- Email: [email protected]
Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development
Mechanisms of Development 122 (2005) 625–634 www.elsevier.com/locate/modo
Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development Rick A. Friedmana,*, Linna Makmuraa, Elzbieta Biesiadaa, Xiaobo Wangb, Elizabeth M. Keithleyb a
Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 W. Third Street, Los Angeles, CA 90057, USA b Division of Otolaryngology, UCSD School of Medicine, 9500 Gilman Dr, La Jolla, CA 92093, USA Received 2 November 2004; received in revised form 22 December 2004; accepted 22 December 2004 Available online 8 January 2005
Abstract Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1K/K inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Eya1; Ngn1; NeuroD; BDNF; NT-3; Tbx1; Otic vesicle; Cochleovestibular ganglion; Neurogenesis
1. Introduction The development of the mammalian ear is directed by a series of tissue movements and inductive interactions that promote spatial patterning and cellular differentiation (Noden and Van de Water, 1992). The identification of genes involved in these processes is facilitating our understanding of the complexity of ear development and the molecular pathogenesis of some forms of genetic hearing loss. Genetic hearing loss can be divided into two broad classes: non-syndromic (occurring in isolation) and syndromic (associated with other abnormalities). A number of forms of syndromic hearing loss demonstrate anomalies of one or more compartments of the ear (outer, middle, and inner), suggesting an early developmental effect (Kalatzis et al., 1998). One such autosomal dominant form, BranchioOto-Renal (BOR) syndrome (MIM 113650), has been well described clinically and genetically.
* Corresponding author. Tel.: C1 213 273 8078; fax: C1 213 273 8088. E-mail address: [email protected] (R.A. Friedman). 0925-4773/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2004.12.011
Although the association of branchial arch anomalies and hearing loss has been recognized for nearly a century, it has only been in the last 25 years that BOR syndrome has been defined. Melnick et al. reported a father and three of six children with mixed hearing loss, abnormally cupped pinnae, preauricular pits, branchial cleft fistulae and renal anomalies (Melnick et al., 1975). BOR syndrome is an autosomal dominant disorder with incomplete penetrance and variable expressivity. Recently, mutations in the human homologue of the Drosophila eyes absent gene (EYA1) were identified in BOR patients (Abdelhak et al., 1997). EYA genes encode a family of proteins (EYA1–4) that are highly conserved in the animal kingdom. The EYA proteins are characterized by a carboxyterminal domain (ED), necessary for cooperative co-factor binding, and a PST rich carboxy-terminal trans-activating domain (Xu et al., 1997). In Drosophila, eyes absent (eya), has been shown to interact synergistically, in a regulatory network including eyeless, sine oculis and dachshund, during compound eye morphogenesis (for reviews see Desplan (1997), and Wawersik and Maas (2000)). The vertebrate homologues of ey, eya, so, and dac, Pax, Eya, Six,
626
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
and Dach, have been characterized and several studies have demonstrated that this regulatory network is conserved in vertebrate eye, limb, kidney, and ear development (Heanue et al., 1999, 2002; Torres et al., 1996; Xu et al., 1999). It has recently been demonstrated that Eya possesses intrinsic phosphatase activity that, when complexed with Dach, Six1 and CBP, permits the expression of target genes essential for precursor cell proliferation and survival (Li et al., 2003; Rayapureddi et al., 2003; Tootle et al., 2003). The effects of mutations in the mouse orthologue, Eya1, on inner ear development have been described by us and others (Johnson et al., 1999; Xu et al., 1999). Targeted deletion of Eya1 in mice suggests that it is involved in the early inductive events of inner ear morphogenesis, including cochleovestibular ganglion (cvg) neurogenesis (Xu et al., 1999). We have described a hypomorphic allele (Eya1bor/bor) in which the adult homozygous phenotype consists of severe cochlear dysmorphogenesis and absence of the cochlear and vestibular nerves (Johnson et al., 1999). Neural progenitor specification is one of the earliest fate determination steps in the developing ear. The Atonalrelated basic helix-loop-helix genes, Neurogenin1 (Ngn1) and NeuroD are essential positive regulators of cvg neurogenesis (Ma et al., 2000). Despite our increasing understanding of the genes governing regional and cellular specification in the developing inner ear, little is known of the genetic mechanisms underlying the regulation of these factors. Targeted and naturally occurring mutations in Eya1 provide excellent model systems for the study of these early genetic events in the developing ear, particularly the process of neural precursor specification. In this manuscript, utilizing the null and hypomorphic Eya1 alleles, we demonstrate that Eya1 influences cvg neurogenesis throughout development through its effects on the expression of Tbx1, Ngn1, NeuroD, BDNF and NT-3.
2. Results 2.1. Eya1 mutant mice display alterations in the expression of proneural genes Ngn1 and NeuroD in a dose dependent fashion Recent evidence suggests that both Eya1 and Six1, as part of a conserved genetic network, are involved in cell fate specification within the otocyst epithelium (Zheng et al., 2003). Early in inner ear development, Eya1 is expressed in the otic placode (E9) and is regionalized to the ventral otocyst (E10.5) (Kalatzis et al., 1998). Evidence from our laboratory and others supports a role for Eya1 in inner ear development. Targeted deletion of Eya1 led to absence of cvg morphogenesis and inner ear developmental arrest at the otocyst stage (E10.5) (Xu et al., 1999). Deficient levels of Eya1 (Eya1bor/bor) led to a number of morphogenetic abnormalities (Johnson et al., 1999). Whole mount preparations of the hypomorphic ear revealed subtle
abnormalities of the pars superior, or vestibular portion. Specifically, the lateral semicircular canal, the last to appear developmentally, is foreshortened with a much narrower diameter than that of the wild-type. Several of the postnatal inner ears studied also revealed an incomplete common crus, the region of the joined non-ampullated ends of the superior and posterior semicircular canals. The abnormalities of the pars inferior, or cochlear portion of the inner ear, were the most severe and constant. All but the most basal one-quarter of the cochlea was absent in the adult mutant inner. Histological analysis demonstrated the rudimentary basal portion of the mutant cochlea with a spiral ligament and no overlying stria vascularis. Additionally, there was complete absence of the organ of Corti, and cochlear and vestibular nerves in the adult hypomorphs. To explore the mechanisms underlying failed neurogenesis in the inner ears of the Eya1 mutants we examined the expression of Ngn1 and NeuroD in Eya1 null (Eya1K/K) and deficient (Eya1bor/bor) mice. Ngn1 (Fig. 1B) and NeuroD (Fig. 1E) transcripts were completely absent in the otic vesicles of E10.5 Eya1K/K embryos in contrast to the their expression in the ventral otic epithelium and delaminating neuroblasts of the wild-type mice (Fig. 1A,D). These data suggest that Eya1, either directly or through the action of another control gene(s), regulates the expression of these proneural genes and/or the specification of the neural precursors in the otocyst that normally express them. As mentioned above, Eya1 expression begins early in the otic placode stage (E8.5–E9). Its expression persists in the otic epithelium and cvg throughout development suggesting an ongoing effect of Eya1 on spiral and vestibular ganglion survival (Kalatzis et al., 1998). We have shown that mice homozygous for a hypomorphic allele of Eya1 (Eya1bor/bor) have a less severe inner ear defect than that present in the null. Cochlear morphogenetic arrest occurs at approximately E12, and the spiral and vestibular ganglion cells are absent in the adult. We hypothesized that cvg developmental arrest would occur at a later stage in the Eya1bor/bor mice than in the Eya1K/K, and that the Eya1bor/bor mice would provide insights into the ongoing effects of Eya1 on cvg development. To investigate the developmental basis for the reduction of the cvg in the Eya1bor/bor adult mice, we surveyed the expression of these same proneural markers at the time of neuroblast specification and delamination (E10.5). Ngn1 expression in the Eya1bor/bor otic vesicle was reduced in the otic epithelium (Fig. 1C) in comparison to the wild-type (Fig. 1A). Similarly, NeuroD expression in the Eya1bor/bor inner ears was detected at reduced levels overall in comparison to the wild-type. Furthermore, much of the NeuroD expression in the hypomorph was confined to the otic epithelium at this stage, with few NeuroD-positive delaminated cells in the cvg rudiment (Fig. 1F) compared to the wild-type (Fig. 1D).
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
627
Fig. 1. Ngn1 and NeuroD expression in the E10.5 otocyst detected by in situ hybridization in wild-type (nZ5), Eya1K/K (nZ5,7, respectively) and Eya1bor/bor (nZ5). Note the ventral epithelial expression in the wild-type (A) and the absence and reduction of expression in the Eya1K/K (B) and Eya1bor/bor (C) otocysts, respectively. NeuroD expression is reduced in the Eya1bor/bor otocyst and fewer delaminated neural precursors are present.
