Developmental Biology 411 (2016) 1–14

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Developmental Biology

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A central to peripheral progression of cell cycle exit and differentiation in the developing mouse cristae

Amber D. Slowik a,b, Olivia Bermingham-McDonogh a,c,n a Department of Biological Structure, University of Washington, Seattle, WA 98195, United States b Graduate Program in Neuroscience, University of Washington, Seattle, WA 98195, United States c Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA 98109, United States article info abstract

Article history: The inner contains six distinct sensory organs that each maintains some ability to regenerate hair Received 22 January 2016 cells into adulthood. In the postnatal , there appears to be a relationship between the develop- Accepted 26 January 2016 mental maturity of a region and its ability to regenerate as postnatal regeneration largely occurs in the Available online 28 January 2016 apical turn, which is the last region to differentiate and mature during development. In the mature cristae there are also regional differences in regenerative ability, which led us to hypothesize that there may be a general relationship between the relative maturity of a region and the regenerative competence of that region in all of the sensory organs. By analyzing adult mouse cristae labeled embry- onically with BrdU, we found that hair cell birth starts in the central region and progresses to the per- iphery with age. Since the peripheral region of the adult cristae also maintains active Notch signaling and some regenerative competence, these results are consistent with the hypothesis that the last regions to develop retain some of their regenerative ability into adulthood. Further, by analyzing embryonic day 14.5 inner we provide evidence for a wave of hair cell birth along the longitudinal axis of the cristae from the central regions to the outer edges. Together with the data from the adult inner ears labeled with BrdU as embryos, these results suggest that hair cell differentiation closely follows cell cycle exit in the cristae, unlike in the cochlea where they are uncoupled. & 2016 Elsevier Inc. All rights reserved.

