The inner ear is a complex sensory organ for balance and hearing. The mammalian inner ear develops from an induced ectodermal placode that first invaginates and then closes to form the epithelial otic vesicle. The vesicle epithelium subsequently undergoes a sequence of morphogenetic steps to give rise to the membranous labyrinth, including the three semicircular ducts, utricle, saccule, endolymphatic duct, and cochlea. The surrounding mesenchyme differentiates in close interaction with the otic epithelium to give rise to the bony otic capsule that closely follows the intricate morphology of the membranous labyrinth (Sher, 1971; Martin and Swanson, 1993).
Gata2 and Gata3 belong to the Gata family of zinc finger domain transcription factors (Gata1–6) in vertebrates that bind to a consensus “GATA” DNA sequence. Gata factors have been shown to be important in cell-fate specification as well as cell differentiation, proliferation, and movement during development. Gata2 and Gata3 are coexpressed in several tissues, and mice with targeted mutations in these genes die usually at embryonic day (E) 10.5–E11.5 with defects in hematopoiesis, T-cell differentiation, urogenital development, and neurogenesis (reviewed in Patient and McGhee, 2002). Gata2 and Gata3 are the only family members expressed in the central nervous system (CNS), and they have been shown to be important for hindbrain and spinal cord development (Pata et al., 1999; Nardelli et al., 1999; Zhou et al., 2000). Interestingly, Gata3 expression is dependent on Gata2 in the developing CNS (Pata et al., 1999; Nardelli et al., 1999).
Gata3 has also been shown to be important for inner ear development. It is one of the earliest marker genes for the developing mouse inner ear, and it is expressed in the entire placode at E8.0 (Lawoko-Kerali et al., 2002). In Gata3−/− embryos, inner ear morphogenesis and growth is strongly inhibited after the initial formation of the otic vesicle (Karis et al., 2001). The molecular basis of this arrest is not understood, and the target genes controlled by Gata3 have not been identified. In addition to its morphogenetic role, Gata3 is thought to participate in hair cell differentiation. It is expressed in inner ear sensory epithelia from early on, and later, its expression becomes down-regulated in cochlear hair cells but is maintained in the supporting cells at least until birth (Rivolta and Holley, 1998; Lawoko-Kerali et al., 2002). Mutations in the human GATA3 result in the HDR syndrome with multiple developmental disorders, including deafness (Van Esch et al., 2001). In contrast, nothing is known of the role or expression of Gata2 during mammalian inner ear development.
In the current study, we have performed a comparison of Gata2 and Gata3 expression patterns during normal inner ear development and investigated their relationship in mice where either Gata3 or Gata2 have been inactivated. The expression domain of Gata2 is within the broader domain of Gata3 at early stages, but later, the expression domains of the two factors became increasingly distinct. Whereas Gata2 is predominant in the dorsal vestibular system, Gata3 was detected mainly in the ventral cochlear duct and ganglion. In contrast to the CNS, Gata2 expression is strongly reduced in Gata3-deficient otic epithelium. No phenotypic abnormalities could be observed in the Gata2−/− inner ear.
Partially Overlapping Expression of Gata2 and Gata3 in the Early Otic Epithelium
The expression of Gata2 and Gata3 overlaps especially in the developing CNS, and there, Gata2 expression precedes Gata3 (Pata et al., 1999; Nardelli et al., 1999; Craven et al., 2004). We undertook a detailed study of Gata2 expression during mouse inner ear development and compared the expression with that of Gata3.
A rather weak Gata2 expression could be detected first after otic vesicle closure at E9.5 in the dorsolateral epithelium wall (Fig. 1A). Gata3 is known to be expressed in the whole otic placode at E8.0 (Lawoko-Kerali et al., 2002), and at E9.5 Gata3 is expressed all over the otic epithelium (Fig. 1B). At E10.5, the expression of Gata2 becomes confined to separate dorsal (strong) and ventral (weaker) domains of the otic epithelium, whereas no expression could be detected in the adjacent mesenchyme (Fig. 1C). Very similar dorsal and ventral epithelial expression domains were observed for Gata3 at this stage, Gata3 expression, however, being stronger in the ventral area (Fig. 1D). In addition, Gata3 was also expressed in the mesenchyme (arrow in Fig. 1D).
