Expression of myosin VIIA in the developing chick inner ear neurons

Gene Expression Patterns xxx (2015) 1e9

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Expression of myosin VIIA in the developing chick inner ear neurons Kristi Nguyen, Amanda L. Hall, Jennifer M. Jones* Department of Biology, Washington College, 300 Washington Ave., Chestertown, MD 21620, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 June 2015 Received in revised form 11 July 2015 Accepted 17 July 2015 Available online xxx

The auditory-vestibular ganglion (AVG) is formed by the division of otic placode-derived neuroblasts, which then differentiate into auditory and vestibular afferent neurons. The developmental mechanisms that regulate neuronal cell fate determination, axonal pathfinding and innervation of otic neurons are poorly understood. The present study characterized the expression of myosin VIIA, along with the neuronal markers, Islet1, NeuroD1 and TuJ1, in the developing avian ear, during HamburgereHamilton (HH) stages 16e40. At early stages, when neuroblasts are delaminating from the otic epithelium, myosin VIIA expression was not observed. Myosin VIIA was initially detected in a subset of neurons during the early phase of neuronal differentiation (HH stage 20). As the AVG segregates into the auditory and vestibular portions, myosin VIIA was restricted to a subset of vestibular neurons, but was not present in auditory neurons. Myosin VIIA expression in the vestibular ganglion was maintained through HH stage 33 and was downregulated by stage 36. Myosin VIIA was also observed in the migrating processes of vestibular afferents as they begin to innervate the otic epithelium HH stage 22/23. Notably, afferents targeting hair cells of the cristae were positive for myosin VIIA while afferents targeting the utricular and saccular maculae were negative (HH stage 26e28). Although previous studies have reported that myosin VIIA is restricted to sensory hair cells, our data shows that myosin VIIA is also expressed in neurons of the developing chick ear. Our study suggests a possible role for myosin VIIA in axonal migration/pathfinding and/or innervation of vestibular afferents. In addition, myosin VIIA could be used as an early marker for vestibular neurons during the development of the avian AVG. © 2015 Elsevier B.V. All rights reserved.

Keywords: Auditory-vestibular ganglion (AVG) Inner ear development Myosin VIIA Unconventional myosin Chicken

1. Introduction The inner ears of terrestrial vertebrates are comprised of the basilar papilla (or cochlea), which is responsible for the sensation of sound, and the vestibular organs, which detect head movements. The avian inner ear contains seven vestibular organs (three cristae and four maculae) that detect angular and linear acceleration, respectively. The auditory organ, the basilar papilla, detects vibrations created by transmitted sound waves. Each sensory organ contains mechanosensory hair cells that provide input to the bipolar afferent neurons of the AVG (Whitehead and Morest, 1985). This information is then conveyed to the auditory and vestibular nuclei in the brainstem (Whitehead and Morest, 1985; Rubel and Fritzsch, 2002). The afferent neurons and associated glial cells within the AVG, are derived from the otic placode and neural crest cells, respectively

* Corresponding author. E-mail addresses: [email protected] (K. Nguyen), [email protected] (A.L. Hall), [email protected] (J.M. Jones).

(D'Amico-Martel, 1982). Development of otic neurons begins with the specification of neuronal precursors within the otic placode. This is followed by the delamination of the neuroblasts from the epithelium into the adjacent mesenchyme, where they coalesce to form the AVG. In the chick, neuroblasts continue to proliferate within the AVG and vestibular neurons begin to differentiate at embryonic (E) days 2e4 (HamburgereHamilton (HH) stages 12e23), while auditory neurons differentiate between E4 e E7 (HH stages 25e30) (D'Amico-Martel and Noden, 1983; D'Amico-Martel, 1982). As these neurons differentiate, they also segregate to form the auditory and vestibular ganglia (D'Amico-Martel and Noden, 1983; Bell et al., 2008). The vestibular ganglion includes anterior and inferior domains, which send projections to the maculae (Bell et al., 2008). In addition, two branches of the vestibular ganglion extend to form the anterior-lateral vestibular nerve, which sends projections to the lateral and anterior cristae and the posterior vestibular nerve, which targets the posterior crista (Bell et al., 2008). Prior to the segregation of the AVG into distinct auditory and vestibular ganglia, otic neurons begin to extend peripheral processes toward the otic epithelium at ~HH stage 17 (Kuratani

