Gata3 directly regulates early inner ear expression of Fgf10

Abstract

The analysis of Fgf10 mouse mutants has demonstrated a critical role for this ligand in neurosensory development of the vertebrate inner ear, and we have been looking to define the direct upstream regulators of Fgf10 in this sensory organ, as part of constructing the programme of early inner ear development. Through the analysis of reporter constructs in transgenic mouse embryos and neonatal mice, in this report we define a minimal 1400 bp enhancer from the 5' flanking region of Fgf10. This enhancer drives reporter transgene expression in a manner that recapitulates endogenous expression of Fgf10, from its initial onset in the invaginating otic placode and onwards throughout gestation, controlling Fgf10 expression in all developing sensory patches and in the developing VIIIth ganglion. This regulatory region includes three putative Gata3 binding sites that we demonstrate directly interacts with Gata3 protein through the DNA binding domain with differing affinities. Site directed mutagenesis of all three sites and functional testing in transgenic embryos using reporter transgenes reveals an absolute requirement for Gata3 in controlling Fgf10 expression. Transgenic analysis of individual Gata3 binding site mutations illustrates that only one of these binding sites is necessary for reporter expression. Together these data demonstrate that Gata3 directly activates Fgf10 in the early inner ear, and does so through a single binding site.

The Ups and Downs of Retinoic Acid Signaling in Early Inner Ear Development

“…Exposure of embryos to RA at a stage where hindbrain patterning is either unaffected by RA exposure [25] or subsequent to hindbrain patterning, revealed that Fgf3 reporter activity is down regulated in the anteroventral otic vesicle but unaltered in the developing posterior hindbrain [26]. These findings support direct regulation of otic Fgf3 expression by RA, and underscore the importance of determining whether the response to RA may be mediated via binding to RA responsive elements located at the Fgf3 locus [16].In common with Fgf3, Fgf10 reporter expression is down regulated in the antero-ventral otic vesicle by exogenous RA during hindbrain patterning, but to a lesser extent after hindbrain patterning [24]. It will thus be interesting to further investigate whether the response to RA post-hindbrain patterning is direct on the otic Fgf10 enhancer or a secondary consequence of alterations in hindbrain pattern that lead to different signals influencing the development of the inner ear.…”

“…An issue is thus raised as to whether regulation of otic Fgf by RA may be direct or secondary to RA-induced changes in hindbrain pattern/specification. A more detailed understanding of the role of RA in regulating Fgf expression has been attained through analyses of reporter expression in mice containing lacZ either under the control of a minimal Fgf3 or Fgf10 enhancer [23,24]. Exposure of embryos to RA at a stage where hindbrain patterning is either unaffected by RA exposure [25] or subsequent to hindbrain patterning, revealed that Fgf3 reporter activity is down regulated in the anteroventral otic vesicle but unaltered in the developing posterior hindbrain [26].…”

“…Of note, changes in localization of Fgf3 and Fgf10 by excess RA also occur within the otocyst epithelium during hindbrain patterning, implicating the ability of the inner ear to reprogram its spatial expression profiles during this developmental time point. However, the precise spatial changes differ between these related signaling molecules, and likely represent differences in the regulatory response of Fgf3 and Fgf10 to RA [24].…”

Inductive and Morphogenetic Responses of the Inner Ear to FGF Signaling

“…It is therefore not surprising that the vestibuloacoustic ganglion is reduced in size in Fgf3 mouse mutants [5,21] and defects in vestibular sensory neurons and hair cells are produced following Fgf10 mutation [22]. Thus within the mouse, Fgf3 and Fgf10 are also required in a non-redundant manner for proper development within the otic epithelium as well as the earlier hindbrain-signalling events outlined above [23]. In addition, epithelial-derived FGF8 and FGF20, expressed in the presumptive epithelial domain of the organ of Corti, function in the specification of distinct cell types, in particular pillar cells, one of the supporting cells found in the developing auditory organ [24,25,26].…”

“…Otic capsule chondrogenesis can, however, be rescued by supplementation of cultures with a combination of FGF3 and FGF10 ligands, suggesting that these FGFs are indeed subject to control through RA signaling. Furthermore, recent studies show that the transcription factor GATA3 is a direct upstream regulator of Fgf10 that activates inner ear transcription [23].…”