1. Introduction differentiation of the various cell types, have suggested several potential strategies to stimulate hair cell regeneration in the inner The sensory modalities of and balance depend on the ear sensory organs (reviewed in Atkinson et al. (2015)). For ex- six sensory organs of the inner ear that are each comprised of the ample, hair cells can be produced through the transdifferentiation same two main cell types, support cells and mechanosensory hair of support cells following inhibition of Notch signaling (Hori et al., cells. The contains the within the 2007; Jung et al., 2013; Lin et al., 2011b; Mizutari et al., 2013; and the contains the gravity sen- Slowik and Bermingham-McDonogh, 2013), which devel- sing utricular and saccular maculae and the three rotation-sensing opmentally determines the precise ratio of support cells and hair cristae ampullaris. There is currently no therapeutic treatment to cells through lateral inhibition (Kiernan et al., 2005; Lanford et al., replace lost sensory hair cells which, depending on the inner ear 1999; Takebayashi et al., 2007; Yamamoto et al., 2006; Zhang et al., organ affected, leads to permanent hearing loss and/or balance 2000; Zheng et al., 2000; Zine et al., 2001). However, just as in disorders such as vertigo. In some cases, such as Usher Syndrome other neural systems, the level of regeneration is low and the Type1, both auditory and vestibular hair cells are affected and determinants for the regenerative competence of individual sup- these individuals have profound deafness and balance disorders at port cells are poorly understood. birth (Cosgrove and Zallocchi, 2014). Here, we define the spatial patterns of hair cell development in Studies of the development of the sensory organs, particularly order to better understand the increasing limitations on re- the specification of the sensory regions and the cues governing the generative ability as the inner ear matures. Developmentally, al- most every support cell in the inner ear can be induced to trans- differentiate (Burns et al., 2012a; Collado et al., 2011; Hayashi et al., n Corresponding author at: Department of Biological Structure, University of Washington, Seattle, WA 98195, United States. 2008; Lanford et al., 1999; White et al., 2006; Yamamoto et al., E-mail address: [email protected] (O. Bermingham-McDonogh). 2006; Zhao et al., 2011; Zine et al., 2001); however, later, only an http://dx.doi.org/10.1016/j.ydbio.2016.01.033 0012-1606/& 2016 Elsevier Inc. All rights reserved. 2 A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 increasingly restricted subset of cells retains the ability to trans- two washes in 0.1 M sodium borate (pH 8.5, EMD Millipore) for differentiate. For example, as the cochlea matures, hair cell re- 10 min to neutralize the acid. The cristae were then rinsed in PBS generation is increasingly restricted until it primarily occurs in the and blocked in a solution containing 10% normal donkey serum apical turn (Bramhall et al., 2014; Cox et al., 2014; Doetzlhofer (EMD Millipore), 4% bovine serum albumin (Sigma-Aldrich), and et al., 2009; Kelly et al., 2012; Li et al., 2015; Liu et al., 2012, 2014; 100 mM glycine (Fisher Scientific) in 0.5% PBSTx for 3 h at RT. Both Maass et al., 2015; Shi et al., 2013; Walters et al., 2014; Yamamoto the primary and secondary antibody solutions were made in the et al., 2006; Zheng and Gao, 2000). The fact that the apical turn is blocking solution and applied O/N at 4 °C. Cristae were washed the last region in the cochlea to differentiate and mature (Chen between solutions using 0.5% PBSTx. Hoechst 33342 (1:10,000, Life et al., 2002; Lanford et al., 2000; Lim and Anniko, 1985; Sher, 1971; Technologies) was added to the secondary antibody solution to Woods et al., 2004) suggests that there is a correlation between label all nuclei. relative maturity and regenerative ability in the cochlea. In the After immunolabeling, samples were dehydrated in 70% etha- adult cristae, there are also regional differences in hair cell re- nol overnight, followed by 95% ethanol for 30 min, and then two generation and in the expression of Notch signaling components incubations with pure ethanol for two hours each. After dehy- (Lopez et al., 1997; Slowik and Bermingham-McDonogh, 2013). In dration, samples were incubated in a 1:1 mixture of methyl sali- particular, peripheral support cells maintain active Notch signaling cylate (Fisher Scientific) and benzyl benzoate (Sigma-Aldrich) and can transdifferentiate in response to Notch inhibition in the (MSBB) (MacDonald and Rubel, 2008) and pure ethanol for four adult. Since the peripheral region maintains some regenerative hours. Samples were then cleared using three changes of MSBB for ability into adulthood, we hypothesized that, similar to the co- 2 h, 4 h, and overnight. For imaging, samples were mounted in the chlea, the relative maturity and regenerative ability of a region MSBB solution on 1 mm thick rubber imaging spacers (Coverwell would be linked in the cristae and that the peripheral region PCI-A-2.0, Grace Bio-Labs). would differentiate last during development. The following primary antibodies were used: BrdU (rat, 1:200, Using embryonic injections of BrdU, we show in all three Accurate), Gfi1 (guinea pig, 1:1000, gift from Dr. Hugo J. Bellen, cristae that hair cell birth largely begins in the central region and Baylor College of Medicine, Houston, TX), Sox2 (goat, 1:400, Santa shifts with age towards the periphery, consistent with previous Cruz), and Myosin7a (rabbit, 1:500, Proteus Biosciences). The fol- data from the rat horizontal (lateral) crista (Sans and Chat, 1982). lowing secondary antibodies were used: donkey anti-rat Alexa In addition, by analyzing hair cell markers in E14.5 inner ears we Fluor 488 (Life Technologies), donkey anti-goat Alexa Fluor 568 provide evidence for a gradient of hair cell differentiation along (Life Technologies), donkey anti-guinea pig DyLight 649 (Jackson the longitudinal axis of the cristae. Together, these data support ImmunoResearch), and donkey anti-rabbit Alexa Fluor 649 (Life our hypothesis that regenerative competence is correlated with Technologies). To preserve fluorescence, samples were mounted in relative maturity and suggest that hair cell differentiation is cou- Fluoromount-G (SouthernBiotech). pled to cell cycle exit in the developing cristae. We used a modified procedure for the analysis of embryonic inner ear. The inner ears were fixed, decalcified in 10% ethylene- diamine tetraacetic acid-disodium salt (EDTA, Fluka Analytical) in 2. Methods PBS, pH 7.4, at 4 °C. Samples were changed into a new solution of 10% EDTA each day. After four days, the samples were rinsed in 2.1. Animals and BrdU injections PBS, immunolabeled and then cleared after immunolabeling in MSSB. Animal housing and care was provided by the Department of Samples were imaged using a Nikon A1R laser scanning con- Comparative Medicine at the University of Washington. All pro- focal mounted on a Nikon TiE inverted microscope. Image stacks cedures were done in compliance with the standards and proto- were taken in NIS Elements at a step size of 0.875 μm using a 20x cols set forth by the University of Washington Institutional Animal dry CFI Plan Apochromat VC objective with a numerical aperture Care and Use Committee. (NA) of 0.75. In order to birthdate cells, a single intraperitoneal injection of 5-bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich, 1.25 mg/25 g mouse) in sterile phosphate buffered saline (PBS, Calbiochem) was 4. Normalization of cristae position administered to time-mated pregnant Swiss Webster females (Harlan Laboratories, stock no. 032) at either 9.5, 12.5, 15.5, or 17.5 For each confocal stack, the standard cell counter plugin in days after their plug-date. The age of the embryos at the time of ImageJ was used to mark the position of the BrdU-labeled hair injection was determined by plug date and confirmed by birth- cells (double positive for BrdU and Myosin7a) in addition to the date, assuming a gestation period of 19 days. Analyses were per- position of the majority of Myosin7a-positive hair cells. This in- formed using both genders. formation was then exported into two separate lists of xyz-co- ordinates, one containing the BrdU-positive hair cells and the other containing the Myosin7a-positive hair cells for that same 3. Immunofluorescence and imaging crista. Once these coordinate lists were obtained, they were run through a custom python script [Python(x,y)] that normalized the Mice that received embryonic BrdU injections were euthanized position of the crista in all three dimensions by rotating all of the either at postnatal day 30 (P30) or embryonic day 14.5 (E14.5). The coordinates in both lists. inner ear capsule was removed from the head, rinsed in PBS, fixed In order to determine the degree of rotation along the z-axis, overnight (O/N) in cold 4% paraformaldehyde (PFA, Electron Mi- the script found the median y-value of the cells in a 40 pixel range croscopy Sciences), and then rinsed in PBS. For the animals sa- around the x-coordinates at the 25th and 75th percentiles (ap- crificed at P30, cristae were dissected from the capsule and stained proximately 0.56 μm/pixel). The slope of the line between the two as free floating whole mounts in mesh tissue baskets. median values was calculated and the angle, theta, between this For the BrdU antibody staining, P30 cristae were permeabilized line and a line with a slope of 0 was determined. If theta was in 0.5% Triton-X in PBS (PBSTx) for 30 min at room temperature greater than þ/0.07°, then the coordinates in both lists were (RT) and then antigen retrieval was performed using 2 M hydro- rotated by theta. chloric acid (HCl, Fisher Scientific) for 30 min at 37 °C followed by Almost identical functions were performed for rotations along A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 3 the y-axis and the x-axis except the script found the median along positive hair cells in each bin was then counted and exported. For the z-axis. For y-axis rotations, the cutoff value for theta was an analysis along the z-axis, which is equivalent to digitally un- þ/0.018°.Forx-axis rotations, the range of cells used to de- folding the cristae and analyzing the y-axis, hair cells were divided termine the two median values was expanded. Here, the hair cells into four evenly distributed bins based on their position along the were divided into two halves along the y-axis and the median z- z-axis. The total number of BrdU-positive hair cells in each bin was value for each half was calculated. In addition, the cutoff value for then counted and exported. theta for rotations along the x-axis was þ/0.034°. All rotations Prism v5.0c (GraphPad, La Jolla, CA) was used to create graphs were visually verified by plotting both the raw coordinates and the and perform statistical analyses. All analyses were two-way AN- rotated coordinates in 2-dimensions by flattening the dimension OVAs with Bonferroni post-tests. The error bars on all graphs are of rotation. standard error of the mean (SEM) and significance is denoted as follows: * for pr0.05, ** for pr0.01, *** for pr0.001, and **** for pr0.0001. The symbol, n, denotes the number of cristae analyzed. 5. Spatial and statistical analyses Data from each developmental stage were obtained from at least one whole litter of pups that received BrdU at that stage. Cristae To analyze the spatial distribution of the BrdU-positive hair dissected from the ears of the litter were processed as a group and cells, a python script was again used in order to divide the hair selected for analysis based on the orientation of the cristae after cells into bins based on their position. For an analysis along the x- mounting on a glass slide. To minimize the effects of normalization axis, hair cells were divided into ten evenly distributed bins based and processing, only cristae with relatively flat orientations were on their position along the x-axis. The total number of BrdU- selected.