At E11.5, the future semicircular ducts appear as two-layered outpocketings. At this stage, the highest Gata2 expression could be detected in the dorsal otic epithelium in the areas that will give rise to the semicircular ducts. Weaker expression was also seen in the mesenchyme surrounding the future superior semicircular duct epithelium (Fig. 1E). Gata2 expression was retained also in the tip of the ventrally extending cochlear duct but seemed weaker than that of Gata3 (Fig. 1F). In contrast to Gata2, Gata3 expression was most prominent in cochlear duct and the nearby periotic mesenchyme and only weak expression could be detected in the dorsal vestibular region (Fig. 1F).
At E12.5, Gata2 expression is strongest in the newly formed semicircular ducts as well as in the dorsal utricle and adjacent mesenchymal cells (arrow in Fig. 1G). Expression could also be detected in the lateral wall of the cochlear duct (Fig. 1G). Highest levels of Gata3 expression were observed in the medial wall of the cochlear duct (Fig. 1H). Expression was also detected in the vestibular sensory patches (not shown) and in the ventral areas of the periotic mesenchyme especially adjacent to the lateral semicircular duct and the cochlear duct (arrows in Fig. 1H). No expression of Gata2 or Gata3 was observed in the dorsally extending endolymphatic duct or in the area that will form the saccule at any of the stages analyzed.
Increasingly Diverging Expression of Gata2 and Gata3 at Later Stages
At E14.5, Gata2 continues to be expressed strongly in the vestibular mesenchyme and epithelium with the exception of the saccule. Expression could also be detected on one side of the cochlear duct (Fig. 2A). In the epithelium, Gata2 expression is strictly restricted to the nonsensory domains, and no expression could be observed in the crista of the semicircular ducts or in the macula of the utricle at E14.5–E18.5 (Fig. 2A,E). Gata3 is expressed in the mesenchyme and the sensory areas of the vestibule at E14.5, but at E18.5, no expression could be observed in the vestibule anymore (Fig. 2B,F). In the developing cochlear duct, Gata3 expression is partially overlapping with Gata2 at E14.5–E18.5 (Fig. 2A–H). At E18.5, Gata3 is expressed together with Gata2 in the stria vascularis and, in addition, its expression extends to the developing organ of Corti while Gata2 expression does not reach this sensory epithelium (Fig. 2G,H). Gata3 is known to be down-regulated in the cochlear hair cells from E15.5 and retained in the adjacent supporting cells (Rivolta and Holley, 1998).
Both Gata genes are expressed in the vestibulocochlear ganglion at E14.5–E18.5. Gata2 expression is rather uniform in the vestibular ganglion (Fig. 2A), whereas in the cochlear ganglion, Gata2 is expressed in only a small subset of cells located on the outer edges (Fig. 2A,C). Gata3 is expressed in scattered cells of the vestibular ganglion (Fig. 2B) and in broader areas of the cochlear spiral ganglion, excluding the Gata2-expressing cells (Fig. 2D).
Early Otic Development Occurs Normally in Gata2−/− Embryos
The expression pattern of Gata2 suggested that this transcription factor might have regulatory roles at the early vesicle stage as well as during later vestibular morphogenesis and/or cell differentiation. Because the targeted mutation of Gata2 leads to lethality at E10.5 (Tsai et al., 1994), we studied the effect of the lack of Gata2 at the early otic vesicle stage. In general, no gross morphological changes could be observed in Gata2−/− otic vesicles at E9.5 or E10.5 (shown for E10.5 in Fig. 3).
