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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Gene Expr Patterns. 2014 Apr 4;15(1):31–37. doi: 10.1016/j.gep.2014.03.001

Comparative expression analysis of POU4F1, POU4F2 and ISL1 in developing mouse cochleovestibular ganglion neurons

Min Deng 1,2,5, Hua Yang 4,5, Xiaoling Xie 1,2, Guoqing Liang 3, Lin Gan 1,2,3,*
PMCID: PMC4065884  NIHMSID: NIHMS583149  PMID: 24709358

Abstract

POU-homeodomain and LIM-homeodomain transcription factors are expressed in developing projection neurons within retina, inner ear, dorsal root ganglion, and trigeminal ganglion, and play synergistic roles in their differentiation and survival. Here, using immunohistochemistry, we present a comparative analysis of the spatiotemporal expression pattern of POU4F1, POU4F2, and ISL1 during the development of cochleovestibular ganglion (CVG) neurons in mouse inner ear. At early stages, when otic neurons are first detected in the otic epithelium (OE) and migrate into periotic mesenchyme to form the CVG, POU4F1 and ISL1 are co-expressed in a majority of the delaminated CVG neurons, which are marked by NEUROD1 expression, but POU4F1 is absent in the otic epithelium. The onset of POU4F2 expression starts after that of POU4F1 and ISL1, and is observed in the NEUROD1-negative, post-mitotic CVG neurons. When the CVG neurons innervate the vestibular and cochlear sensory organs, the expression of POU4F1, POU4F2, and ISL1 continues in both vestibular and spiral ganglion cells. Later in development, POU4F1 expression becomes down-regulated in a majority of spiral ganglion (SG) neurons and more neurons express POU4F2 expression while ISL1 expression is maintained. The differential as well as overlapping expression of POU4F1, POU4F2, and ISL1 combined with previous studies suggests possible functional interaction and regulatory relationship of these transcription factors in the development of inner ear neurons.

Keywords: inner ear, CVG, neurogenesis, LIM-homeodomain, POU-homeodomain, transcription factor

The inner ear is one of the most elaborate structures among mammalian organs. It consists of two different but relevant parts, the cochlea to detect hearing and the vestibule to contribute to balance and to the sense of spatial orientation. Published studies reveal that both the cochleovestibular ganglion (CVG) and the sensory epithelium of the inner ear are derived from the common otocyst, which is originated from the otic placode, a thickened patch of the ectoderm during embryogenesis (Anniko and Wikstrom, 1984; Fritzsch, 2003; Fritzsch et al., 2002). In mice, neurogenesis initiates as CVG neuronal progenitors emanate from the otic placode at E9.5 and finishes at E15.5 to form the CVG, which later separates into the spiral and vestibular ganglia (Fritzsch et al., 2002; Fritzsch et al., 1999; Morsli et al., 1998). Studies have implicated the essential roles of several transcription factor genes in the development of CVG neurons, such as the bHLH genes Neurog1 (Ma et al., 1998) and Neurod1 (Kim et al., 2001; Liu et al., 2000), and the GATA family gene Gata3 (Appler et al., 2013; Duncan and Fritzsch, 2013; Duncan et al., 2011; Economou et al., 2013; Jones and Warchol, 2009; Lawoko-Kerali et al., 2002; Lawoko-Kerali et al., 2004; Luo et al., 2013).