Fig. 1. Birthdating hair cells using embryonic injections of BrdU. A) A diagram of the experimental paradigm showing when the embryos received BrdU and when the inner ear sensory tissue was harvested. Swiss Webster females were given a single injection of BrdU between the 9th and 18th days of their gestation, and harvested at P30. B) The age of the embryos at time of injection shown against a replotting of data from a birthdating study of the inner ear by Ruben (1967) showing when hair cells in each of the three cristae exit the cell cycle. C) A plot of the Myo7a-positive hair cells and the BrdU-positive hair cells in a posterior crista (from panel G, includes the ‘bridge’ of hair cells spanning the normally non-sensory region) showing the regions referred to as central (gray band) and peripheral. D) For the longitudinal axis, medial refers to the regions near the crux eminentia while lateral refers to the regions near the planum semilunatum. E–H) Cristae for each age were immunolabeled for BrdU (yellow), hair cells (Myosin7a, cyan) and nuclei (Hoechst 33342, magenta). Examples of posterior cristae from each age are shown in three different views. The single z-slice shows the amount of BrdU labeling within the sensory epithelium; white arrowhead points to an incomplete crux eminentia and white arrow shows a complete crux eminentia. The bottom panels show the coordinate pairs for the Myosin7a-positive hair cells (cyan) and the BrdUþ hair cells (yellow) exported from the standard cell counter plugin in ImageJ. 4 A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14