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et al., 1988). Vestibular afferents then innervate the vestibular sensory organs, followed by auditory afferents, which innervate the basilar papilla (Ginzberg and Gilula, 1980; Von Bartheld et al., 1991; Goodyear et al., 2010). How peripheral processes are able to navigate their way to innervate the appropriate sensory organ is an important, yet unresolved question. One possible mechanism is for peripheral processes to retrace the migratory pathway taken by the delaminating neuroblasts (Carney and Silver, 1983). More recent data indicates axonal guidance molecules may be responsible for successful navigation of peripheral processes to the appropriate sensory organs of the inner ear (for reviews see Webber and Raz, 2006; Fekete and Campero, 2007, Coate and Kelley, 2013). The twelve classes of unconventional myosins serve a broad range of functions in many cell phenotypes (Hasson et al., 1997; Hasson and Mooseker, 1997). Within the ear, Myosin VI and VIIA, two members of the unconventional myosin family, have been shown to play a critical role in the morphogenesis and function of sensory hair cells (Avraham et al., 1995; Gibson et al., 1995; Weil et al., 1995; Self et al., 1998). Mutations in these genes result in genetic abnormalities such as Usher syndrome type IB in humans and Snell's waltzer and shaker-1 phenotypes in mice (Mercer et al., 1991; Avraham et al., 1995; Gibson et al., 1995; Weil et al., 1995). While myosin VI and VIIA are involved in cell development and function, myosin II and V may play a role in the development and function of inner ear neurons. Previous data have shown that myosin V is present in the spiral and vestibular ganglia and in the afferent synaptic terminals in mature guinea pigs (Hasson et al., 1997). Interestingly, myosin V may play a role in the function of growth cones in dorsal root ganglion neurons of chick, (Wang et al., 1996). Myosin II has also been observed in axonal growth cones, and found to play a role in axonal growth (Rochlin et al., 1995; Burnette et al., 2008). Currently, myosin VIIA is accepted as a hair cell specific protein in the mammalian inner ear (Hasson et al., 1995; Sahly et al., 1997). In this study, we characterized the spatiotemporal expression of myosin VIIA in the developing chick ear during otic neuron delamination, differentiation, migration, segregation and innervation. We utilized immunohistochemistry to compare the expression of myosin VIIA with that of other well-characterized neuronal markers, such as Islet1, NeuroD1 and TuJ1 (Li et al., 2004; Alsina et al., 2004; Stone et al., 2003). In addition, we utilized the TuJ1 antibody, which labels neuronal axons (Stone et al., 2003) to analyze the expression of myosin VIIA during innervation of the otic sensory epithelia. Our analysis indicates that myosin VIIA expression begins at stage 20 (E3) in post-mitotic neurons of the AVG. Expression of myosin VIIA is maintained through stage 33 (E8), when the AVG divisions are distinct and innervation is complete. Myosin VIIA expression is restricted to the vestibular neurons that target the three cristae. These results suggest a possible role for myosin VIIA during neuronal differentiation, axon guidance and/or innervation. 2. Results and discussion 2.1. Myosin VIIA is expressed in post-mitotic AVG neurons To characterize the expression pattern of myosin VIIA during the development of chicken inner ear neurons, we used immunohistochemistry to compare the expression of myosin VIIA with that of various neuronal markers (Islet1, NeuroD1 and TuJ1). Otic neuroblasts delaminate from the anteroventral otocyst (stage 13e21) and migrate into the surrounding mesenchyme, where they will form the AVG (Hemond and Morest, 1991; Adam et al., 1998). Neuroblasts continue to proliferate within the AVG until vestibular neurons undergo terminal mitosis (HH stages 12e25), followed by auditory