2.2. Eya1bor/bor inner ears have fewer cells committed to the neuroblast lineage To quantitate numbers of neural precursors and young neurons in Eya1bor/bor inner ears, we examined expression of the nuclear factor Islet1. Eya1bor/bor inner ears demonstrated significantly fewer cells staining for the neuroblast lineage marker Islet1 at E10.5. Qualitatively, the volume of the cvg was consistently reduced in the hypomorphs in comparison to the controls (Fig. 2). Using an image analysis
system we counted the Islet1 positive cells from four wildtype and six Eya1bor/bor ears at E10.5. The number of Islet1 positive cells in the Eya1bor/bor otocysts was approximately 20% of the controls (Fig. 3, t-test, 2-tailed distribution, 2-sample unequal variance, PZ0.01). From these data it is clear that deficient but not absent Eya1 mRNA levels result in a reduced population of committed neuroblasts in the developing ear. These findings are an extension of those in the Eya1K/K inner ears and demonstrate a dose-dependent effect of Eya1 on
Fig. 2. Islet-1 immunostaining in the E10.5 otocyst of wild-type (A) and Eya1bor/bor (B) mice. This marker of developing neuroblasts demonstrates a significantly reduced population of cells committed to the neural lineage in the mutant inner ears.
628
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Fig. 3. Histogram demonstrating the significant reduction of committed neuroblasts in the Eya1bor/bor otocyst epithelium.
the neuroblast lineage possibly as a controller gene upstream of Ngn1 and NeuroD. 2.3. Abnormal cell death likely contributes to the early and late cvg loss in Eya1bor/bor mice The reduction of neural precursor numbers described above cannot, by itself, account for the absence of cochlear and vestibular neurons in the adult Eya1bor/bor mice. Complete loss of Eya1 is associated with abnormal cell death in the developing otic epithelium (Xu et al., 1999). Six1, a known Eya1 cofactor in inner ear development, is also required for otocyst epithelial cell proliferation and survival (Zheng et al., 2003). We began by exploring the potential role for impaired otocyst epithelial proliferation in the Eya1bor/borcvg phenotype. BrdU incorporation in the otocyst epithelium of the Eya1bor/bor otocysts (E10.5) was not different from the wild-type suggesting abnormal cell death as the predominant mechanism for progressive cvg cell loss. TUNEL staining was used to identify whether precursors to the cochlear and spiral ganglion cells are undergoing enhanced apoptosis. Sections containing the otic pit (E9.5) or the otocyst (E10.5–14.5) and the adjacent cvg were evaluated for TUNEL labeled cells. Wild-type (Eya1C/C) mouse embryos (nZ14, 9.5–14.5 embryonic days) did not contain labeled cells in auditory
precursor structures with the exception of one 12.5-day-old embryo that did have five labeled cells in the cvg (Fig. 4). In Eya1bor/bor, at E9.5, five labeled cells/section (nZ2) were seen among the epithelial cells of the otic pit and the ectodermal cells adjacent to the pit. As the pit became invaginated to form the otocyst (E10.5 days, nZ3), a mean of two labeled cells/section were seen among the epithelial cells. Between 0 and 30 TUNEL-positive cells/section were present within the cvg of these embryos. Between E12.5 and E14.5 (nZ8 embryos) 0–75 labeled cells/section were identified on the medial side of the otocyst. At E14.5, the otocyst begins to form a thickening and out-pouching that will develop into the coiled cochlea. Many of these labeled cells were within and adjacent to the thickening. These embryos also had between 0 and 80 TUNEL-positive cells/section among the cvg. The older embryos had more labeled cells than the younger ones. TUNEL positive cells were also present within the facioacoustic neuronal complex in Eya1bor/bor embryos at E10.5 that is beginning to develop at that time. The number of labeled cells within this complex increased with embryonic age. As the three ganglia begin to separate from each other, the label remains mostly in the cochlear ganglion, but is also present in the vestibular ganglion. Other structures containing 1–2 labeled cells/section were the trigeminal ganglion and mesoderm adjacent to the optic stalk. Cells in positive control sections exposed to TACS nuclease prior to the TUNEL assay were all labeled. Cells in negative control sections in which PBS was used in place of the terminal transferase enzyme were not labeled. The continued cvg cell loss resulting from abnormal apoptosis was reminiscent of the developmental abnormalities reported in the NeuroD mutant cvg (Liu et al., 2000). We therefore hypothesized a similar mechanism for a continued wave of neuronal cell death in the hypomorph, a lack of neurotrophic support. 2.4. Lack of neurotrophic support and consequent cvg neuronal cell death contributes to the adult phenotype of the Eya1bor/bor inner ear Both BDNF and NT-3 are expressed in the epithelia of the inner ear throughout development and into the postnatal period (Ernfors et al., 1995; Farinas et al., 2001; Fritzsch et al., 1997). Their receptors, TrkB and TrkC show overlapping temporal expression in the developing cvg (Ernfors et al., 1995; Farinas et al., 2001). Repeated survey of the expression of TrkB and TrkC in the delaminated cvg early in development (E10.5–11.5) revealed no qualitative differences between the Eya1bor/bor mice and the wildtype. In contrast to their receptors, the expression of both BDNF and NT-3 were greatly reduced throughout the inner ears of the Eya1bor/bor mice at E10.5 through E14.5. At E10.5, NT-3 expression in the ventrolateral domain of the Eya1bor/bor otocysts was reduced (Fig. 5B) compared to the control (Fig. 5A). BDNF expression was similarly reduced
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
629
Fig. 4. Hematoxylin and eosin stained sections of the E12.5 wild-type (A, B) and Eya1bor/bor (C, D) inner ears. Note the presence of cvg neuroblasts in the Eya1bor/bor inner ear (B). TUNEL-labeled cells were identified in the ventrolateral otic epithelium and cvg throughout development in the Eya1bor/bor mice (D). Scale barZ100 mm in (A and C), and 50 mm in (B and D); sa: sacculus.
in both the anterior and posterior regions of the Eya1bor/bor otocysts (Fig. 6B,D) compared to the controls (Fig. 6A,C). At E12.5, little to no NT-3 expression was observed in the developing cochlear epithelium of the Eya1bor/bor mice
(Fig. 5D,F,H) in contrast to robust expression in the controls (Fig. 5C,E,G). BDNF expression was reduced in the developing vestibular sensory epithelia (crista ampullaris) of the Eya1bor/bor mice (Fig. 6F,H,J) compared to
Fig. 5. NT-3 expression detected by in-situ hybridization in the E10.5 and E12.5 mouse otocyst (nZ6). NT-3 expression is absent ventrally in the Eya1bor/borotocyst (A, B). At E12.5, the time of cochlear morphogenetic arrest in the Eya1bor/bor mouse, reduced expression is evident in the ventral structures of the Eya1bor/bor inner ears in comparison to controls at the level of the sacculus (C, D), the cochlear base (E, F) and the cochlear hook region (G, H).
630
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Fig. 6. BDNF expression detected by in-situ hybridization in the E10.5 and E12.5 mouse otocyst (nZ8). BDNF expression is reduced in the ventral domain of the Eya1bor/bor otocyst (A, B). Expression is reduced in the dorsal structures of the Eya1bor/bor inner ears in comparison to controls.
the controls (Fig. 6E,G,I). At later stages (E14.5) both vestibular and cochlear epithelial BDNF expression was reduced in the Eya1bor/bor mice (data not shown). Similarly, NT-3 expression was greatly reduced in the cochlear epithelium of the Eya1bor/bor mice when compared to the control, a finding partially explained by the reduced cochlear duct size in these animals at E14.5 (data not shown). 2.5. Absent and deficient levels of Eya1 alter the expression of Tbx1 Tbx1 is a negative regulator of Ngn1 and NeuroD expression in the otic epithelium and thereby controls the
numbers of cvg neural precursors and the size of the cvg rudiment. To investigate a role for Tbx1 in the cvg abnormalities in the Eya1 mutants, we analyzed its expression in Eya1K/K and Eya1bor/bor ears (Fig. 7). At E10.5 Tbx1 expression is in the anterior otocyst confined to a lateral domain with a sharp border at the level of the anterior cardinal vein (Fig. 7A). In the Eya1K/K otocyst, this regionalization is lost and Tbx1 expression extends ventrally and medially within the epithelium (Fig. 7B). The sharp regionalization was altered to a lesser degree in the Eya1bor/bor mice; however, more ventral expression can be detected in these mice when compared to controls (Fig. 7C, see arrowhead). This alters the normally complimentary expression patterns of Tbx1 and
Fig. 7. Tbx1 expression in the E10.5 otocysts of wild-type (A), Eya1K/K (B) and Eya1bor/bor (C) mice detected by in situ hybridization. Laterally regionalized expression of Tbx1 in the anterior otocyst of normals (A) shifts ventrally in the Eya1K/K otocyst and, to a lesser degree, in the Eya1bor/bor otocyst at this early stage.