The Class IV family of POU-HD transcription factors play crucial roles in the differentiation and survival of sensory neurons (DeCarvalho et al., 2004; Erkman et al., 1996; Gan et al., 1999; Gan et al., 1996; Huang et al., 2001; Pan et al., 2008). Targeted deletion of Pou4f1 results in defective axonal growth in the dorsal root ganglion (DRG) and trigeminal ganglia (TG) and abnormal expression of developmental sensory markers (Eng et al., 2007; Eng et al., 2001; Eng et al., 2004). In the retina, POU4F2 expression precedes that of POU4F1 and functions redundantly with POU4F1 to regulate the differentiation and survival of retinal ganglion cells (Gan et al., 1999; Gan et al., 1996; Pan et al., 2008; Pan et al., 2005). In mouse inner ear, POU4F1 controls the soma size of CVG cells and regulates the target field innervations as well as the axon path-finding (Huang et al., 2001). Similarly, the expression of ISL1 is also essential for sensory neurogenesis (Elshatory et al., 2007a; Elshatory et al., 2007b; Korzh et al., 1993; Li et al., 2004; Pan et al., 2008; Pfaff et al., 1996; Radde-Gallwitz et al., 2004; Sun et al., 2008). Interestingly, ISL1 and POU4F proteins are often co-expressed and function synergistically in regulating neuronal differentiation and survival. In the developing retina, POU4F2 and ISL1 are co-expressed in the nascent RGCs. While targeted deletion of either Pou4f2 or Isl1 results in the partial loss of RGCs, nearly all RGCS undergo apoptosis in mice lacking both Pou4f2 and Isl1 (Mu et al., 2008; Pan et al., 2008). Likewise, nearly all neurons of the developing mouse DRG and TG at E9.5–10.5 co-express ISL1 and POU4F1 (Anderson, 1999). Similar to the phenotype of Pou4f1-null mice, initial neurogenesis appears normal in Isl1-null ganglia. However, by embryonic day 12.5, the ganglion neurons undergo excess apoptosis by E12.5 (Sun et al., 2008). Moreover, in Pou4f1/Isl1 double knock-out embryos, sensory neurons display a more profound loss of all early markers of sensory subtypes that in mice lack either Pou4f1 or Isl1. Examination of global gene expression in the E12.5 DRG of single and double knockout embryos shows that POU4F1 and ISL1 are synergistically required for nearly all aspects of sensory-specific gene expression (Dykes et al., 2011). In the developing mouse inner ear, ISL1 is expressed in both the neuronal and sensory lineages (Radde-Gallwitz et al., 2004). Its expression first appears in the developing otic epithelium and is maintained throughout the development of the inner ear neurons. In the developing cochlea, ISL1 expression starts in the ventral portion of the cochlear epithelium and down-regulated in the sensory epithelia once hair cell begins to differentiate.

Currently, whether ISL1 plays an essential role in inner ear development and whether POU4F and ISL1 function together to regulate inner ear neurogenesis remain unknown. In this study, we investigated the expression profile of POU4F and ISL1 in reference to other well-studied neuronal markers, such as NEUROD1 and NeuN during the development of mouse CVG cells. Our data demonstrated that POU4F1 and ISL1 are mostly co-expressed throughout the CVG neurogenesis. ISL1 is expressed in the late epithelial CVG precursors, the delaminated neuroblasts as well as mature CVG neurons. Interestingly, the onset of POU4F1 starts slightly after that of ISL1 but precedes POU4F2 expression. Compared to POU4F1 expression that is confined to the post-delaminated CVG cells, POU4F2 is only expressed in the differentiated and mature CVG neurons. Thus, the temporal expression order of these known transcription factors in the development of mouse CVG neurons is NEUROD1>ISL1>POU4F1>POU4F2.

MATERIALS AND METHODS

Animals

Neurod1-null and Pou4f1-null, mice were described previously (Naya et al., 1997; Xiang et al., 1996). C57BL/6J mice were obtained from The Jackson Laboratory (Stock #000664). The mice were time mated and embryos were designated as E0.5 at noon on the day the vaginal plugs were first observed. The expression studies of each time point were performed at least three times, each time with a different embryo or pup. All animal procedures described in this study were in accordance to NIH guidelines and were approved by the University Committee of Animal Resources (UCAR) at the University of Rochester.

Tissue preparation, immunohistochemistry, and BrdU labeling

Heads of staged embryos and neonatal mice were dissected in cold phosphate-buffered saline (PBS), pH 7.2 and immediately fixed in 4% paraformaldehyde (PFA) in PBS for 2 hours. After fixation, the samples were washed in PBS and were cryoprotected in 20% sucrose in PBS for overnight at 4°C. The specimens were subsequently embedded in OCT compound (Tissue-Tek, Torrance, CA) and stored at −80°C prior to and after cryosectioning. Serial cryosections were cut at 20 μm thickness and collected on superfrost plus microslides (VWR, Westchester, PA).

For immunolabeling, slides with cryosections were dried at 37°C for 30–45 minutes. Non-specific binding was blocked with 10% normal horse serum and 0.3% Triton X-100 in PBS at room temperature for 30 minutes. The sections were incubated in 5% normal horse serum and 0.3% Triton X-100 in PBS with diluted primary antibodies at 4°C for overnight. After 6 washes over 45 minutes in 0.3% Triton X-100 in PBS, secondary antibodies were added and incubated in 0.3% Triton X-100 in PBS for one hour at room temperature. The slides were washed twice in PBS, mounted in Mowiol mounting media for examination under Zeiss LSM510 META confocal microscope.