neurons (HH stages 25e30) (D'Amico-Martel, 1982). First, we observed that myosin VIIA was not detected within the auditoryvestibular ganglion (AVG) from HH stages 16e19, (Fig. 1B and data not shown). Next, we compared the expression of myosin VIIA and Islet1 in transverse sections of the chick otic vesicle at HH stages 20e22 (representative data shown for HH stage 21 only, Fig. 1). Previous studies have shown that Islet1 labels neuroblasts within the otic epithelium and the AVG and its expression is maintained throughout the development of inner ear neurons (Li et al., 2004; Radde-Gallwitz et al., 2004). We initially observed myosin VIIA positive-cells within the AVG at stage 20 (data not shown). At HH stage 21, immunoreactivity for both myosin VIIA and Islet1 was observed within the AVG (Fig. 1D, E). Notably, myosin VIIA and Islet1 are expressed in the same cells, indicating that these are developing neurons (Fig. 1F, inset). Next, we compared the expression of myosin VIIA with that of NeuroD1. It has been previously reported that NeuroD1 labels neuroblasts within the otic epithelium and AVG early in development and is down-regulated following terminal mitosis (Alsina et al., 2004; Davies, 2007; Deng et al., 2014). NeuroD1 was observed in a subset of the AVG (Fig. 1G). Myosin VIIA was expressed in a subset of cells in the AVG, which corresponded to the NeuroD negative region (Fig. 1I). However, there were a few NeuroD positive cells within the myosin VIIA positive region (Fig. 1I, inset). We continued to examine this complimentary expression pattern, by comparing myosin VIIA and NeuroD1 at developmental HH stages 23 and 25, (Fig. 1JeL and data not shown). By HH stage 25, only a few cells in the AVG were NeuroD1 positive, suggesting that neural precursors lose NeuroD1 expression as they continue to develop (compare Fig. 1G, J; Deng et al., 2014). At HH stage 25, myosin VIIA expression continued to be restricted to a NeurodD-negative subset of cells within the AVG (Fig. 1L). The fact that we did not observe expression of myosin VIIA and NeuroD1 within the same AVG cells indicates that myosin VIIA does not label neuroblasts. To further explore this notion, we compared the expression of myosin VIIA with immunoreactivity for TuJ1, at HH stage 21. TuJ1 has been shown to label neurons following terminal mitosis (Molea et al., 1999; Stone et al., 2003; Deng et al., 2014). Our co-labeling data show that myosin VIIApositive cells within the AVG also express TuJ1, indicating that these are post-mitotic neurons (Fig. 1O, inset). Together, these data show that myosin VIIA expression is first observed at HH stage 20 in early differentiating neurons within the AVG, indicating that this protein is unlikely to be playing a role in the delamination, migration or proliferation of inner ear neuroblasts. 2.2. Myosin VIIA is expressed in afferents near the onset of innervation To investigate the expression of myosin VIIA in otic afferents as they begin to innervate sensory receptors, we co-labeled samples at HH stages 22 and 23 for myosin VIIA and TuJ1. We observed TuJ1 to label both the cell soma as well as the axonal projections of AVG neurons (Fig. 2A, D, G). Neurites begin to extend from AVG neurons as early as E2.5 (~stage 17) and these projections first reach the otic epithelium at E3 (~stage 19) prior to the onset of hair cell differentiation (Kuratani et al., 1988; Hemond and Morest, 1991; Bartolami et al., 1991; Goodyear et al., 1995). Previous studies have reported that afferents begin to innervate the vestibular epithelia at E4 (stage 23), and auditory afferents begin to innervate the basilar papilla at E5 (stage 26) (von Bartheld et al., 1991; Whitehead and Morest, 1985). At stage 22, immunoreactivity for TuJ1 was observed in the AVG and in neuronal projections innervating the otic epithelia (Fig. 2A). Myosin VIIA labeling was present in a subset of cells within the AVG as well as their neuronal projections (Fig. 2B). Notably, these myosin VIIA-positive neuronal

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Fig. 1. Myosin VIIA is expressed in the post-mitotic neurons of the developing chick AVG. Expression of Myosin VIIA co-labeled with Islet1, NeuroD or TuJ1 in transverse sections at stages 17, 21, or 25. Islet1, stage 17 (AeC) A. Islet1 labels neurons of the AVG. B. Myosin VIIA labeling is not present. C. Myosin VIIA is not present in the Islet1 positive AVG. Islet1, stage 21 (DeF) D. Islet1 labels neurons of the AVG. Note Islet1 is a nuclear label. E. Myosin VIIA is expressed in a subset of cells within the AVG. Note Myosin VIIA is a cytoplasmic label. F. Myosin VIIA positive cells are also Islet1 positive, see inset (* region shown in inset). NeuroD1, stage 21 (GeI) G. NeuroD labels neuroblasts in the AVG. Note NeuroD is a nuclear label. H. Myosin VIIA labels a subset of cells in the AVG. I. Myosin VIIA positive cells are present in the NeuroD negative region of the AVG. NeuroD1 positive cells are occasionally present in the Myosin VIIA positive region, see inset (* region shown in inset). NeuroD1, stage 25 (JeL) J. NeuroD positive cells are present at the dorsomedial edge of the AVG. K. Myosin VIIA labels a subset of cells the AVG. L. Myosin VIIA positive cells are present in the NeuroD negative region of the AVG. TuJ1, stage 21 (MeO) M. TuJ1 labels neurons of the AVG. N. Myosin VIIA labels a subset of cells in the AVG. O. Myosin VIIA positive cells are TuJ1 positive, see inset (* region shown in inset). Auditory-vestibular ganglion (AVG). Orientation: Dorsal, D; Medial, M. Scale bar in (AeC; GeI; M
O) ¼ 11 mm; (DeF) ¼ 10 mm; (JeL) ¼ 20um.