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Ngn1/NeuroD in this location providing a potential mechanism for the neural suppression seen in the Eya1 null and hypomorphic mutants.
3. Discussion The mouse inner ear begins its development as a thickening of surface ectoderm called the otic placode (E8.5), which invaginates to form the otic cup (E9). The otic cup then pinches off into the surrounding mesenchyme to form the otic vesicle (E9.5). During the next several days, the epithelium of the otocyst undergoes significant proliferation and differentiation, ultimately establishing the sensory and supporting cells of the auditory and vestibular systems, as well as the cochleovestibular ganglion (cvg) (D’AmicoMartel and Noden, 1983). Several lines of evidence support the existence of compartmental boundaries of gene expression within the otocyst governing the divergence of epithelial cell lineages (Fekete and Wu, 2002). Examples include the expression of Dlx5 in the dorsal epithelium of the otocyst and its responsibility for development of the semicircular canals and the expression of Otx1 in the ventral otocyst and its essential role in cochlear morphogenesis (Acampora et al., 1996; Depew et al., 1999). Specification of neural progenitors is the earliest identifiable fate determination event in the developing otocyst, beginning around E9. This subpopulation of ventral otic epithelial cells is identifiable by their expression of the Atonal-related basic helix-loop-helix (Cau et al., 1997) genes, Neurogenin1 (Ngn1) and NeuroD. Ngn1 is necessary for neural progenitor determination and formation of the cvg. Supporting its role in inner ear development, studies in Ngn1 deficient mice show complete absence of the cvg (Ma et al., 1998, 2000). Ngn1 regulates another gene in this cascade, NeuroD. It is expressed in a spatially and temporally overlapping pattern with Ngn1 and promotes neuroblast delamination into the ventral mesenchyme and growth factor mediated neuronal survival (Kim et al., 2001; Liu et al., 2000). Tbx1 has recently been shown to act upstream of Ngn1 and NeuroD as a negative regulator of neural fate specification in the otocyst (Raft et al., 2004). The cvg initially forms as one aggregate, but by E11.5 it begins to separate into a medial cochlear portion and a lateral vestibular portion. At E13.5, the primordial spiral ganglion terminates in the structures of the pars inferior, the saccule and the newly forming cochlea (one-half turn) (Altman and Bayer, 1982). It is at approximately this developmental period that cochlear and vestibular neurons are penetrating the epithelia of their respective end organs (Galinovic-Schwartz et al., 1991). This process is highly dependent upon the neurotrophins, BDNF and NT-3 (Ernfors et al., 1995; Fritzsch, 2003).
631
BDNF and NT-3 are expressed in the otic epithelium throughout development in largely non-overlapping domains with no known connection to Ngn1 or NeuroD. BDNF and NT-3 expression can also be detected outside of the sensory epithelium in delaminating cells suggesting an early source of neurotrophic support for the forming cvg cells (Farinas et al., 2001). BDNF and NT-3 deficient mice begin to show loss of vestibular and cochlear ganglion fibers, respectively, after E13 suggesting a critical time-point of cvg dependence on neurotrophins at or prior to this time (Ernfors et al., 1995; Farinas et al., 2001). Eya1 and Six1 are expressed in a spatially and temporally overlapping pattern throughout inner ear development (Kalatzis et al., 1998; Ozaki et al., 2004). A genetic interaction between Eya1 and Six1 has been established in the developing inner ear of mice and humans (BOR syndrome) (Ruf et al., 2004; Zheng et al., 2003). Furthermore, targeted deletion of Eya1 and Six1 result in early developmental arrest of the otocyst with no identifiable cvg (Ozaki et al., 2004; Xu et al., 1999). Increased apoptosis in the Eya1K/K mice and a combination of abnormal cell death and proliferation in the Six1K/K mice is the underlying cellular mechanism for the phenotype. The molecular mechanisms underlying failed neurogenesis in these mutants has, to date, remained a mystery. The expression of Fgf3 and Lfng, both markers of inner ear sensory precursors and neuroblasts is altered in both Eya1K/K and Six1K/K mice (Ozaki et al., 2004; Xu et al., 1999). Fgf3, Lfng, Ngn1 and NeuroD have overlapping expression with Eya1 and Six1 in the developing otocyst (Mansour et al., 1993; Morsli et al., 1998). Tbx1, a suppressor of neural fate in the inner ear has complimentary expression to these proneural genes and its regionalized expression posterolaterally in the early otocyst begins at the lateral border of proneural gene expression (Raft et al., 2004). We have shown that with absent or reduced levels of Eya1 expression, Tbx1 regionalization is altered leading to more ventral otocyst expression and either absent (Eya1K/K) or reduced (Eya1bor/bor) numbers of cells fated for neurogenesis. In the Eya1bor/bor mice the reduced population of neural precursors is followed by a second wave of cvg cell loss. Abnormal apoptosis occurs in association with a reduction in BDNF and NT-3 expression in the mutant inner ears beginning at the earliest time points (E10.5) and continuing on through spiral and vestibular ganglion development. A similar second wave of cvg loss was reported in NeuroD deficient mice in association with reduced neurotrophin receptor expression (TrkB and TrkC) (Liu et al., 2000). This supports an ongoing effect of Eya1 signaling regulating the levels of expression of these neurotrophins either directly or indirectly and provides a mechanism for the cvg cell death in the Eya1bor/bor mice leading to the adult phenotype.
632
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
4. Experimental procedures 4.1. Animals and genotyping The EyaC/K animals were kindly provided by Richard Maas (Harvard Medical School). For the staging of embryos, midday of the day of the vaginal plug was considered as E0.5. Embryos were harvested at E10.5. The EyaC/bor animals were obtained from Jackson Laboratories. Genotype was determined using the following PCR primers: KO-Fwd: 5 0 -TTTGGTGTGTGGTTCGGCAAGCAAT30; KO-wt-Rev: 5 0 -CATCATCTATATGGACTTGGTCACACT-3 0 ; KO-mut-Rev: 5 0 -ATCTCTCGTGGGATCATTGTTT TTCTA-3 0 ; Bor-wt-Fwd: 5 0 -TAAGCAGACCTGACAAGAGGCATTGTTT-3 0 ; Bor-mut-Fwd: 5 0 -GAACGCGTCGAATAACAGGTAGGAAAGT-3 0 ; Bor-Rev: 5 0 -TTGGGTCCTAGGTCTACCTTATCCACCAC-3 0 . 4.2. Probes Protein sequences of Neurogenin1 (Ngn1) and NeuroD are encoded in a single exon (Cau et al., 1997; Oda et al., 2000). A fragment of 352 bp of Ngn1 (containing the 5 0 UTR and the first 88 amino acids, excluding the bHLH domain) and 282 bp of NeuroD (containing the first 94 amino acids, excluding the bHLH domain) were generated by PCR from the mouse genomic DNA with the primers: Ngn1: 5 0 -TAGCCCCGGGTCGTACGGACAGTAA-3 0 and 5 0 -AGTGCAGCACCTCGGA-3 0 ; NeuroD: 5 0 -ATGACCAAATCGTACAGCGAGAGT3 0 , and 5 0 -AGCCTTAGTCATCTTCTTC-3 0 . BDNF, NT-3, trkB, and trkC probes were directly transcribed from the cloned plasmids supplied by Mary Ellen Palko from the Neural Development Group of the Department of Health and Human Services. 4.3. In situ hybridization In situ hybridization on tissue sections was performed with modifications as described (Groves and BronnerFraser, 2000). Briefly, antisense digoxigenin-labeled RNA probes were synthesized according to the Riboprobew System protocol (Promega Co., Madison, WI). Sections were treated with 2 mg/ml proteinase K in PK buffer (pH 7.5) at room temperature for 10 min. The tissue sections were then delipidated and dehydrated before hybridization. Hybridization with digoxigenin-labeled riboprobes was
performed at 60 8C overnight in a hybridization buffer solution. After hybridization, sections were incubated in 20% heat-inactivated sheep serum in PBT to block nonspecific binding sites. To visualize the hybrids, the sections were incubated with an anti-digoxigenin antibody (conjugated with alkaline phosphatase). The slides were developed in the dark for 24 h at room temperature after the addition of a chromogenic substrate, BCIP using NBT as a catalyst. Three to five embryos of each genotype were analyzed for every probe. 4.4. Immunohistochemistry Embryos were fixed in 4% PFA in PBS at 4 8C overnight, then in 30% sucrose/PBS solution at 4 8C until the embryos sink down to the bottom of the tube. Embryos were then embedded and freeze in OCT. Compound by Tissue-Tekw and were cryo-sectioned into 20 mm thick serial sections. Sections containing the otic pit or cyst and the cochleovestibular ganglion were air dried onto coated slides. Sections were incubated in 10% sheep serum in PBT at room temperature for 1 h to block non-specific binding sites, and then they were incubated in Islet1 primary antibody diluted in blocking solution at 4 8C overnight. The next day the sections were washed three times for 10 min each in PBT. Then, the sections were incubated with Rhodamine Rede fluorescent secondary antibody diluted in blocking solution at room temperature for 45 min. Sections were then washed three times for 5 min each in PBT, and a final rinse for 5 min with water before view. 4.5. TUNEL Apoptotic cells were identified in cryostat sections (6 mm) of embryos aged 9.5–14.5 days using TUNEL assay (Trevigen, Gaithersburg, MD, TACSe in situ apoptosis detection kit, ca. 4810–30-k). The whole embryo was immersion fixed in 4% buffered paraformaldehyde, washed in phosphate-buffered saline and then saturated with 30% sucrose overnight. The tissue was embedded in OCT for cryo-sectioning in the horizontal plane. Sections containing the otic pit or cyst and the cochleovestibular ganglion were air dried onto coated slides and subsequently hydrated in PBS and exposed to proteinase K (0.02 mg/ml) to digest DNA binding proteins. Hydrogen peroxide was used to inactivate endogenous peroxidases. The sections were then incubated according to the kit instructions with a mixture of terminal deoxynucleotidyl transferase (TdT), biotinylated dUTP, and divalent cation for 1 h at 37 8C. Streptavidin–horseradish peroxidase and diaminobenzidine (DAB) were used to detect labeled DNA fragments. The tissue was not counter-stained. Adjacent sections were stained with hematoxylin and eosin. Controls, run with each assay, included wild-type and heterozygote embryonic sections. The positive reagent control was pre-incubation
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
of sections with TACS-nuclease that results in label of all nuclei and a negative reagent control was incubation with PBS in place of TdT.