For bromodeoxyuridine (BrdU) pulse-labeling, pregnant females were injected intraperitoneally with 100 μg BrdU/gram body weight one hour before they were sacrificed. Embryo processing and anti-BrdU labeling were performed on serial sections as previously described (Deng et al., 2010).

The working dilutions and sources of primary antibodies used in this study were: mouse anti-BrdU (1:50, DSHB #G3G4), mouse anti-ISL1/2 (1:500, DSHB #39.4D5), mouse anti-Ki67 (1:200, BD Pharmingen #550609), goat anti-NEUROD1 (1:500, Santa Cruz #SC-1084), mouse anti-NEUN (1:200, Chemicon #MAB377), mouse anti-POU4F1 (1:500, Santa Cruz #SC-8429), goat anti-POU4F2 (1:500, Santa Cruz #SC-6026), mouse anti-β-Tubulin (1:1000, Covance #MMS-435P). Alexa Fluro-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were used at a dilution of 1:1,000.

RESULTS

Spatiotemporal expression of POU4F1 and POU4F2 in the developing CVG cells

To characterize the expression patterns of POU4F1 and POU4F2 during the development of mouse CVG cells, we performed immunolabeling experiments with anti-POU4F1 and anti-POU4F2 antibodies. When the neuronal progenitors derived from the otic placode began to migrate and delaminate into CVG cells at E9.5, POU4F1 expression was detected in the delaminated CVG cells. Strong POU4F1 expression was visible in many of CVG and a few of facial ganglion (FG) cells (Fig. 1A). The onset of POU4F2 expression occurred after that of POU4F1 and only a very few of POU4F2-expressing cells were found amongst the POU4F1 positive cells in the CVG (Fig. 1C, arrows). At E10.5–11.5, more POU4F1-expressing cells were observed in the delaminated CVG cells and the POU4F2-positive cells also increased in number (Fig. 1D–I). While POU4F1 was expressed in a majority of the delaminated CVG neurons but not in the otic epithelium cells from E9.5–E11.5, POU4F2 expression started in the kernel of CVG cells and co-localized with POU4F1 positive cells (Fig. 1A–I). At E12.5, CVG cells begin to segregate into the vestibular ganglia (VG) and spiral ganglia (SG), and the basic structure and ganglia are settled by E14.5 (Fritzsch et al., 1999). In addition to its expression in the center of the CVG, POU4F2 was detected in a small group of the spiral ganglion neurons near the cochlear duct at E12.5 (Fig. 1L, arrow). Later, POU4F2 expression expanded to almost all spiral ganglion neurons, co-localizing with the POU4F1-expressing cells (Fig. 1O), and was maintained at high levels in both vestibular and spiral ganglia at least P0 (Fig. 1P–X). In contrast, peak POU4F1 expression was detected in nearly all SG cells until E15.5, then the number of POU4F1-expressing cells were gradually reduced (Fig. 1R, arrowheads). At P0, only a few POU4F1-positive cells were visible in the SG compared to the widespread POU4F2 expression in the SG (Fig. 1S–U). In contrast, POU4F1 expression persisted in the VG neurons and was co-localized with POU4F2 expressing (Fig. 1V–X).

Figure 1.

Figure 1

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Expression of POU4F1 and POU4F2 in the developing CVG.

Co-immunolabeling of mouse inner ear cryosections at indicated stages with anti-POU4F1 and anti-POU4F2. A–I: transverse sections; J–X: sagittal sections. POU4F1 is expressed in a majority of the delaminated CVG neurons but not in the otic epithelium from E9.5–E11.5. Co-localization of POU4F1 and POU4F2 is detected at early stages. At E12.5, POU4F1 expression persists in almost all CVG. POU4F2 is observed in a small number of SG cells that also express POU4F1. At E15.5, Prominent expression of POU4F2 in SG is detected. At E17.5 and P0, POU4F1 expression is reduced in the SG while POU4F2 expression is strong in the SG. POU4F1 and POU4F2 are co-expressed in the VG. Scale bars are 50 μm.

