Identification of Synergistic Signals Initiating Inner Ear Development

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Science  08 Dec 2000:
Vol. 290, Issue 5498, pp. 1965-1967
DOI: 10.1126/science.290.5498.1965


Tissue manipulation experiments in amphibians more than 50 years ago showed that induction of the inner ear requires two signals: a mesodermal signal followed by a neural signal. However, the molecules mediating this process have remained elusive. We present evidence for mesodermal initiation of otic development in higher vertebrates and show that the mesoderm can direct terminal differentiation of the inner ear in rostral ectoderm. Furthermore, we demonstrate the synergistic interactions of the extracellular polypeptide ligands FGF-19 and Wnt-8c as mediators of mesodermal and neural signals, respectively, initiating inner ear development.

In amphibians, induction of the otic placode, which forms the inner ear, has been shown to require a mesodermal signal followed by a neural signal (1, 2). Neuroectodermal signals have also been shown to be involved in chick and mouse, but definitive proof for mesodermal signaling has yet to be demonstrated (2–5). In all systems, the signaling factors involved have remained elusive. Fibroblast growth factors (FGFs) have been implicated in otic development. In particular, FGF-3, which is essential for inner ear development, is crucial for the later stages of otic induction, acting within the otic ectoderm and for otic maintenance (2, 68). In addition to roles in otic development, FGFs are involved in caudalization of the neuroectoderm, partly by signaling through the paraxial mesoderm (9). Because otic development is dependent on mesodermal and neural signals, this raises the possibility that FGFs in the paraxial mesoderm may also play a role in otic induction. By low-stringency screening of a cDNA library, we identified a new chick FGF: FGF-19 (10). In situ expression analysis suggested a potential role for FGF-19 in otic development, which we have characterized here (11).

During early chick embryogenesis, Fgf-19 is expressed transiently (stages 6 to 9) in a restricted region of paraxial mesoderm, initially underlying the neural plate (stages 6 and 7), whereas later it is in contact with neural and nonneural ectoderm (Fig. 1, A to C and F to I). From stage 9, Fgf-19 is also expressed in the endoderm and transiently in the developing neural tube until stage 9+ (Fig. 1, C to E and H to K) [Web fig. 1 (12)]. Fate mapping of Fgf-19–expressing mesoderm at stage 7 and neuroectoderm at stage 9 (13) showed that these regions colocalize with the presumptive otic placode along the rostro-caudal axis (Web fig. 2). The otic placode arises lateral to the neural plate and initially at stage 7 does not overlieFgf-19–expressing mesoderm, but will later be in contact with or in close proximity to Fgf-19–expressing mesoderm as the neural groove closes (Web fig. 2).

Figure 1

Fgf-19 expression is associated with otic development. (A through K) Whole-mount in situ hybridizations show the expression of Fgf-19 in chick embryos at stages 6 (A and F), 8+ (B and G), 9 (C, H, and I), 9+ (D and J), and 10 (E and K). [(F) through (K)] Section analyses through the levels indicated show that Fgf-19 is expressed in the paraxial mesoderm between stages 6 to 9 [(F) through (I)], the developing caudal hindbrain between stages 9 to 9+ [(I) and (J)], and endoderm from stage 9 until at least stage 24 [(I) through (K); see Web fig. 1] (16). Scale bars: 500 μm (A), 250 μm [(B) and (C)], and 125 μm [(D) and (E)].

Coincident expression of Fgf-19 in the mesoderm and neural tube with the position of the presumptive placode made FGF-19 a candidate to direct otic development. Therefore, we tested the ability of Fgf-19–expressing mesoderm at stage 7 (regions a and b,Fig. 2) versus nonexpressing mesoderm (regions c and d, Fig. 2) to direct otic development in stage 5 ectoderm (Et) (14). Otic development was assessed using markers that are expressed at various stages of inner ear development from the presumptive placode to terminal differentiation (15). Fgf-19–expressing mesoderm induced all otic markers tested, whereas nonexpressing mesoderm did not (Fig. 2, A through D, and Web fig. 3) (16). At stage 9+, the equivalent mesoderm (a′, Fig. 2), which now does not express Fgf-19, could not induce otic development, as assessed by Pax-2 and Nkx5.1 expression (Fig. 2E and Web fig. 3). Only when adjacent neuroectoderm (N, Fig. 2), which expresses Fgf-19, was included could otic development proceed (Fig. 2F and Web fig. 3). This supports work in amphibians demonstrating that mesodermal signaling acts over a defined period and is followed by a neural signal (1, 2).

