hammond and Whitfield, 2011;Kwak et al, 2002). Exposure of zebrafish embryos to the FGF receptor inhibitor SU5402 after the formation of the otic placode causes a dramatic loss of anterior markers and duplication of posterior otocyst markers, leading to an inner ear with two mirror-image posterior domains (Hammond and Whitfield, 2011).…”

“…These mutants have an expansion of anterior otic markers, whereas the fgf3 mutant limabsent (lia) displays a partial loss of anterior otic markers (Hammond and Whitfield, 2011;Kwak et al, 2002). Exposure of zebrafish embryos to the FGF receptor inhibitor SU5402 after the formation of the otic placode causes a dramatic loss of anterior markers and duplication of posterior otocyst markers, leading to an inner ear with two mirror-image posterior domains (Hammond and Whitfield, 2011). Conversely, heat-shock activation of fgf3 in embryos containing ten somites or more led to the opposite phenotype -a downregulation of posterior otic markers and an inner ear bearing two mirror-image anterior domains (Hammond and Whitfield, 2011).…”

“…Exposure of zebrafish embryos to the FGF receptor inhibitor SU5402 after the formation of the otic placode causes a dramatic loss of anterior markers and duplication of posterior otocyst markers, leading to an inner ear with two mirror-image posterior domains (Hammond and Whitfield, 2011). Conversely, heat-shock activation of fgf3 in embryos containing ten somites or more led to the opposite phenotype -a downregulation of posterior otic markers and an inner ear bearing two mirror-image anterior domains (Hammond and Whitfield, 2011). Since the anterior regions of the zebrafish otocyst lie immediately adjacent to rhombomere 4, it is likely that Fgf3 and other FGF family members provide anterior patterning signals to the otocyst.…”

Fate map of the chicken otic placode

“…The gradient-governed interaction of DV and AP organizing centers would establish a Cartesian coordinate positional system for differential molecular signaling in the otic placodal field (Meinhardt, 2008). However, we cannot rule out other possibilities that have been suggested for DV and AP patterning, in particular the relevance of Shh and retinoic acid signals (Bok et al, 2007(Bok et al, , 2011Brown and Epstein, 2011;Hammond and Whitfield, 2011;Riccomagno et al, 2005;Wu et al, 1998). Restricted spot-like signaling centers from the segmented hindbrain, or even from the pharyngeal endoderm and subjacent mesoderm, might also be involved directly or indirectly in otic placode specification, representing potential sources of additional differential AP signals (Abelló et al, 2010;Alsina et al, 2009;Baker et al, 2008;Begbie et al, 1999;Chen and Streit, 2013;Graham, 2008;Grocott et al, 2012;Ladher et al, 2010;Lleras-Forero and Streit, 2012;McCabe and Bronner-Fraser, 2009;Schlosser, 2006Schlosser, , 2010Schneider-Maunoury and Pujades, 2007;Whitfield and Hammond, 2007).…”