Spatiotemporal expression of ISL1 in the developing CVG

Previous studies have shown that in the developing inner ear, ISL1 is expressed throughout neurogenesis and that its expression in the sensory lineage starts at high level and fades out upon the initiation of hair cell differentiation (Li et al., 2004; Radde-Gallwitz et al., 2004). To better characterize ISL1 expression in reference to other transcription factors, we compared the expression of ISL1 and POU4F1 in the neuronal lineage of the developing mouse inner ear. From E9.5–12.5, strong ISL1 expression was detected in all migrating and delaminated CVG cells as well as the FG cells (Fig. 2B,C,E,F,H,I,K,L). ISL1 was also expressed in the epithelial CVG precursors (Fig. 2C,F, arrows) while POU4F1 was only expressed in the delaminated ganglion cells (Fig. 2A,C,D,F). The expression domain of ISL1 was broader than that of POU4F1, particularly in the newly delaminated CVG cells near the otocyst and in the ventral FG, and encompassed all POU4F1-positive CVG cells (Fig. 2A–L).

Figure 2.

Figure 2

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Co-expression of POU4F1 and ISL1 in the developing CVG.

Immunofluorescent co-localization of POU4F1 and ISL1 during the development of the mouse CVG. Inner ear transverse sections (A–I) and sagittal sections (J–U) from embryos at the indicated stages were immunostained with anti-POU4F1 (A, D, G, J, M, P, S) and anti-ISL1(B, E, H, K, N, Q, T). ISL1 is expressed in the CVG precursors in the otic epithelium (arrows in C and F) and in almost all delaminated CVG neuroblasts and differentiated neurons. POU4F1 is co-expressed with ISL1 in the delaminated CVG cells but is not expressed in neural progenitors in the otic epithelium. At later stage (E17.5), POU4F1 expression is down-regulated in the SG (S and U). Scale bars equal to 50 μm.

At E13.5, the SG cell mass and the VG cell mass were completely separated and clearly distinguishable, ISL1 and POU4F1 were co-expressed in all SG and VG cells (Fig. 2M–O). Interestingly, at this stage, ISL1 expression level was lower in the SG than in the VG. On the contrary, POU4F1 expression level was higher in the SG than in the VG (Fig. 2M–O). At E15.5, strong POU4F1 and ISL1 expression was seen in the SG cells (Fig. 2P–R). ISL1 expression was also detected in the cochlear sensory region (Fig. 2Q, 2T). Then, at later stages, POU4F1 expression became down-regulated in the SG cells while strong ISL1 expression was maintained in almost all SG cells (Fig. 2S–U). The above data demonstrate that ISL1 is expressed relatively earlier than POU4F1 in the developing CVG and FG while mostly co-localizing with POU4F1 at later stages in the CVG.

ISL1 and POU4F1 are expressed in both proliferating and post-mitotic CVG cells

We further compared the expression of ISL1 to that of NEUROD1, one of the earliest CVG markers (Kim et al., 2001), during the development of the inner ear neurons. At E10.5, NEUROD1 was expressed in the epithelial CVG precursors and the delaminated CVG cells (Fig. 3B). Its expression was down-regulated when CVG cells exit the cell cycle (Davies, 2007) (arrow in Fig. 3B). ISL1 is co-expressed with NEUROD1 at the base of the otic epithelium and in the delaminated CVG cells but not in the NEUROD1-positive cells located more apically within the otic epithelium (arrows in Fig. 3C). Furthermore, using cell proliferation marker Ki67, we showed that ISL1 and POU4F1 are expressed in proliferating cells as well as in post-mitotic cells in the CVG (Fig. 3D–I), which differs than their restricted expression in the post-mitotic RGC in the retina (Pan et al., 2008).

Figure 3.

Figure 3

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(A–C) Immunolabeling for ISL1 and NEUROD1 in mouse CVG at E10.5 reveals that NEUROD1 is expressed in the CVG precursors in the otic epithelium and delaminated CVG, and is down-regulated when CVG neurons begin to mature (arrow in B). ISL1 is co-expressed with NEUROD1 in cells at the base of the otic epithelium and in the delaminated CVG, but is not expressed in the NEUROD1-positive cells located more apically within the otic epithelium (arrows in C). (D–I) Co-localization of ISL1 (D) and POU4F1 (G) with cell proliferation marker Ki67 (E, H) in mouse CVG at E10.5. ISL1 and POU4F1 are expressed in both proliferating and non-proliferating cells. (J–M) Anti-ISL1 immunolabeling reveals ISL1 expression in the control (J, L), Neurod1-null (K), and Pou4f1-null (M) CVGs at E10.5. Loss of Neurod1 results in a significant reduction in the number of CVG cells. However, the remaining CVG cells in Neurod1-null express ISL1. A–M: transverse sections.