Gata3 expression is lost in the hindbrain of Gata2−/− embryos (Pata et al., 1999; Nardelli et al., 1999). We studied the expression of Gata3 in the dorsal and ventral otic vesicle domains where it overlapped with that of Gata2 in Gata2−/− embryos and observed no change at E10.5 (Fig. 3A,B). This finding indicates that, in contrast to hindbrain, both the activation and maintenance of Gata3 expression is independent of Gata2 in the otic region.
The expression of Pax2, Eya1, and Six1 genes is activated in response to early inducing signals, and they seem to be instrumental in otic placode territory specification and/or determination (Reviewed in Riley and Phillips, 2003). Dlx5 is also expressed very early in the otic placode and its inactivation leads to disturbed vestibular morphogenesis (Acampora et al., 1999; Depew et al., 1999). In wild-type embryos at E10.5, Pax2, Eya1, and Six1 are expressed in the ventralmost domain of the otic vesicle with some variation in how far the expression extends laterally or medially (Fig. 3C,E,G). The expression of these three genes overlaps with the ventral expression domain of Gata2 (Fig. 1C). However, no change in the expression of these three early markers was observed in Gata2−/− otic epithelium (Fig. 3D,F,H), indicating that the early specification of the ventral otic vesicle cells had occurred normally without Gata2. We also verified the expression of Dlx5 known to be expressed in the vestibular area, including the endolymphatic duct. Dlx5 expression is unchanged in Gata2−/− embryos (Fig. 3I,J), indicating that also the dorsal otic epithelium specification is normal.
At E10.5 BMP4 is expressed in the otic sensory anlagen that will give rise to the semicircular duct cristae (reviewed in Fekete and Wu, 2002). We observed BMP4 expression in both wild-type and Gata2−/− otic epithelium, suggesting that the cell fate determination toward the sensory lineage had occurred normally in the absence of Gata2 (Fig. 3K,L).
Gata2 Expression Is Strongly Down-Regulated in Gata3−/− Otic Epithelium
The targeted mutation of Gata3 leads to an early arrest in inner ear growth and morphogenesis that affects both the vestibular and cochlear development. The only outgrowing part is the endolymphatic duct (Karis et al., 2001).
We could not detect any sign of Gata2 expression at E9.5 in the Gata3−/− otic vesicles (data not shown). At E10.5, we observed a very weak Gata2 expression in Gata3−/− otic epithelium compared with the wild-type littermate (Fig. 4A,B). Similarly, a clearly reduced Gata2 expression could be observed at the most dorsal and ventral tips of the mutant otic epithelium at E11.5, corresponding most likely to the remnants of vestibular and cochlear compartments (Fig. 4C,D). In the hindbrain, no difference in Gata2 expression could be detected between the wild-type and Gata3−/− embryos (Fig. 4A,B). These results suggest that, in contrast to hindbrain, Gata3 is upstream of Gata2 in the ear.
The expression of Pax2, Eya1, and Six1 was unaffected in Gata3−/− otic epithelium (Fig. 4E–J), indicating that either the two Gata factors act downstream of these early effectors or they function in an independent pathway.
Members of the Gata transcription factor family control important phenomena during the development of many organs. Gata2 and Gata3 have been implicated especially in the development of the nervous system, hematopoietic lineage, and T-cells, and the inactivation of either family member leads to lethality at E10.5–E11.5 (Patient and McGhee, 2002). Their expression overlaps in a restricted domain of the developing hindbrain, and there, Gata2 expression is initiated earlier than that of Gata3. Gata3 expression is lost in Gata2−/− hindbrain, suggesting that Gata3 is downstream of Gata2 (Pata et al., 1999; Nardelli et al., 1999).