Acknowledgements We wish to thank Dr Richard Maas for the Eya1 mutant mice, Drs Andy Groves and Steven Raft for their thoughtful comments, Kate Blair for her technical assistance, and Mary Ellen Palko and Lino Tessarollo for the BDNF, NT-3, trkB, and trkC probes. This work was supported by NIDCD DCO4796 (R.A.F.) and the Research Service of the Department of Veterans Affairs (E.M.K.).
References Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C., et al., 1997. A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nat. Genet. 15, 157–164. Acampora, D., Mazan, S., Avantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., et al., 1996. Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat. Genet. 14, 218–222. Altman, J., Bayer, S.A., 1982. Development of the cranial nerve ganglia and related nuclei in the rat. Adv. Anat. Embryol. Cell Biol. 74, 1–90. Cau, E., Gradwohl, G., Fode, C., Guillemot, F., 1997. Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors. Development 124, 1611–1621. D’Amico-Martel, A., Noden, D.M., 1983. Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am. J. Anat. 166, 445–468. Depew, M.J., Liu, J.K., Long, J.E., Presley, R., Meneses, J.J., Pedersen, R.A., Rubenstein, J.L., 1999. Dlx5 regulates regional development of the branchial arches and sensory capsules. Development 126, 3831–3846. Desplan, C., 1997. Eye development: governed by a dictator or a junta? Cell 91, 861–864. Ernfors, P., Van De, W.T., Loring, J., Jaenisch, R., 1995. Complementary roles of BDNF and NT-3 in vestibular and auditory development. Neuron 14, 1153–1164. Farinas, I., Jones, K.R., Tessarollo, L., Vigers, A.J., Huang, E., Kirstein, M., et al., 2001. Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression. J. Neurosci. 21 (16), 6170–6180. Fekete, D.M., Wu, D.K., 2002. Revisiting cell fate specification in the inner ear. Curr. Opin. Neurobiol. 12 (1), 35–42. Fritzsch, B., 2003. Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia. Brain Res. Bull. 60 (5–6), 423–433. Fritzsch, B., Silos-Santiago, I., Bianchi, L.M., Farinas, I., 1997. The role of neurotrophic factors in regulating the development of inner ear innervation. Trends Neurosci. 20, 159–164. Galinovic-Schwartz, V., Peng, D., Chiu, F.C., Van de Water, T.R., 1991. Temporal pattern of innervation in the developing mouse inner ear: an immunocytochemical study of a 66-kD subunit of mammalian neurofilaments. J. Neurosci. Res. 30, 124–133. Groves, A.K., Bronner-Fraser, M., 2000. Competence, specification and commitment in otic placode induction. Development 127, 3489–3499. Heanue, T.A., Reshef, R., Davis, R.J., Mardon, G., Oliver, G., Tomarev, S., et al., 1999. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. Genes Dev. 13, 3231–3243.
633
Heanue, T.A., Davis, R.J., Rowitch, D.H., Kispert, A., McMahon, A.P., Mardon, G., Tabin, C.J., 2002. Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes. Mech. Dev. 111 (1–2), 75–87. Johnson, K.R., Cook, S.A., Erway, L.C., Matthews, A.N., Sanford, L.P., Paradies, N.E., Friedman, R.A., 1999. Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eya1 gene in a mouse model of BOR syndrome. Hum. Mol. Genet. 8, 645–653. Kalatzis, V., Sahly, I., El Amraoui, A., Petit, C., 1998. Eya1 expression in the developing ear and kidney: towards the understanding of the pathogenesis of Branchio-Oto-Renal (BOR) syndrome. Dev. Dyn. 213, 486–499. Kim, W.Y., Fritzsch, B., Serls, A., Bakel, L.A., Huang, E.J., Reichardt, L.F., et al., 2001. NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development 128 (3), 417–426. Li, X., Oghi, K.A., Zhang, J., Krones, A., Bush, K.T., Glass, C.K., et al., 2003. Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426 (6964), 247–254. Liu, M., Pereira, F.A., Price, S.D., Chu, M.J., Shope, C., Himes, D., et al., 2000. Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev. 14 (22), 2839– 2854. Ma, Q., Chen, Z., del, B.B.I., de la Pompa, J.L., Anderson, D.J., 1998. Neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20, 469–482. Ma, Q., Anderson, D.J., Fritzsch, B., 2000. Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. J. Assoc. Res. Otolaryngol. 1 (2), 129– 143. Mansour, S.L., Goddard, J.M., Capecchi, M.R., 1993. Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear. Development 117, 13–28. Melnick, M., Bixler, D., Silk, K., Yune, H., Nance, W.E., 1975. Autosomal dominant branchiootorenal dysplasia. Birth Defects Orig. Artic. Ser. 11, 121–128. Morsli, H., Choo, D., Ryan, A., Johnson, R., Wu, D.K., 1998. Development of the mouse inner ear and origin of its sensory organs. J. Neurosci. 18, 3327–3335. Noden, D.M., Van de Water, T.R., 1992. Genetic analyses of mammalian ear development. Trends Neurosci. 15, 235–237. Oda, H., Iwata, I., Yusanami, M., Ohkubo, H., 2000. Structure of the mouse NDRF gene and its regulation during neuronal differentiation on P19 cells. Brain Res. Mol. Brain Res. 77 (1), 37–46. Ozaki, H., Nakamura, K., Funahashi, J., Ikeda, K., Yamada, G., Tokano, H., et al., 2003. Six1 controls patterning of the mouse otic vesicle. Development 131 (3), 551–562. Raft, S., Nowotschin, S., Liao, J., Morrow, B.E., 2004. Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development 131 (8), 1801–1812. Rayapureddi, J.P., Kattamuri, C., Steinmetz, B.D., Frankfort, B.J., Ostrin, E.J., Mardon, G., Hegde, R.S., 2003. Eyes absent represents a class of protein tyrosine phosphatases. Nature 426 (6964), 295–298. Ruf, R.G., Xu, P.X., Silvius, D., Otto, E.A., Beekmann, F., Muerb, U.T., et al., 2004. SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes. Proc. Natl Acad. Sci. USA 101 (21), 8090–8095. Tootle, T.L., Silver, S.J., Davies, E.L., Newman, V., Latek, R.R., Mills, I.A., et al., 2003. The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426 (6964), 299–302. Torres, M., Gomez-Pardo, E., Gruss, P., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122, 3381–3391.
634
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Wawersik, S., Maas, R.L., 2000. Vertebrate eye development as modeled in Drosophila. Hum. Mol. Genet. 9 (6), 917–925. Xu, P.X., Woo, I., Her, H., Beier, D.R., Maas, R.L., 1997. Mouse Eya homologues of the Drosophila eyes absent gene require Pax6 for expression in lens and nasal placode. Development 124, 219–231.
Xu, P.X., Adams, J., Peters, H., Brown, M.C., Heaney, S., Maas, R., 1999. Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23, 113–117. Zheng, W., Huang, L., Wei, Z.B., Silvius, D., Tang, B., Xu, P.X., 2003. The role of Six1 in mammalian auditory system development. Development 130 (17), 3989–4000.
Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development Rick A. Friedmana,*, Linna Makmuraa, Elzbieta Biesiadaa, Xiaobo Wangb, Elizabeth M. Keithleyb a
Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 W. Third Street, Los Angeles, CA 90057, USA b Division of Otolaryngology, UCSD School of Medicine, 9500 Gilman Dr, La Jolla, CA 92093, USA Received 2 November 2004; received in revised form 22 December 2004; accepted 22 December 2004 Available online 8 January 2005
Abstract Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1K/K inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Eya1; Ngn1; NeuroD; BDNF; NT-3; Tbx1; Otic vesicle; Cochleovestibular ganglion; Neurogenesis
1. Introduction The development of the mammalian ear is directed by a series of tissue movements and inductive interactions that promote spatial patterning and cellular differentiation (Noden and Van de Water, 1992). The identification of genes involved in these processes is facilitating our understanding of the complexity of ear development and the molecular pathogenesis of some forms of genetic hearing loss. Genetic hearing loss can be divided into two broad classes: non-syndromic (occurring in isolation) and syndromic (associated with other abnormalities). A number of forms of syndromic hearing loss demonstrate anomalies of one or more compartments of the ear (outer, middle, and inner), suggesting an early developmental effect (Kalatzis et al., 1998). One such autosomal dominant form, BranchioOto-Renal (BOR) syndrome (MIM 113650), has been well described clinically and genetically.
* Corresponding author. Tel.: C1 213 273 8078; fax: C1 213 273 8088. E-mail address: [email protected] (R.A. Friedman). 0925-4773/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2004.12.011
Although the association of branchial arch anomalies and hearing loss has been recognized for nearly a century, it has only been in the last 25 years that BOR syndrome has been defined. Melnick et al. reported a father and three of six children with mixed hearing loss, abnormally cupped pinnae, preauricular pits, branchial cleft fistulae and renal anomalies (Melnick et al., 1975). BOR syndrome is an autosomal dominant disorder with incomplete penetrance and variable expressivity. Recently, mutations in the human homologue of the Drosophila eyes absent gene (EYA1) were identified in BOR patients (Abdelhak et al., 1997). EYA genes encode a family of proteins (EYA1–4) that are highly conserved in the animal kingdom. The EYA proteins are characterized by a carboxyterminal domain (ED), necessary for cooperative co-factor binding, and a PST rich carboxy-terminal trans-activating domain (Xu et al., 1997). In Drosophila, eyes absent (eya), has been shown to interact synergistically, in a regulatory network including eyeless, sine oculis and dachshund, during compound eye morphogenesis (for reviews see Desplan (1997), and Wawersik and Maas (2000)). The vertebrate homologues of ey, eya, so, and dac, Pax, Eya, Six,
626
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
and Dach, have been characterized and several studies have demonstrated that this regulatory network is conserved in vertebrate eye, limb, kidney, and ear development (Heanue et al., 1999, 2002; Torres et al., 1996; Xu et al., 1999). It has recently been demonstrated that Eya possesses intrinsic phosphatase activity that, when complexed with Dach, Six1 and CBP, permits the expression of target genes essential for precursor cell proliferation and survival (Li et al., 2003; Rayapureddi et al., 2003; Tootle et al., 2003). The effects of mutations in the mouse orthologue, Eya1, on inner ear development have been described by us and others (Johnson et al., 1999; Xu et al., 1999). Targeted deletion of Eya1 in mice suggests that it is involved in the early inductive events of inner ear morphogenesis, including cochleovestibular ganglion (cvg) neurogenesis (Xu et al., 1999). We have described a hypomorphic allele (Eya1bor/bor) in which the adult homozygous phenotype consists of severe cochlear dysmorphogenesis and absence of the cochlear and vestibular nerves (Johnson et al., 1999). Neural progenitor specification is one of the earliest fate determination steps in the developing ear. The Atonalrelated basic helix-loop-helix genes, Neurogenin1 (Ngn1) and NeuroD are essential positive regulators of cvg neurogenesis (Ma et al., 2000). Despite our increasing understanding of the genes governing regional and cellular specification in the developing inner ear, little is known of the genetic mechanisms underlying the regulation of these factors. Targeted and naturally occurring mutations in Eya1 provide excellent model systems for the study of these early genetic events in the developing ear, particularly the process of neural precursor specification. In this manuscript, utilizing the null and hypomorphic Eya1 alleles, we demonstrate that Eya1 influences cvg neurogenesis throughout development through its effects on the expression of Tbx1, Ngn1, NeuroD, BDNF and NT-3.
2. Results 2.1. Eya1 mutant mice display alterations in the expression of proneural genes Ngn1 and NeuroD in a dose dependent fashion Recent evidence suggests that both Eya1 and Six1, as part of a conserved genetic network, are involved in cell fate specification within the otocyst epithelium (Zheng et al., 2003). Early in inner ear development, Eya1 is expressed in the otic placode (E9) and is regionalized to the ventral otocyst (E10.5) (Kalatzis et al., 1998). Evidence from our laboratory and others supports a role for Eya1 in inner ear development. Targeted deletion of Eya1 led to absence of cvg morphogenesis and inner ear developmental arrest at the otocyst stage (E10.5) (Xu et al., 1999). Deficient levels of Eya1 (Eya1bor/bor) led to a number of morphogenetic abnormalities (Johnson et al., 1999). Whole mount preparations of the hypomorphic ear revealed subtle
abnormalities of the pars superior, or vestibular portion. Specifically, the lateral semicircular canal, the last to appear developmentally, is foreshortened with a much narrower diameter than that of the wild-type. Several of the postnatal inner ears studied also revealed an incomplete common crus, the region of the joined non-ampullated ends of the superior and posterior semicircular canals. The abnormalities of the pars inferior, or cochlear portion of the inner ear, were the most severe and constant. All but the most basal one-quarter of the cochlea was absent in the adult mutant inner. Histological analysis demonstrated the rudimentary basal portion of the mutant cochlea with a spiral ligament and no overlying stria vascularis. Additionally, there was complete absence of the organ of Corti, and cochlear and vestibular nerves in the adult hypomorphs. To explore the mechanisms underlying failed neurogenesis in the inner ears of the Eya1 mutants we examined the expression of Ngn1 and NeuroD in Eya1 null (Eya1K/K) and deficient (Eya1bor/bor) mice. Ngn1 (Fig. 1B) and NeuroD (Fig. 1E) transcripts were completely absent in the otic vesicles of E10.5 Eya1K/K embryos in contrast to the their expression in the ventral otic epithelium and delaminating neuroblasts of the wild-type mice (Fig. 1A,D). These data suggest that Eya1, either directly or through the action of another control gene(s), regulates the expression of these proneural genes and/or the specification of the neural precursors in the otocyst that normally express them. As mentioned above, Eya1 expression begins early in the otic placode stage (E8.5–E9). Its expression persists in the otic epithelium and cvg throughout development suggesting an ongoing effect of Eya1 on spiral and vestibular ganglion survival (Kalatzis et al., 1998). We have shown that mice homozygous for a hypomorphic allele of Eya1 (Eya1bor/bor) have a less severe inner ear defect than that present in the null. Cochlear morphogenetic arrest occurs at approximately E12, and the spiral and vestibular ganglion cells are absent in the adult. We hypothesized that cvg developmental arrest would occur at a later stage in the Eya1bor/bor mice than in the Eya1K/K, and that the Eya1bor/bor mice would provide insights into the ongoing effects of Eya1 on cvg development. To investigate the developmental basis for the reduction of the cvg in the Eya1bor/bor adult mice, we surveyed the expression of these same proneural markers at the time of neuroblast specification and delamination (E10.5). Ngn1 expression in the Eya1bor/bor otic vesicle was reduced in the otic epithelium (Fig. 1C) in comparison to the wild-type (Fig. 1A). Similarly, NeuroD expression in the Eya1bor/bor inner ears was detected at reduced levels overall in comparison to the wild-type. Furthermore, much of the NeuroD expression in the hypomorph was confined to the otic epithelium at this stage, with few NeuroD-positive delaminated cells in the cvg rudiment (Fig. 1F) compared to the wild-type (Fig. 1D).
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
627
Fig. 1. Ngn1 and NeuroD expression in the E10.5 otocyst detected by in situ hybridization in wild-type (nZ5), Eya1K/K (nZ5,7, respectively) and Eya1bor/bor (nZ5). Note the ventral epithelial expression in the wild-type (A) and the absence and reduction of expression in the Eya1K/K (B) and Eya1bor/bor (C) otocysts, respectively. NeuroD expression is reduced in the Eya1bor/bor otocyst and fewer delaminated neural precursors are present.