The earlier expression onset of NEUROD1 than ISL1 suggests that Neurod1 might act upstream of Isl1. To test this, we compared ISL1 expression in the CVG cells of wild type and Neurod1-null mice. As is shown in Fig. 3, a severe loss of the CVG cells was observed in the Neurod1-null compared to the control. However, the remaining CVG cells in the Neurod1-null mice expressed ISL1, indicating that ISL1 expression does not exclusively depend on NEUROD1 (Fig. 3J, K). The slightly later onset of POU4F1 expression than ISL1 expression in the developing CVGs suggests that Isl1 is either upstream of or in parallel with Pou4f1 during the CVG development. We thus test if loss of Pou4f1 affects ISL1 expression. As is shown in Fig. 3L and 3M, we found no change in ISL1 expression in the Pou4f1-null CVG in comparison to the control. This result is similar to the regulatory relationship between ISL1 and POU4F2 in the developing RGCs (Pan et al., 2008).

POU4F2 is expressed in NEUROD1-negative differentiated neural cells

The late onset of POU4F2 expression and the down-regulation of NEUROD1 in the center of the CVG (Fig. 1B,E,H and 3B) imply that POU4F2 expression highlights neuronal differentiation and maturation starting in the central CVG. Thus, we further examined the differential expression of NEUROD1 and POU4F2. Co-immunolabeling experiments showed that POU4F2 is only expressed in NEUROD1-negative CVG cells (Fig. 4A–I, arrows). The monoclonal antibody against neuronal class III β-Tubulin (Tuj1) recognizes the differentiated neural processes when tissue is heavily fixed with paraformaldehyde (Molea et al., 1999; Stone et al., 2003) and is useful to identify the differentiated CVG neurons. As is shown in Fig. 4J–L, the differentiated CVG neurons were Tuj1-positive and NEUROD1-negative and were positioned in the center of the CVG, which contrasts the relatively undifferentiated Tuj1-negative neural precursors and migrating CVG cells. These results demonstrate that NEUROD1 expression is robust in neural precursor as they travel through the mesoderm and wanes as the neurons begin to mature.

Figure 4.

Figure 4

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POU4F2 is expressed in NEUROD1-negative CVG cells.

(A–I) Co-immunolabeling of transverse sections across the otic vesicles for POU4F2 and NEUROD1 at E9.5 (A–C), E10.5 (D–F), and E11.5 (G–I) shows that during early development of mouse CVG, POU4F2 expression is present in NEUROD1-negative, differentiated neural cells. (J–L) Co-immunolabeling for βIII tubulin (J) and NEUROD1 (K) in transverse sections across the mouse otocyst at E11.5. NEUROD1 immunoreactivity is highest in the nuclei of neural progenitors in the otic epithelium and in delaminating CVG cells, and is down-regulated as the neurons within the center of the CVG mature and express βIII tubulin (K, L). (M–O) Co-labeling with anti-POU4F2 and anti-BrdU in transverse sections reveals that POU4F2 is not expressed in proliferating cells. Scale bars are 50 μm.

In contrast to NEUROD1 expression pattern, POU4F2 expression is post-mitotic (BrdU-negative) and is only detected in differentiated, maturing CVG neurons (Fig. 4M–O, arrow). Thus, POU4F2 can be served as an excellent marker for the post-mitotic differentiated CVG neurons. Since NeuN is a mature neurons marker throughout the nervous system of mice (Cardona et al., 2006; Hafezparast et al., 2003; Karsten et al., 2006; Mullen et al., 1992), we also compared the spatiotemporal expression pattern of POU4F2 and NeuN during CVG development. Our results showed that POU4F2 and NeuN are co-localized within the center of CVG cells at early stages. When CVGs were separated to two groups, POU4F2 and NeuN are co-expressed in the vestibular and spiral ganglion neurons throughout development (Fig. 5).

Figure 5.

Figure 5

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Co-expression of POU4F2 and NeuN in the developing CVG.

Double immunostaining of E11.5 (A–C), E15.5 (D–F), E17.5 (G–I), P1 (J–O) sections across the inner ear with anti-POU4F2 and anti-NeuN shows that throughout the development of mouse CVG, POU4F2 is co-expressed with NeuN in CVG neurons. A–C: transverse sections; D–O: sagittal sections.