projections were also immunoreactive for TuJ1 (Fig. 2C, inset). It is likely that these myosin VIIA/TuJ1 positive projections innervating the otic epithelium are extensions of vestibular neurons, since the first afferents to arrive at the otic epithelium are vestibular (von

Bartheld et al., 1991). At stage 23, TuJ1 labeled cells in the AVG and neuronal projections innervating the sensory epithelia of the presumptive anterior crista and saccular macula (Fig. 2D, E). Myosin VIIA

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Fig. 2. Myosin VIIA is expressed in neuronal projections of the AVG in the developing chick. Expression of Myosin VIIA and TuJ1 in transverse sections at stages 22 and 23.5. Stage 22 (AeC). A. TuJ1 labels cells in the AVG and peripheral projections innervating the presumptive anterior crista (AC). B. Myosin VIIA labels a subset of cells in the AVG and the peripheral projections innervating the presumptive AC. C. In the AVG, myosin VIIA positive cells are within the TuJ1 positive domain. Myosin VIIA positive neuronal projections co-express TuJ1, see inset (*region shown in the inset). Stage 23.5, anterior region of the otocyst (DeF). D. TuJ1 labels cells in the AVG as well as peripheral projections innervating the presumptive anterior cristae (AC). E. Myosin VIIA labels a subset of cells in the AVG, neuronal projections innervating the presumptive AC as well as presumptive sensory hair cells (arrow). F. In the AVG, myosin VIIA positive cells are within the TuJ1 positive domain. Myosin VIIA positive neuronal projections innervating the presumptive AC co-express TuJ1, see inset (* region shown in the inset). Stage 23.5, posterior region of the otocyst (GeI). G. TuJ1 labels cells in the AVG as well as the peripheral projections innervating the presumptive saccular macula (S). H. Myosin VIIA labels a majority of cells in the AVG as well as the sensory hair cells (arrow) in the presumptive saccular macula (S). I. In the AVG, myosin VIIA positive cells are within the TuJ1 positive domain. TuJ1 positive neuronal projections do not express Myosin VIIA, see inset, (* region shown in the inset). Auditory-vestibular ganglion (AVG); Otic epithelium (OE), Anterior Crista (AC); Saccular Macula (S). Orientation: Dorsal, D; Medial, M. Scale bar ¼ 20 mm.

expression was present in a subset of cells within the AVG and those neuronal projections innervating the presumptive anterior crista (Fig. 2E). These myosin VIIA-positive neuronal projections were co-labeled with TuJ1 and extend to the region of developing hair cells (Fig. 2F, inset). In contrast, TuJ1-positive neuronal projections that targeted the presumptive saccular macula were myosin VIIA-negative (Fig. 2I, inset). Finally, myosin VIIA labeled hair cell precursors in the presumptive anterior cristae and saccular macula (Fig. 2E, H arrows). This labeling was analogous to the expression of Cath1, the chicken homologue of atonal, which demarcates early post-mitotic hair cells within the developing sensory organs of the inner ear (Stone et al., 2003). These data indicate that myosin VIIA is expressed by the subset of AVG neurons that target the anterior cristae, but not in those that target the saccular macula (Fig. 2E, H). Although myosin VIIA is a well-characterized hair cell marker, our data show that myosin VIIA is also expressed in a subset of afferent inner ear neurons. 2.3. Myosin VIIA labels a subset of neurons in the AVG We next compared the expression of myosin VIIA with that of Islet1 or TuJ1, in order to investigate developmental changes in their expression patterns. First, we compared labeling for myosin VIIA and Islet1 in anterior and posterior regions of the otocyst. Our data were consistent with previous reports showing that Islet1 labels cells in the AVG and weakly labels cells demarcating the