Here, we have compared the spatiotemporal expression patterns of Gata2 and Gata3 in the developing mouse inner ear. Although it has been reported that Gata3 is expressed in the whole mouse otic placode at E8.0 (Lawoko-Kerali et al., 2002), we showed that Gata2 expression could be detected only after otic vesicle closure at E9.5. The expression domains of these two factors is highly overlapping in the otic epithelium at E10.5 but becomes more distinct as development progresses. While Gata2 expression is mainly confined to the nonsensory vestibular epithelium and vestibular ganglion, Gata3 expression becomes restricted to the cochlea and spiral ganglion. Neither factor was detected in the outgrowing endolymphatic duct or in the saccule. Our expression data suggest that the two Gata factors might have overlapping roles during early otic development and that, later, Gata2 would be more important for vestibular development, whereas Gata3 would be required for the maturation of the cochlea.
In the hematopoietic system, Gata2 is required at an early phase for the development of blood progenitors and its expression has to be down-regulated later to allow erythroid differentiation (Tsai et al., 1994). In the inner ear, Gata2 is expressed at all vestibular developmental stages and no down-regulation was observed until after birth, suggesting a different role for Gata2 in otic epithelium.
We could not detect any morphological defects in the otic vesicles at E10.5 in Gata2 mutants. The absence of any detectable phenotype in Gata2−/− otic vesicles might be due to the fact that Gata3 is expressed in these vesicles. Several Gata-factors can bind to the same “GATA”-containing binding sites (Ko and Engel, 1993; Merika and Orkin, 1993) and different Gata factors are known to compensate for each other's functions (Pevny et al., 1995). Therefore, Gata3 might compensate for the loss of Gata2 during early otic development. A conditional mutagenesis approach will be required to address the role of Gata2, especially during vestibular morphogenesis at later stages.
The early expression of Gata3 in the dorsal and ventral otic compartments and in the adjacent mesenchyme appears to be important for the outgrowth and morphological development of both the semicircular ducts and the cochlea, because no further development can be observed at E11.5. We show here that this developmental arrest is accompanied by a strong delay and decrease in Gata2 expression. Furthermore, this weak Gata2 expression is not sufficient to compensate for the loss of Gata3. The down-regulation of Gata2 in Gata3−/− otic vesicles suggests that these two factors act in the same genetic pathway and that, in contrast to hindbrain, Gata2 is downstream of Gata3 in the inner ear.
Mouse Breeding and Genotyping
The generation of Gata2−/− and Gata3−/− (Gata3nlslacZ) mice has been described previously (Tsai et al., 1994; Hendriks et al., 1999). Gata2 genotyping was performed with the following polymerase chain reaction (PCR) primers: neo503 5′-GAT CTC CTG TCA TCT CAC CTT GCT-3′, M39 5′-GGA ACG CCA ACG GGG AC-3′, and m208 5′-GCT GGA CAT CTT CCG ATT CCG GGT-3′. Gata3 genotyping was performed with the following PCR primers: G3ex2-F 5′-CCT CCG ACG GCA GGA GTC-3′, G3ex2-R 5′-ACC GTA GCC CTG ACG GAG TTT-3′, and LacZ-R1 5′-ACG GCG GAT TGA CCG TAA TG-3′. The embryos were taken from timed matings, and the day on which a vaginal plug was detected was assigned as E0.5.
RNA In Situ Hybridization
Radioactive in situ hybridization was performed on 10-μm serial paraffin sections as previously described (Salminen et al., 2000). The preparation of the radioactive antisense and sense probes were performed as described in Salminen et al. (2000). No specific signals were detected with any of the sense probes. The red pseudocolor images were reproduced by overlaying the original brightfield and darkfield pictures of the sections in Adobe Photoshop and by replacing the white signal in darkfield with red color.
We thank Giovanni Levi, Peter Gruss, Illar Pata, Ali Imam, and Richard Maas for some of the in situ probes we used. We also thank Luydmula Rasskazova, Christel Pussinen, and Mariana Loto-Peña for expert technical assistance.