Figure 2

Fgf-19–expressing mesoderm directs otic development. Diagram shows the region of stage 5 ectoderm, Et (test ectoderm) and stage 7 mesendoderm (regions a through d) or stage 9+ mesendoderm a′, which were recombined in collagen gel culture. The Fgf-19–expressing regions are purple. The otic placode arises from region c. Whole-mount in situ hybridizations and antibody staining show that only regions a and b, which includeFgf-19–expressing mesoderm, and not regions c, d, or a′, can induce the expression of Pax-2 (A,C, and E) and development of inner hair cells (B and D) (Pax-2, region a,n = 5/5; b, n = 5/5; c,n = 0/5; d, n = 0/5, a′,n = 0/5. Inner hair cells, a, n = 4/4; b, n = 4/4; c, n = 0/4; d,n = 0/4). However, a′ can induce the expression ofPax-2 when the overlying neural tube N, which expressesFgf-19, is included [(F), n = 5/5]. The inset in (A) confirms that Pax-2 expression is confined to the ectoderm while the insets in (E) and (F) show section analysis following antibody staining to determine the position of the quail cells (stained brown). Scale bars: 100 μm (A, C, E, and F) and 10 μm (B and D).

Because Fgf-19 expression correlated spatially and temporally with mesodermal and neural otic-inducing signals, we tested whether FGF-19 alone could induce expression of otic markers in the presumptive, but uncommitted, otic region (region c, Fig. 2) and nonotic regions (regions d and e and Et, Fig. 2) (17). FGF-19 alone did not induce any of the otic markers tested when added to the presumptive otic (c) or nonotic (e) regions but could induce otic markers in nonotic tissue if neural (d) or presumptive neural tissue (Et) was included (region c:Dlx-5, Fgf-3, Nkx5.1,Pax-2, and SOHo-1; region e: Pax-2 and 0/4; Fig. 3 and Web figs. 4 and 6) (16). These data suggest that under certain conditions one molecule can initiate several components of the otic signaling cascade, provided that a neural signal is also present.

Figure 3

FGF-19 can induce components of the otic signaling cascade. Whole-mount in situ hybridizations show that FGF-19 (B, F, D, and H) but not control (A, E, C, and G) beads can induce the expression of otic markers Fgf-3 (A through D), Pax-2 (D through H) in stage 5 ectoderm (A, B, E, and F); Et, Fig. 2] and in region d (Fig. 2) at stage 7 (C, D, G, and H). For additional data, see Web fig. 4. Although these genes are expressed in other ectodermal derivatives (15), the combinatorial expression of these genes is consistent with the induction of otic pathway components. Stage 5 ectoderm:Fgf-3, n = 3/4; Pax-2,n = 4/5. Region d: Fgf-3, n= 4/10; Pax-2, n = 4/5. Scale bar: 100 μm.

In Xenopus laevis embryos, cooperative Wnt and FGF signaling modifies cell fate, and it has been proposed that FGFs mediate neural crest production via Wnt signaling (18–20). Thus, we investigated the role of Wnts, which are expressed in the neuroectoderm (21), during early otic development. We focused on Wnt-8c because it is expressed in the neural plate, initially overlying Fgf-19–expressing mesoderm (Web fig. 5) (22). Later, both are coexpressed in the caudal hindbrain until stage 10, after which the otic inductive capabilities of the hindbrain are lost (Web fig. 5) (5, 22). Furthermore, FGF-19 can induce the expression of Wnt-8c in preneural stage 5 ectoderm (Fig. 4, A and B) but not in presumptive otic tissue (region c, Fig. 2;n = 12; 6 to 18 hours) (16, 23). This raised the possibility that mesodermal FGF-19 initiates and/or maintainsWnt-8c expression in the neural plate, acting via or in cooperation with Wnt-8c to direct otic development.