presumptive sensory epithelium (Fig. 3A, D; Li et al., 2004). Myosin VIIA expression persisted in a subset of cells within the AVG as well as in hair cell precursors (arrows) in the presumptive saccular macula and anterior crista (Fig. 3B, E). Myosin VIIA-positive cells within the AVG co-expressed the neuronal marker Islet1 (Fig. 3C, F, inset). These data further identify the myosin VIIA-positive cells in the AVG as neurons. Co-labeling with Tuj1 also allowed us to investigate the expression of myosin VIIA in the neuronal projections. Neuronal projections innervating the presumptive saccular macula (posterior sections) and anterior crista (anterior sections) were labeled with TuJ1 (Fig. 3G, J respectively), and myosin VIIA was not expressed in neuronal projections targeting the saccular macula (Fig. 3H, I, inset). In contrast, we did observe myosin VIIA expression in the neuronal projections innervating the anterior crista (Fig. 3K, L, inset). This is consistent with the expression pattern at HH stage 23 (compare Figs. 2F, I and 3I, L). The presence of myosin VIIA within the AVG and neuronal projections suggests a possible role for myosin VIIA in axonal guidance. A number of guidance molecules are expressed by cells of the developing AVG and play a role in directing the peripheral projections toward their correct targets (for review see Fekete and Campero, 2007). For example, previous data show that Slit2 is present within the vestibular ganglion and may be involved in guiding vestibular afferents to the otic epithelium (Battisti and Fekete, 2008; Battisti et al., 2014). Our data show a similarly restricted expression of myosin VIIA presumptive vestibular

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Fig. 3. Myosin VIIA is expressed in the developing chick AVG. Co-immunolabeling with Myosin VIIA and Islet1 (AeF) or TuJ1 (DeL) in transverse sections at stage 24. Islet1, posterior region of the otocyst (AeD) A. Islet1 labels the cells in the AVG and weakly labels cells in the otic epithelium (OE), which corresponds to the developing sensory epithelium. B. Myosin VIIA is expressed in a subset of cells in the AVG as well as the presumptive sensory hair cells (arrow) where the saccular macula (S) will develop. C. In the AVG, myosin VIIA positive cells are also Islet1 positive, see inset (* region shown in the inset). Islet1, anterior region of the otocyst (DeF). D. Islet1 labels the cells in the AVG and weakly labels cells in the developing sensory epithelium. E. Myosin VIIA is expressed in cells in the AVG as well as presumptive sensory hair cells (arrow) where the anterior crista (AC) will develop. F. In the AVG, myosin VIIA positive cells are Islet1 positive, see inset (* region that is shown in the inset). TuJ1, posterior region of the otocyst (GeI). G. TuJ1 labels cells in the AVG and peripheral projections innervating the presumptive saccular macula (S). H. Myosin VIIA labels a subset of cells in the dorsolateral AVG and presumptive saccular macula hair cells (arrow). I. Myosin VIIA positive cells in the AVG are present in the TuJ1 positive domain. The neuronal projections express TuJ1 but not myosin VIIA, see inset (* region that is shown in the inset). TuJ1, anterior region of the otocyst (JeL). J. TuJ1 labels cells in the AVG and neuronal projections innervating the presumptive anterior crista (AC). K. Myosin VIIA labels a subset of cells in the AVG, neuronal projections innervating the presumptive AC as well as presumptive saccular hair cells (arrow). L. In the AVG, myosin VIIA positive cells are present in the TuJ1 positive domain. The neuronal projections innervating the AC express both myosin VIIA and TuJ1, see inset (* region shown in the inset). Auditory-vestibular ganglion (AVG); otic epithelium (OE); anterior crista (AC); saccular macula (S). Orientation: Dorsal, D; Medial, M. Scale bar in (AeC) ¼ 20 mm; (DeF) ¼ 35 mm; (GeI) ¼ 25 mm; (JeL) ¼ 30 mm.

neurons at HH stage 24 (Fig. 3). It would be interesting to directly compare the expression of myosin VIIA with slit/robo to investigate whether they may coordinate in the guidance of growing neurites. 2.4. Myosin VIIA is expressed in vestibular but not auditory neurons We further characterized immunoreactivity for myosin VIIA during and following the segregation of the auditory and vestibular ganglia (HH stages 26e33). Neuroblasts initially migrate from the otic epithelium and coalesce to form the AVG, which is a mixed

population of presumptive auditory and vestibular neurons. As development progresses, the AVG will segregate into separate auditory and vestibular ganglia. This initial segregation is first observed at HH stages 23/24 (E4) (D'Amico-Martel and Noden, 1983). However, the individual branches projecting to discrete sensory organs can be better distinguished at HH stage 30 (E7) (Bell et al., 2008). During the process of segregation, the vestibular ganglion resides in the dorsal-most region (adjacent to the developing vestibular organs), while the auditory ganglion is located ventrally, adjacent to the basilar papilla. We compared labeling for