Prism was also used to create the visual representations of the was performed using tritiated-thymidine (Ruben, 1967). According data. In order to visually compare cristae, they were aligned using to this study, embryonic day 9.5 (E9.5) is well before the onset of a python script that created a scaled dataset for each crista with an cell cycle exit for hair cells in the cristae, making it a good baseline arbitrary height of 500 pixels and an arbitrary width of 1000 control. The onset of hair cell birth is covered by the injection at pixels. These scaled datasets were then plotted as xy points in E12.5 while the injection at E15.5 occurs after the peak of hair cell Prism and colored based on z-depth or to distinguish individual birth. The peak of hair cell birth was deliberately avoided because samples. This rescaling of the data only affects the visual depic- many of the regions of the cristae would likely have hair cells born tions of the data and does not affect the analyses as the bins were during that time. The last injection at E17.5 occurs after most of evenly distributed along the analyzed axis, making the size of the the hair cells are born and identifies the regions of the cristae that bins relative to the size of the crista. are the last to exit the cell cycle (Fig. 1B). Examples of the BrdU labeling in the posterior cristae are shown in Fig. 1E–H. In the cristae that received the BrdU injection 6. Results at E9.5, there were no BrdU-positive hair cells in the sensory epithelium and very few BrdU-positive cells in the surrounding 6.1. BrdU injections to birthdate hair cells tissue (Fig. 1E). The BrdU injection at E12.5 resulted in substantial numbers of BrdU-positive hair cells in the central regions of the In order to determine the spatial pattern of hair cell develop- cristae (Fig. 1F). When the BrdU injection was given three days ment in the mouse cristae, cells were birthdated with a single later, at E15.5, the labeled hair cells were predominantly located in embryonic injection of 5-bromo-2′-deoxyuridine (BrdU) and ana- the peripheral regions of the cristae (Fig. 1G). Lastly, injections at lyzed once the animals reached one month of age (Fig. 1A). The E17.5 resulted in few BrdU-positive cells in the sensory epithelium embryonic ages for injection were chosen to highlight specific (as seen in the single z-slice), although there were many labeled stages of crista development based on data from a birthdating cells in the surrounding tissue (as seen in the maximum intensity study of the inner ear in which a similar experimental paradigm projection) (Fig. 1H). While examining the posterior cristae, we

Fig. 2. Qualitative group data for all three cristae. A) The number of BrdU-positive hair cells for each cristae type (colored circles) follows the same trend with age as published by Ruben (1967) using tritiated-thymidine (gray fill, replotted from original publication). Error bars depict standard error of the mean (SEM). B–D) Plots of example data for each age for the posterior (B), anterior (C), and horizontal (D) cristae show the overall trends of hair cell birth. The gray points show all of the Myosin7a-positive hair cells from the cristae of that type (posterior, anterior, or horizontal) irrespective of age, while the colored points represent data from a single crista of that type and age (4 posterior cristae are plotted (green, orange, blue and violet), and 3 anterior and horizontal cristae are plotted (green, orange, and blue). A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 5

Table 1 A summary of the numerical data for the comparison of the total number of birthdated cells here using BrdU versus the study by Ruben (1967) using tritiated-thymidine.

The total number of BrdUþ hair cells by age in each crista type

Posterior Anterior Horizontal

Mean SEM n Mean SEM n Mean SEM n

E9.5 0.00 7 0.00 6 0.00 7 0.00 6 0.00 7 0.00 5 E12.5 135.75 7 7.32 12 173.50 7 7.01 4 94.00 7 15.24 4 E15.5 111.00 7 7.49 6 147.20 7 10.81 5 146.50 7 10.83 6 E17.5 40.75 7 5.20 4 74.33 7 5.61 3 69.00 7 5.51 3