2.2. Eya1bor/bor inner ears have fewer cells committed to the neuroblast lineage To quantitate numbers of neural precursors and young neurons in Eya1bor/bor inner ears, we examined expression of the nuclear factor Islet1. Eya1bor/bor inner ears demonstrated significantly fewer cells staining for the neuroblast lineage marker Islet1 at E10.5. Qualitatively, the volume of the cvg was consistently reduced in the hypomorphs in comparison to the controls (Fig. 2). Using an image analysis
system we counted the Islet1 positive cells from four wildtype and six Eya1bor/bor ears at E10.5. The number of Islet1 positive cells in the Eya1bor/bor otocysts was approximately 20% of the controls (Fig. 3, t-test, 2-tailed distribution, 2-sample unequal variance, PZ0.01). From these data it is clear that deficient but not absent Eya1 mRNA levels result in a reduced population of committed neuroblasts in the developing ear. These findings are an extension of those in the Eya1K/K inner ears and demonstrate a dose-dependent effect of Eya1 on
Fig. 2. Islet-1 immunostaining in the E10.5 otocyst of wild-type (A) and Eya1bor/bor (B) mice. This marker of developing neuroblasts demonstrates a significantly reduced population of cells committed to the neural lineage in the mutant inner ears.
628
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Fig. 3. Histogram demonstrating the significant reduction of committed neuroblasts in the Eya1bor/bor otocyst epithelium.
the neuroblast lineage possibly as a controller gene upstream of Ngn1 and NeuroD. 2.3. Abnormal cell death likely contributes to the early and late cvg loss in Eya1bor/bor mice The reduction of neural precursor numbers described above cannot, by itself, account for the absence of cochlear and vestibular neurons in the adult Eya1bor/bor mice. Complete loss of Eya1 is associated with abnormal cell death in the developing otic epithelium (Xu et al., 1999). Six1, a known Eya1 cofactor in inner ear development, is also required for otocyst epithelial cell proliferation and survival (Zheng et al., 2003). We began by exploring the potential role for impaired otocyst epithelial proliferation in the Eya1bor/borcvg phenotype. BrdU incorporation in the otocyst epithelium of the Eya1bor/bor otocysts (E10.5) was not different from the wild-type suggesting abnormal cell death as the predominant mechanism for progressive cvg cell loss. TUNEL staining was used to identify whether precursors to the cochlear and spiral ganglion cells are undergoing enhanced apoptosis. Sections containing the otic pit (E9.5) or the otocyst (E10.5–14.5) and the adjacent cvg were evaluated for TUNEL labeled cells. Wild-type (Eya1C/C) mouse embryos (nZ14, 9.5–14.5 embryonic days) did not contain labeled cells in auditory
precursor structures with the exception of one 12.5-day-old embryo that did have five labeled cells in the cvg (Fig. 4). In Eya1bor/bor, at E9.5, five labeled cells/section (nZ2) were seen among the epithelial cells of the otic pit and the ectodermal cells adjacent to the pit. As the pit became invaginated to form the otocyst (E10.5 days, nZ3), a mean of two labeled cells/section were seen among the epithelial cells. Between 0 and 30 TUNEL-positive cells/section were present within the cvg of these embryos. Between E12.5 and E14.5 (nZ8 embryos) 0–75 labeled cells/section were identified on the medial side of the otocyst. At E14.5, the otocyst begins to form a thickening and out-pouching that will develop into the coiled cochlea. Many of these labeled cells were within and adjacent to the thickening. These embryos also had between 0 and 80 TUNEL-positive cells/section among the cvg. The older embryos had more labeled cells than the younger ones. TUNEL positive cells were also present within the facioacoustic neuronal complex in Eya1bor/bor embryos at E10.5 that is beginning to develop at that time. The number of labeled cells within this complex increased with embryonic age. As the three ganglia begin to separate from each other, the label remains mostly in the cochlear ganglion, but is also present in the vestibular ganglion. Other structures containing 1–2 labeled cells/section were the trigeminal ganglion and mesoderm adjacent to the optic stalk. Cells in positive control sections exposed to TACS nuclease prior to the TUNEL assay were all labeled. Cells in negative control sections in which PBS was used in place of the terminal transferase enzyme were not labeled. The continued cvg cell loss resulting from abnormal apoptosis was reminiscent of the developmental abnormalities reported in the NeuroD mutant cvg (Liu et al., 2000). We therefore hypothesized a similar mechanism for a continued wave of neuronal cell death in the hypomorph, a lack of neurotrophic support. 2.4. Lack of neurotrophic support and consequent cvg neuronal cell death contributes to the adult phenotype of the Eya1bor/bor inner ear Both BDNF and NT-3 are expressed in the epithelia of the inner ear throughout development and into the postnatal period (Ernfors et al., 1995; Farinas et al., 2001; Fritzsch et al., 1997). Their receptors, TrkB and TrkC show overlapping temporal expression in the developing cvg (Ernfors et al., 1995; Farinas et al., 2001). Repeated survey of the expression of TrkB and TrkC in the delaminated cvg early in development (E10.5–11.5) revealed no qualitative differences between the Eya1bor/bor mice and the wildtype. In contrast to their receptors, the expression of both BDNF and NT-3 were greatly reduced throughout the inner ears of the Eya1bor/bor mice at E10.5 through E14.5. At E10.5, NT-3 expression in the ventrolateral domain of the Eya1bor/bor otocysts was reduced (Fig. 5B) compared to the control (Fig. 5A). BDNF expression was similarly reduced
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
629
Fig. 4. Hematoxylin and eosin stained sections of the E12.5 wild-type (A, B) and Eya1bor/bor (C, D) inner ears. Note the presence of cvg neuroblasts in the Eya1bor/bor inner ear (B). TUNEL-labeled cells were identified in the ventrolateral otic epithelium and cvg throughout development in the Eya1bor/bor mice (D). Scale barZ100 mm in (A and C), and 50 mm in (B and D); sa: sacculus.
in both the anterior and posterior regions of the Eya1bor/bor otocysts (Fig. 6B,D) compared to the controls (Fig. 6A,C). At E12.5, little to no NT-3 expression was observed in the developing cochlear epithelium of the Eya1bor/bor mice
(Fig. 5D,F,H) in contrast to robust expression in the controls (Fig. 5C,E,G). BDNF expression was reduced in the developing vestibular sensory epithelia (crista ampullaris) of the Eya1bor/bor mice (Fig. 6F,H,J) compared to
Fig. 5. NT-3 expression detected by in-situ hybridization in the E10.5 and E12.5 mouse otocyst (nZ6). NT-3 expression is absent ventrally in the Eya1bor/borotocyst (A, B). At E12.5, the time of cochlear morphogenetic arrest in the Eya1bor/bor mouse, reduced expression is evident in the ventral structures of the Eya1bor/bor inner ears in comparison to controls at the level of the sacculus (C, D), the cochlear base (E, F) and the cochlear hook region (G, H).
630
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Fig. 6. BDNF expression detected by in-situ hybridization in the E10.5 and E12.5 mouse otocyst (nZ8). BDNF expression is reduced in the ventral domain of the Eya1bor/bor otocyst (A, B). Expression is reduced in the dorsal structures of the Eya1bor/bor inner ears in comparison to controls.
the controls (Fig. 6E,G,I). At later stages (E14.5) both vestibular and cochlear epithelial BDNF expression was reduced in the Eya1bor/bor mice (data not shown). Similarly, NT-3 expression was greatly reduced in the cochlear epithelium of the Eya1bor/bor mice when compared to the control, a finding partially explained by the reduced cochlear duct size in these animals at E14.5 (data not shown). 2.5. Absent and deficient levels of Eya1 alter the expression of Tbx1 Tbx1 is a negative regulator of Ngn1 and NeuroD expression in the otic epithelium and thereby controls the
numbers of cvg neural precursors and the size of the cvg rudiment. To investigate a role for Tbx1 in the cvg abnormalities in the Eya1 mutants, we analyzed its expression in Eya1K/K and Eya1bor/bor ears (Fig. 7). At E10.5 Tbx1 expression is in the anterior otocyst confined to a lateral domain with a sharp border at the level of the anterior cardinal vein (Fig. 7A). In the Eya1K/K otocyst, this regionalization is lost and Tbx1 expression extends ventrally and medially within the epithelium (Fig. 7B). The sharp regionalization was altered to a lesser degree in the Eya1bor/bor mice; however, more ventral expression can be detected in these mice when compared to controls (Fig. 7C, see arrowhead). This alters the normally complimentary expression patterns of Tbx1 and
Fig. 7. Tbx1 expression in the E10.5 otocysts of wild-type (A), Eya1K/K (B) and Eya1bor/bor (C) mice detected by in situ hybridization. Laterally regionalized expression of Tbx1 in the anterior otocyst of normals (A) shifts ventrally in the Eya1K/K otocyst and, to a lesser degree, in the Eya1bor/bor otocyst at this early stage.