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Fig. 4. Myosin VIIA is expressed in the developing chick vestibular ganglion. Expression of myosin VIIA and TuJ1 in transverse sections at stages 26e33. Stage 26 (AeC). A. TuJ1 labels cells in the VG and AG. B. Myosin VIIA labels a subset of the neurons in the VG, a group of cells on the edge of the AG (arrowhead), and sensory hair cells (arrow) of the saccular macula (S). C. Myosin VIIA positive cells in the VG and periphery of the AG are present in the TuJ1 positive regions. Stage 28, posterior region of the otocyst (DeF). D. TuJ1 labels cells in the IVG, AG, GG as well as the ALVN. E. Myosin VIIA labels cells in the IVG, GG, ALVN, hair cells (arrows) of the saccule (S) and utricule (U) as well as a group of neurons located on the peripheral edge of the AG (arrowhead). F. Myosin VIIA positive cells in the IVG, peripheral edge of the AG (arrowhead), and GG are present in the TuJ1 positive regions. Stage 28, anterior region of the otocyst (GeI). G. TuJ1 labels neurons in the SVG, AG, ALVN and GG. H. Myosin VIIA is present in subset of the cells in the SVG, ALVN and GG. I. Myosin VIIA positive cells in the SVG, ALVN and GG are present within the TuJ1 positive regions. Stage 30, (JeL). J. TuJ1 labels cells in the VG and AG. K. Myosin VIIA labels the cells in the VG, a group of cells on the edge of the AG (arrowhead), and sensory hair cells (arrow) of the saccule (S). L. Myosin VIIA positive cells in the VG and periphery of the AG are present in the TuJ1 positive domains. Stage 33, (MeO). M. TuJ1 labels cells in the VG and AG. N. Myosin VIIA labels a subset of the cells in the VG and the hair cells in the basilar papilla and saccule. O. In the VG, myosin VIIA positive cells are also TuJ1 positive, see inset (* region that is shown in the inset). Auditory ganglion (AG) vestibular ganglion (VG); Basilar Papilla (BP), Saccular Macula (S), Utricular Macula (U), Anterolateral Vestibular Nerve (ALVN), Inferior Vestibular Ganglion (IVG), Superior Vestibular Ganglion (SVG), Geniculate Ganglion (GG). Orientation: Dorsal, D; Medial, M. Scale bar in (AeC, GeI) ¼ 65 mm; (DeF, JeL) ¼ 70 mm; (MeO) ¼ 90 mm.

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myosin VIIA and TuJ1 at stage 26, early in the process of ganglion segregation. Immunoreactivity for TuJ1 highlights the ventral, auditory (AG) and dorsal, vestibular (VG) ganglia (Fig. 4A). In addition to the sensory hair cells of the saccular macula (arrow), myosin VIIA was expressed in a subset of cells in the dorsal most portion of the ganglion, corresponding to the region of vestibular neurons (Fig. 4B). Labeling for myosin VIIA was not observed in the ventral auditory region, corresponding to the auditory neurons, except for a few cells along its outer edge (arrowhead) (Fig. 4B). The

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position of those myosin VIIA- positive cells in the auditory ganglion is near the group of neurons that will eventually contact the lagenar macula (Fischer et al., 1994). As development proceeds, the vestibular ganglion will eventually form distinct domains including the anterior-lateral vestibular nerve (ALVN), posterior vestibular nerve (PVN), superior (SVG) and inferior (IVG) vestibular ganglia, which will innervate discrete vestibular organs (Bell et al., 2008). At HH stage 28, it was possible to discern these anatomical domains of the vestibular ganglion