Figure 4

FGF-19 may initiate otic development by the induction of Wnt-8c. Whole-mount in situ hybridizations show that FGF-19–soaked beads (B), but not control beads (A), induce the expression of Wnt-8c in rostral stage 5 ectoderm (n = 5/7; Et, Fig. 2). (Cthrough N) Whole-mount in situ hybridizations show that Wnt-8c alone can induce Fgf-3 expression, while FGF-19 and Wnt-8c synergize to induce expression of other otic markers. Stage 7 region c (Fig. 2) explants do not express otic markers when cultured with beads soaked in a control solution (C, G, and K) or FGF-19 (D, H, and L). Similarly, with the exception of Fgf-3 [(M);n = 3/3], Wnt-8c–expressing COS cells do not strongly induce otic markers Pax-2 (E) andDlx-5 (I). However, region c (Fig. 2) explants strongly express the otic markers Pax-2 [(F); n = 4/4] and Dlx-5 [(J); n = 3/3] when co-cultured with both Wnt-8c–expressing cells and beads soaked in FGF-19. Scale bars in (A) through (C): 200 μm. For additional information, see Web fig. 6 (12).

To test this, we cultured the uncommitted presumptive otic region (region c, Fig. 2) in the presence of one or both of these factors (17, 24). As previously determined, FGF-19 alone could not induce expression of otic markers (Fig. 4, D, H, and L, and Web fig. 6). Wnt-8c alone induced strong expression of Fgf-3(Fig. 4M); however, only weak or negligible expression ofDlx-5 (n = 1/3), Nkx5.1(n = 3/4), and SOHo-1 (n = 3/5) was observed (Web fig. 6). The combination of both FGF-19 and Wnt-8c consistently induced strong expression of Pax-2(Fig. 4F), Dlx-5 (Fig. 4J), and Nkx5.1 andSOHo-1 (Web fig. 6). Furthermore, section analysis identified regions of thickened ectoderm characterized by otic gene expression (n = 8/11), and in one case, with vesicular morphology indicative of otic placode development (16). Otic induction does not occur through the prior induction of neural tissue because FGF-19 or Wnt-8c, together or alone, do not induce neural characteristics in the presumptive otic region (region c, Fig. 2), as assessed by lack of Sox-2,Wnt-8c, and Fgf-19 expression following treatment (16, 25). Additionally, FGF-19 and Wnt-8c can cooperate to induce at least one otic marker, Pax-2, in nonotic ectoderm (region e, Fig. 2; n = 3/3) (16), suggesting that these molecules are sufficient to initiate otic development.

We propose that FGF-19 in the paraxial mesoderm induces and/or maintains Wnt-8c expression in the competent neuroectoderm (26). Subsequently, Wnt-8c induces Fgf-3in the presumptive otic placode but cooperates with FGF-19 to induce the other otic markers investigated: Nkx5.1,Pax-2, SOHo-1, and Dlx-5. Wnt-8c alone can also induce weak expression of some otic markers, either directly or possibly via the induction and subsequent synergy withFgf-3 (8). The inability to induce inner hair cell development (n = 0/11) may suggest that additional independent signals are required, or that the temporal expression of Wnt-8c and FGF-19 used in these studies is insufficient, or alternatively, inhibitory for differentiation.

Embryological studies in amphibians (1) and chicks (5) indicate that inner ear induction starts during or after late gastrulation, coincident with expression of Fgf-19 in chicks. We propose FGF-19 as an inducer of otic development, acting in part by patterning the neuroectoderm. This suggests that, in general, localized expression of mesodermal signals may be the key components to pattern the tissue layers. Furthermore, we have characterized a novel function of Wnt signaling. This will facilitate the elucidation of additional signaling interactions controlling otic development. Otic development is characterized not only by differentiation of several cell types, but also by complex morphogenetic changes.

  • * To whom correspondence should be addressed. E-mail: pfrancis{at}


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