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Ngn1/NeuroD in this location providing a potential mechanism for the neural suppression seen in the Eya1 null and hypomorphic mutants.
3. Discussion The mouse inner ear begins its development as a thickening of surface ectoderm called the otic placode (E8.5), which invaginates to form the otic cup (E9). The otic cup then pinches off into the surrounding mesenchyme to form the otic vesicle (E9.5). During the next several days, the epithelium of the otocyst undergoes significant proliferation and differentiation, ultimately establishing the sensory and supporting cells of the auditory and vestibular systems, as well as the cochleovestibular ganglion (cvg) (D’AmicoMartel and Noden, 1983). Several lines of evidence support the existence of compartmental boundaries of gene expression within the otocyst governing the divergence of epithelial cell lineages (Fekete and Wu, 2002). Examples include the expression of Dlx5 in the dorsal epithelium of the otocyst and its responsibility for development of the semicircular canals and the expression of Otx1 in the ventral otocyst and its essential role in cochlear morphogenesis (Acampora et al., 1996; Depew et al., 1999). Specification of neural progenitors is the earliest identifiable fate determination event in the developing otocyst, beginning around E9. This subpopulation of ventral otic epithelial cells is identifiable by their expression of the Atonal-related basic helix-loop-helix (Cau et al., 1997) genes, Neurogenin1 (Ngn1) and NeuroD. Ngn1 is necessary for neural progenitor determination and formation of the cvg. Supporting its role in inner ear development, studies in Ngn1 deficient mice show complete absence of the cvg (Ma et al., 1998, 2000). Ngn1 regulates another gene in this cascade, NeuroD. It is expressed in a spatially and temporally overlapping pattern with Ngn1 and promotes neuroblast delamination into the ventral mesenchyme and growth factor mediated neuronal survival (Kim et al., 2001; Liu et al., 2000). Tbx1 has recently been shown to act upstream of Ngn1 and NeuroD as a negative regulator of neural fate specification in the otocyst (Raft et al., 2004). The cvg initially forms as one aggregate, but by E11.5 it begins to separate into a medial cochlear portion and a lateral vestibular portion. At E13.5, the primordial spiral ganglion terminates in the structures of the pars inferior, the saccule and the newly forming cochlea (one-half turn) (Altman and Bayer, 1982). It is at approximately this developmental period that cochlear and vestibular neurons are penetrating the epithelia of their respective end organs (Galinovic-Schwartz et al., 1991). This process is highly dependent upon the neurotrophins, BDNF and NT-3 (Ernfors et al., 1995; Fritzsch, 2003).
631
BDNF and NT-3 are expressed in the otic epithelium throughout development in largely non-overlapping domains with no known connection to Ngn1 or NeuroD. BDNF and NT-3 expression can also be detected outside of the sensory epithelium in delaminating cells suggesting an early source of neurotrophic support for the forming cvg cells (Farinas et al., 2001). BDNF and NT-3 deficient mice begin to show loss of vestibular and cochlear ganglion fibers, respectively, after E13 suggesting a critical time-point of cvg dependence on neurotrophins at or prior to this time (Ernfors et al., 1995; Farinas et al., 2001). Eya1 and Six1 are expressed in a spatially and temporally overlapping pattern throughout inner ear development (Kalatzis et al., 1998; Ozaki et al., 2004). A genetic interaction between Eya1 and Six1 has been established in the developing inner ear of mice and humans (BOR syndrome) (Ruf et al., 2004; Zheng et al., 2003). Furthermore, targeted deletion of Eya1 and Six1 result in early developmental arrest of the otocyst with no identifiable cvg (Ozaki et al., 2004; Xu et al., 1999). Increased apoptosis in the Eya1K/K mice and a combination of abnormal cell death and proliferation in the Six1K/K mice is the underlying cellular mechanism for the phenotype. The molecular mechanisms underlying failed neurogenesis in these mutants has, to date, remained a mystery. The expression of Fgf3 and Lfng, both markers of inner ear sensory precursors and neuroblasts is altered in both Eya1K/K and Six1K/K mice (Ozaki et al., 2004; Xu et al., 1999). Fgf3, Lfng, Ngn1 and NeuroD have overlapping expression with Eya1 and Six1 in the developing otocyst (Mansour et al., 1993; Morsli et al., 1998). Tbx1, a suppressor of neural fate in the inner ear has complimentary expression to these proneural genes and its regionalized expression posterolaterally in the early otocyst begins at the lateral border of proneural gene expression (Raft et al., 2004). We have shown that with absent or reduced levels of Eya1 expression, Tbx1 regionalization is altered leading to more ventral otocyst expression and either absent (Eya1K/K) or reduced (Eya1bor/bor) numbers of cells fated for neurogenesis. In the Eya1bor/bor mice the reduced population of neural precursors is followed by a second wave of cvg cell loss. Abnormal apoptosis occurs in association with a reduction in BDNF and NT-3 expression in the mutant inner ears beginning at the earliest time points (E10.5) and continuing on through spiral and vestibular ganglion development. A similar second wave of cvg loss was reported in NeuroD deficient mice in association with reduced neurotrophin receptor expression (TrkB and TrkC) (Liu et al., 2000). This supports an ongoing effect of Eya1 signaling regulating the levels of expression of these neurotrophins either directly or indirectly and provides a mechanism for the cvg cell death in the Eya1bor/bor mice leading to the adult phenotype.
632
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
4. Experimental procedures 4.1. Animals and genotyping The EyaC/K animals were kindly provided by Richard Maas (Harvard Medical School). For the staging of embryos, midday of the day of the vaginal plug was considered as E0.5. Embryos were harvested at E10.5. The EyaC/bor animals were obtained from Jackson Laboratories. Genotype was determined using the following PCR primers: KO-Fwd: 5 0 -TTTGGTGTGTGGTTCGGCAAGCAAT30; KO-wt-Rev: 5 0 -CATCATCTATATGGACTTGGTCACACT-3 0 ; KO-mut-Rev: 5 0 -ATCTCTCGTGGGATCATTGTTT TTCTA-3 0 ; Bor-wt-Fwd: 5 0 -TAAGCAGACCTGACAAGAGGCATTGTTT-3 0 ; Bor-mut-Fwd: 5 0 -GAACGCGTCGAATAACAGGTAGGAAAGT-3 0 ; Bor-Rev: 5 0 -TTGGGTCCTAGGTCTACCTTATCCACCAC-3 0 . 4.2. Probes Protein sequences of Neurogenin1 (Ngn1) and NeuroD are encoded in a single exon (Cau et al., 1997; Oda et al., 2000). A fragment of 352 bp of Ngn1 (containing the 5 0 UTR and the first 88 amino acids, excluding the bHLH domain) and 282 bp of NeuroD (containing the first 94 amino acids, excluding the bHLH domain) were generated by PCR from the mouse genomic DNA with the primers: Ngn1: 5 0 -TAGCCCCGGGTCGTACGGACAGTAA-3 0 and 5 0 -AGTGCAGCACCTCGGA-3 0 ; NeuroD: 5 0 -ATGACCAAATCGTACAGCGAGAGT3 0 , and 5 0 -AGCCTTAGTCATCTTCTTC-3 0 . BDNF, NT-3, trkB, and trkC probes were directly transcribed from the cloned plasmids supplied by Mary Ellen Palko from the Neural Development Group of the Department of Health and Human Services. 4.3. In situ hybridization In situ hybridization on tissue sections was performed with modifications as described (Groves and BronnerFraser, 2000). Briefly, antisense digoxigenin-labeled RNA probes were synthesized according to the Riboprobew System protocol (Promega Co., Madison, WI). Sections were treated with 2 mg/ml proteinase K in PK buffer (pH 7.5) at room temperature for 10 min. The tissue sections were then delipidated and dehydrated before hybridization. Hybridization with digoxigenin-labeled riboprobes was
performed at 60 8C overnight in a hybridization buffer solution. After hybridization, sections were incubated in 20% heat-inactivated sheep serum in PBT to block nonspecific binding sites. To visualize the hybrids, the sections were incubated with an anti-digoxigenin antibody (conjugated with alkaline phosphatase). The slides were developed in the dark for 24 h at room temperature after the addition of a chromogenic substrate, BCIP using NBT as a catalyst. Three to five embryos of each genotype were analyzed for every probe. 4.4. Immunohistochemistry Embryos were fixed in 4% PFA in PBS at 4 8C overnight, then in 30% sucrose/PBS solution at 4 8C until the embryos sink down to the bottom of the tube. Embryos were then embedded and freeze in OCT. Compound by Tissue-Tekw and were cryo-sectioned into 20 mm thick serial sections. Sections containing the otic pit or cyst and the cochleovestibular ganglion were air dried onto coated slides. Sections were incubated in 10% sheep serum in PBT at room temperature for 1 h to block non-specific binding sites, and then they were incubated in Islet1 primary antibody diluted in blocking solution at 4 8C overnight. The next day the sections were washed three times for 10 min each in PBT. Then, the sections were incubated with Rhodamine Rede fluorescent secondary antibody diluted in blocking solution at room temperature for 45 min. Sections were then washed three times for 5 min each in PBT, and a final rinse for 5 min with water before view. 4.5. TUNEL Apoptotic cells were identified in cryostat sections (6 mm) of embryos aged 9.5–14.5 days using TUNEL assay (Trevigen, Gaithersburg, MD, TACSe in situ apoptosis detection kit, ca. 4810–30-k). The whole embryo was immersion fixed in 4% buffered paraformaldehyde, washed in phosphate-buffered saline and then saturated with 30% sucrose overnight. The tissue was embedded in OCT for cryo-sectioning in the horizontal plane. Sections containing the otic pit or cyst and the cochleovestibular ganglion were air dried onto coated slides and subsequently hydrated in PBS and exposed to proteinase K (0.02 mg/ml) to digest DNA binding proteins. Hydrogen peroxide was used to inactivate endogenous peroxidases. The sections were then incubated according to the kit instructions with a mixture of terminal deoxynucleotidyl transferase (TdT), biotinylated dUTP, and divalent cation for 1 h at 37 8C. Streptavidin–horseradish peroxidase and diaminobenzidine (DAB) were used to detect labeled DNA fragments. The tissue was not counter-stained. Adjacent sections were stained with hematoxylin and eosin. Controls, run with each assay, included wild-type and heterozygote embryonic sections. The positive reagent control was pre-incubation
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
of sections with TACS-nuclease that results in label of all nuclei and a negative reagent control was incubation with PBS in place of TdT.