Fig. 5. Myosin VIIA is expressed in the vestibular afferents of the cristae in the developing chick. Expression of Myosin VIIA and TuJ1 in transverse sections of the lateral crista and utricule at HH stages 28 and 33. Lateral crista, stage 28 (AeD). A. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the epithelium (above line) of the lateral crista. Myosin VIIA is present in the hair cells and cytoplasmic tails (arrowheads). B. TuJ1 is present in the neuronal fibers of the ALVN (below line) and projections innervating the epithelium (above line) of the lateral crista (*, TuJ1 positive projections). C. Myosin VIIA is present in the neuronal fibers of the ALVN and a subset of the projections innervating the epithelium of the lateral crista (arrows, myosin VIIA positive projections, *, myosin VIIA negative). D. Myosin VIIA and TuJ1 are co-expressed in the neuronal fibers of the ALVN and several of the projections innervating the lateral crista sensory epithelium (arrows). Lateral crista, stage 33 (EeH). E. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the epithelium of the lateral crista (above line). Myosin VIIA is present in the hair cells and cytoplasmic tails (arrowheads). F. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the epithelium (above line) of the lateral crista. G. Myosin VIIA is present in the hair cells and cytoplasmic tails (arrowheads). H. Myosin VIIA and TuJ1 are not co-expressed in neuronal projections innervating the sensory epithelium of the lateral crista. Utricule, stage 28 (IeL). I. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the epithelium (above line) of the utricule. Myosin VIIA is present in the hair cells and a few cells within the ALVN (arrowheads). J. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the utricule (above line). K. Myosin VIIA is present in the hair cells of the utricule. L. Myosin VIIA and TuJ1 are not co-expressed in projections innervating the utricule. Utricule, stage 33 (MeP). M. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the epithelium (above line) of the utricule. Myosin VIIA labels the hair cells of the utricule. N. TuJ1 labels the neuronal fibers of the ALVN (below line) and projections innervating the utricule (above line). O. Myosin VIIA is present in the hair cells of the utricule. P. Myosin VIIA and TuJ1 are not co-expressed in projections innervating the utricule. Anterior-lateral vestibular nerve (ALVN); Lateral crista (LC), Utricule (U). Scale bar in (A) ¼ 13 mm; (BeC) ¼ 8 mm; (EeH) ¼ 11 mm; (I,M) ¼ 20 mm; (JeL, NeP) ¼ 7 mm.

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when observing anterior and posterior regions of the TuJ-1-labeled otocyst (Fig. 4D, G). Expression of myosin VIIA was present in the sensory hair cells of the saccular and utricular maculae and in the four domains of the vestibular ganglion: IVG, SVG, ALVN and PVN (Fig. 4E, H and data not shown). In addition, immunoreactivity for myosin VIIA was also noted in the geniculate ganglion (Fig. 4E). At HH stage 30, myosin VIIA continued to be expressed in hair cells (arrow), a subset of cells in the vestibular ganglion, and a few cells in the outer edge of the auditory ganglion (Fig. 4K; arrowhead; possible lagenar neurons). At HH stage 33, myosin VIIA was observed in hair cells of both the vestibular organs and the basilar papilla (Fig. 4N, arrows). Similar to previous ages, myosin VIIA continued to be expressed in a subset of TuJ1-labeled cells within the vestibular ganglion (Fig. 4O, inset), and this expression pattern was observed until HH stage 36 (data not shown). Since expression of myosin VIIA within the ganglion is restricted to vestibular neurons, it could serve as a useful developmental marker to distinguish vestibular from auditory afferents. 2.5. Myosin VIIA expression is restricted to vestibular afferents innervating the cristae In order to determine whether myosin VIIA continued to be expressed during the later maturation of vestibular neurons, we compared immunoreactivity for both myosin VIIA and TuJ1 at HH stages 26, 28, 30 and 33 (data shown are for the lateral crista and utricular macula at HH stages 28 and 33). At HH stage 28, the TuJ1labeled neuronal projections could be observed in the ALVN and innervating the lateral crista (Fig. 5A, B below and above line respectively). Myosin VIIA was present in the bodies of hair cells and in their cytoplasmic tails, which are characteristic of immature hair cells (Fig. 5A, arrowheads). Higher magnification images confirmed that myosin VIIA and TuJ1 were co-expressed in a subset of the neuronal projections innervating the lateral cristae (Fig. 5D, arrows), and in neuronal fibers of the ALVN (Fig. 5D, below line). Notably, myosin VIIA expression in the ALVN and neuronal projections to the lateral crista was lost by HH stage 30, (Fig. 5E, H). A similar loss of expression was seen in the anterior and posterior cristae (data not shown). These observations indicate that, as development progresses, myosin VIIA expression is downregulated in the afferents that innervate the cristae. Also, we did not observe myosin VIIA-positive neuronal projections innervating the sensory epithelium of the saccule or utricle at any of the stages investigated (Fig. 5L, P and data not shown). Together these data demonstrate that myosin VIIA is transiently present in neuronal projections that innervate the cristae, but is absent in those that contact the utricular and saccular macula. Early synaptic components are first observed at HH stage 28 in the vestibular sensory organs, followed by more defined chemical synapses at HH stage 32 (Ginzberg and Gilula, 1980). Since we observed downregulation of myosin VIIA within neuronal projections innervating the cristae by HH stage 30, it seems unlikely that this protein plays a role in synaptogenesis. In summary, our study reveals a dynamic expression of myosin VIIA during inner ear neuron development (Table 1). Myosin VIIA is expressed in some post-mitotic neurons of the AVG. This expression persistents until HH stage 33 and is downregulated by HH stage 36. Neuronal expression of myosin VIIA is restricted to afferents that innervate the cristae, and is not present in those afferents that innervate the utricle or saccule. In addition, myosin VIIA expression does not appear to be expressed by afferents of the acoustic ganglion or its projections to the basilar papilla. Although myosin VIIA is a well-known hair cell marker, our data show that myosin VIIA can also identify a subpopulation of developing vestibular neurons. Based on the timing of its expression, myosin VIIA may play a role in axonal pathfinding and/or innervation of the