In summary, our study demonstrates a sequential expression order of NEUROD1>ISL1>POU4F1>POU4F2 during the inner ear neurogenesis (Fig. 6). Our data confirmed that NEUROD1 is expressed in the CVG precursors in the otic epithelium and in the delaminated CVG cells. NEUROD1 expression becomes down-regulated when neurons in the central CVG begins to mature. ISL1 expression starts in the otic epithelium and overlaps with NEUROD1 in cells at the base of the otic epithelium and in the delaminated CVG but not in the NEUROD1-positive cells located more apically within the otic epithelium. POU4F1 expression largely overlaps with ISL1 in the delaminated CVG neuroblasts at early stages. Unlike ISL1’s persistent expression in the CVG lineage at later stages, POU4F1 expression is maintained in the VG neurons but is down-regulated in a majority of the SG neurons at later stages. The expression of POU4F2 starts behind POU4F1 in the central CVG and is expressed only in the post-mitotic, differentiated CVG cells. POU4F2-expressing neurons are NEUROD1-negative but co-express NeuN in the early CVG cells and the later SG as well as the VG cells. Therefore, our expression study shows that the differential expression of NEUROD1, ISL1, POU4F1, and POU4F2 serves as excellent biomarkers to identify CVG neurons through various developmental stages.

Figure 6.

Figure 6

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Schematic summary of NEUROD1, ISL1, POU4F1, and POU4F2 expression in the developing mouse CVG.

When neuronal cells emanate from the otic epithelium and delaminate to form CVG at E9.5–E11.5, NEUROD1 (yellow) is expressed transiently in the epithelial CVG precursors and in delaminated CVG neuroblasts, but is not expressed in terminally differentiated CVG.

ISL1 (red) is expressed in almost all delaminated CVG neuroblasts and is co-expressed with NEUROD1 in the nuclei at the base of the epithelium and delaminated CVG, but not in the NEUROD1-positive cells located more apically within the otic epithelium.

POU4F1 (green) is not expressed in the epithelial CVG precursors but is co-expressed with ISL1 in the delaminated CVG.

Expression of POU4F2 (blue) is only observed in the NEUROD1-negative, post-mitotic CVG neuron.

Furthermore, our genetic analysis suggests that Isl1 and Pou4f1 might function in parallel during CVG neurogenesis. In the retina, ISL1 and POU4F2 are co-expressed in the differentiating RGCs and both play essential roles in RGCs (Elshatory et al., 2007; Pan et al., 2005; Badea et al., 2009; Pan et al., 2008). Targeted deletion of Isl1 or Pou4f2 each results in the loss about 70% RGCs loss during development in mice while knocking out both Isl1 and Pou4f2 abolishes RGC development, demonstrating a synergistic function of ISL1 and POU4F2 (Gan et al., 1996; Pan et al., 2008). While it is known that POU4F1 is essential for the target field innervations and path-finding of CVG neurons (Huang et al., 2001), the role of ISL1 in CVG neurogenesis is not clear. It is possible that similar to its role in RGC development, ISL1 could function synergistically with POU4F1 to regulate CVG differentiation and survival. Future experiments to conditionally delete Isl1 specifically in the inner ear will help address this issue.

Highlights.

  • A comparative analysis of the spatiotemporal expression of POU4F1, POU4F2, and ISL1 during mouse inner ear neurogenesis;

  • ISL1 expression starts in the otic epithelium and precedes POU4F1 expression in cochleovestibular ganglion (CVG) neurons;

  • POU4F1 is co-expressed with ISL1 and NEUROD1 in delaminated CVG neurons;

  • POU4F2 expression starts after that of POU4F1 and ISL1, and is expressed in post-mitotic, NEUROD1-negative CVG neurons;

  • The temporal expression order of these transcription factors in mouse CVG neurogenesis is NEUROD1>ISL1>POU4F1>POU4F2.

Acknowledgments

We thank Drs. Amy Kiernan, Richard Libby, Patricia White and the other members of the Gan laboratory for helpful discussions and technical assistance. This work was supported by National Institutes of Health Grant DC008856, National Natural Science Foundation of China grant 81271006, Hangzhou City Health Science Foundation grant No. 20120633B25, Zhejiang Province Science Grant No. 2012C13023-1, and the Research to Prevent Blindness Challenge Grant to the Department of Ophthalmology at the University of Rochester.

Footnotes

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