Acknowledgements We wish to thank Dr Richard Maas for the Eya1 mutant mice, Drs Andy Groves and Steven Raft for their thoughtful comments, Kate Blair for her technical assistance, and Mary Ellen Palko and Lino Tessarollo for the BDNF, NT-3, trkB, and trkC probes. This work was supported by NIDCD DCO4796 (R.A.F.) and the Research Service of the Department of Veterans Affairs (E.M.K.).
References Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C., et al., 1997. A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nat. Genet. 15, 157–164. Acampora, D., Mazan, S., Avantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., et al., 1996. Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat. Genet. 14, 218–222. Altman, J., Bayer, S.A., 1982. Development of the cranial nerve ganglia and related nuclei in the rat. Adv. Anat. Embryol. Cell Biol. 74, 1–90. Cau, E., Gradwohl, G., Fode, C., Guillemot, F., 1997. Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors. Development 124, 1611–1621. D’Amico-Martel, A., Noden, D.M., 1983. Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am. J. Anat. 166, 445–468. Depew, M.J., Liu, J.K., Long, J.E., Presley, R., Meneses, J.J., Pedersen, R.A., Rubenstein, J.L., 1999. Dlx5 regulates regional development of the branchial arches and sensory capsules. Development 126, 3831–3846. Desplan, C., 1997. Eye development: governed by a dictator or a junta? Cell 91, 861–864. Ernfors, P., Van De, W.T., Loring, J., Jaenisch, R., 1995. Complementary roles of BDNF and NT-3 in vestibular and auditory development. Neuron 14, 1153–1164. Farinas, I., Jones, K.R., Tessarollo, L., Vigers, A.J., Huang, E., Kirstein, M., et al., 2001. Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression. J. Neurosci. 21 (16), 6170–6180. Fekete, D.M., Wu, D.K., 2002. Revisiting cell fate specification in the inner ear. Curr. Opin. Neurobiol. 12 (1), 35–42. Fritzsch, B., 2003. Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia. Brain Res. Bull. 60 (5–6), 423–433. Fritzsch, B., Silos-Santiago, I., Bianchi, L.M., Farinas, I., 1997. The role of neurotrophic factors in regulating the development of inner ear innervation. Trends Neurosci. 20, 159–164. Galinovic-Schwartz, V., Peng, D., Chiu, F.C., Van de Water, T.R., 1991. Temporal pattern of innervation in the developing mouse inner ear: an immunocytochemical study of a 66-kD subunit of mammalian neurofilaments. J. Neurosci. Res. 30, 124–133. Groves, A.K., Bronner-Fraser, M., 2000. Competence, specification and commitment in otic placode induction. Development 127, 3489–3499. Heanue, T.A., Reshef, R., Davis, R.J., Mardon, G., Oliver, G., Tomarev, S., et al., 1999. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. Genes Dev. 13, 3231–3243.
633
Heanue, T.A., Davis, R.J., Rowitch, D.H., Kispert, A., McMahon, A.P., Mardon, G., Tabin, C.J., 2002. Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes. Mech. Dev. 111 (1–2), 75–87. Johnson, K.R., Cook, S.A., Erway, L.C., Matthews, A.N., Sanford, L.P., Paradies, N.E., Friedman, R.A., 1999. Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eya1 gene in a mouse model of BOR syndrome. Hum. Mol. Genet. 8, 645–653. Kalatzis, V., Sahly, I., El Amraoui, A., Petit, C., 1998. Eya1 expression in the developing ear and kidney: towards the understanding of the pathogenesis of Branchio-Oto-Renal (BOR) syndrome. Dev. Dyn. 213, 486–499. Kim, W.Y., Fritzsch, B., Serls, A., Bakel, L.A., Huang, E.J., Reichardt, L.F., et al., 2001. NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development 128 (3), 417–426. Li, X., Oghi, K.A., Zhang, J., Krones, A., Bush, K.T., Glass, C.K., et al., 2003. Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426 (6964), 247–254. Liu, M., Pereira, F.A., Price, S.D., Chu, M.J., Shope, C., Himes, D., et al., 2000. Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev. 14 (22), 2839– 2854. Ma, Q., Chen, Z., del, B.B.I., de la Pompa, J.L., Anderson, D.J., 1998. Neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20, 469–482. Ma, Q., Anderson, D.J., Fritzsch, B., 2000. Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. J. Assoc. Res. Otolaryngol. 1 (2), 129– 143. Mansour, S.L., Goddard, J.M., Capecchi, M.R., 1993. Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear. Development 117, 13–28. Melnick, M., Bixler, D., Silk, K., Yune, H., Nance, W.E., 1975. Autosomal dominant branchiootorenal dysplasia. Birth Defects Orig. Artic. Ser. 11, 121–128. Morsli, H., Choo, D., Ryan, A., Johnson, R., Wu, D.K., 1998. Development of the mouse inner ear and origin of its sensory organs. J. Neurosci. 18, 3327–3335. Noden, D.M., Van de Water, T.R., 1992. Genetic analyses of mammalian ear development. Trends Neurosci. 15, 235–237. Oda, H., Iwata, I., Yusanami, M., Ohkubo, H., 2000. Structure of the mouse NDRF gene and its regulation during neuronal differentiation on P19 cells. Brain Res. Mol. Brain Res. 77 (1), 37–46. Ozaki, H., Nakamura, K., Funahashi, J., Ikeda, K., Yamada, G., Tokano, H., et al., 2003. Six1 controls patterning of the mouse otic vesicle. Development 131 (3), 551–562. Raft, S., Nowotschin, S., Liao, J., Morrow, B.E., 2004. Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development 131 (8), 1801–1812. Rayapureddi, J.P., Kattamuri, C., Steinmetz, B.D., Frankfort, B.J., Ostrin, E.J., Mardon, G., Hegde, R.S., 2003. Eyes absent represents a class of protein tyrosine phosphatases. Nature 426 (6964), 295–298. Ruf, R.G., Xu, P.X., Silvius, D., Otto, E.A., Beekmann, F., Muerb, U.T., et al., 2004. SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes. Proc. Natl Acad. Sci. USA 101 (21), 8090–8095. Tootle, T.L., Silver, S.J., Davies, E.L., Newman, V., Latek, R.R., Mills, I.A., et al., 2003. The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426 (6964), 299–302. Torres, M., Gomez-Pardo, E., Gruss, P., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122, 3381–3391.
634
R.A. Friedman et al. / Mechanisms of Development 122 (2005) 625–634
Wawersik, S., Maas, R.L., 2000. Vertebrate eye development as modeled in Drosophila. Hum. Mol. Genet. 9 (6), 917–925. Xu, P.X., Woo, I., Her, H., Beier, D.R., Maas, R.L., 1997. Mouse Eya homologues of the Drosophila eyes absent gene require Pax6 for expression in lens and nasal placode. Development 124, 219–231.
Xu, P.X., Adams, J., Peters, H., Brown, M.C., Heaney, S., Maas, R., 1999. Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23, 113–117. Zheng, W., Huang, L., Wei, Z.B., Silvius, D., Tang, B., Xu, P.X., 2003. The role of Six1 in mammalian auditory system development. Development 130 (17), 3989–4000.