Table 1 Summary of the expression of myosin VIIA in inner ear neurons during embryonic development of the chick. Stage

20 22 23 24 26 28 30 33 36 40

Approximate embryonic day 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 10 14

AVG

a

þ þa þa þa n/a n/a n/a n/a n/a n/a

AG

n/a n/a n/a n/a e e e e e e

VG

n/a n/a n/a n/a þa þa þa þa e e

Peripheral projections SM

UM

LC

AC

PC

nd nd e e e e e e e e

nd nd nd e e e e e e e

nd nd þ þ þb þb e e e e

nd þ þ þ þb þb e e e e

nd nd nd nd þb þb e e e e

n/a e not applicable before or after segregation of the ganglion. nd e not determined. a Staining observed in a subset of cells. b Staining observed in a subset of projections.

vestibular neurons. Future gain- or loss-of-function studies targeting myosin VIIA will help elucidate its specific role in these processes. 3. Experimental procedures 3.1. Tissue preparation Fertilized White Leghorn chick eggs were incubated at 37.8  C in a humidified incubator with automatic rotation. Embryos were staged according to Hamburger and Hamilton (1992) and fixed overnight in 4% paraformaldehyde solution (0.2 M Phosphate Buffer pH 7.4) at 4  C. Specimens were cryoprotected in 15% sucrose solution (PBS) until the tissue sank, followed by 30% sucrose solution (PBS), until the tissue sank. The tissue was then placed in cryomolds and submersed in optimal cutting temperature compound (OCT). The cryomolds were frozen in 2-Methylbutane mixed with dry ice and stored at
20  C. Tissue cross-sections of 18 mm thickness were collected onto slides (Tru Scientific Trubond) and allowed to dry overnight at room temperature and stored at
80  C until processing. 3.2. Immunohistochemistry Slides with tissue sections were washed for 10 min in phosphate buffer saline (PBS) with 0.2% Triton X-100 (PBT). Slides were blocked from non-specific binding with 5% normal horse serum in PBT for 60 min at room temperature. Slides were incubated in the following primary antibodies diluted in PBT: Myosin VIIA (1:500; Proteus Biosciences); Islet1 (1:20; Developmental Studies Hybridoma Bank); NeuroD1 (1:20; R&D Systems); TuJ1 (1:500; BioLegend) overnight at 4  C. To remove unbound primary antibodies slides were washed 3 times for 10 min with PBS. Secondary antibodies diluted in PBS at 1:500 (Alexa Fluor-conjugated; Molecular Probes) were applied to the slides and allowed to incubate for 90 min at room temperature. Next, the slides were washed 3 times in PBS and mounted using glycerol mounting media (90% glycerol and 10% PBS). Negative controls, in which primary antibodies were omitted were processed as stated above. We did not observe specific labeling in any of the negative control samples. 3.3. Confocal analysis and image processing The mounted slides were analyzed under a Zeiss LSM700 confocal microscope. Confocal stacks were converted to an

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Please cite this article in press as: Nguyen, K., et al., Expression of myosin VIIA in the developing chick inner ear neurons, Gene Expression Patterns (2015), http://dx.doi.org/10.1016/j.gep.2015.07.001