SEM – Standard error of the mean. noticed that a minority of the posterior cristae had an incomplete frequency of approximately 1:3–4 and may be unique to Swiss crux eminentia with hair cells spanning the entire length of the Webster mice, as we have not observed this phenomenon in cristae on one side (Fig. 1E arrowhead vs Fig. 1H arrow). Normally, C57BL/6 or CBA/CaJ mice (data not shown). Further, the in- the crux eminentia of the posterior and anterior cristae is a com- complete crux appears early in development, as immunolabeling pletely hair cell free region that divides the cristae into two se- of E14.5 inner ears with the nuclear hair cell marker, Gfi1, also parate hemicristae. This incomplete crux eminentia occurred at a shows an incomplete crux eminentia in a similar subset of

Fig. 3. Hair cell birth shifts from the central to peripheral regions with age. A) Cristae were divided into four evenly distributed bins along the z-axis using the Myosin7a- positive hair cells shown in gray. The number of BrdU-positive hair cells (shown by color based on z-depth with cooler colors at the top of the cristae and warmer colors at the bottom) was counted for each bin. B) Plots of the cristae color-coded for z-depth. C–E) The same plots from Fig. 2A–C showing example data for each age for the posterior (C), anterior (D), and horizontal (E) cristae here color coded for z-bin in the color order from top to bottom of blue, green, orange, red. F–H) Graphs of the mean number of BrdU-positive hair cells in each bin show the pattern of hair cell birth with age in the posterior (F), anterior (G), and horizontal (H) cristae. Error bars depict SEM. I-K) Graphs depicting the distribution of BrdU-positive hair cells in each bin as a percentage of the total number of BrdU-positive hair cells in that crista. Asterisks in panels F-K indicate significance from two-way ANOVAs with Bonferroni post-tests comparing the bins of E12.5 to E15.5 and of E15.5 to E17.5 (po0.05 – *, po0.01 – **, po0.001 – ***). 6 A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 posterior cristae (data not shown). cristae into four evenly distributed bins along the z-axis using the The summaries of the qualitative trends for both the number Myosin7a-positive hair cells and counted the number of BrdU- and the spatial pattern of BrdU-labeled cells for each age of BrdU positive hair cells in each bin. injection are shown in Fig. 2. The pattern of BrdU-positive hair In each type of crista, the quantitative analysis supports the cells labeled at each age agreed with previous data using tritiated- qualitative assessment that there is a central to peripheral pro- thymidine (Ruben, 1967)(Table 1)(Fig. 2A, colored dots plotted gression of hair cell birth during development. In the posterior against Ruben's data shown by the gray shaded area). The spatial cristae, the hair cells born at E12.5 were found largely in the most patterns of hair cell birth can be seen in the normalized group data central bins (Fig. 3C). At E15.5, hair cell birth shifted more per- from a select number of crista for each age and crista type (pos- ipherally into the more intermediate bins, indicated by the de- terior, anterior, and horizontal) shown in Fig. 2B–D. The gray dots crease in BrdU-positive hair cells in the central bins and the con- represent all of the Myosin7a-positive cells for all cristae of that comitant increase in BrdU-positive hair cells in the peripheral bins type (posterior, anterior, or horizontal) irrespective of age, while (Tables 2 and 3)(Fig. 3F and I). At E17.5, the overall number of the colored dots represent data from an individual crista of that BrdU-positive hair cells was decreased, with significant reductions age and type. For example, in Fig. 2B, the gray background is in the number of hair cells in the intermediate bins and a sig- identical for each of the three ages because it encompasses the nificant increase in the percentage of BrdU-positive hair cells in hair cell data for all posterior cristae. The colored data for each the most peripheral bin (Tables 2 and 3)(Fig. 3F and I). crista is then overlaid on top to allow for a comparison of the The anterior cristae showed a very similar trend. At E12.5, the spatial patterns in posterior cristae between ages. Qualitatively, it majority of the BrdU-positive hair cells were in the central bins appears that hair cells are born in the more central regions in all (Fig. 3D). At E15.5, there was a decrease in the BrdU-positive hair three cristae types at E12.5 and cell generation shifts to the more cells in the most central bin and an increase in the BrdU-positive peripheral regions at both E15.5 and E17.5 (Fig. 2B–D). hair cells in the peripheral bin (Tables 2 and 3)(Fig. 3G and J). At E17.5, there was a shift from the intermediate to the peripheral 6.2. Hair cell birth on the central to peripheral axis regions with a significant decrease in cells in the central bin and a significant increase in cells in the most peripheral bin To quantitatively analyze the shift in regional hair cell devel- (Tables 2 and 3)(Fig. 3G and J). opment from the central to peripheral regions of the cristae, we Lastly, the horizontal cristae showed a similar trend to both the performed an analysis along the z-axis. Due to the highly curved anterior and posterior cristae, but in general, had fewer BrdU-labeled shape of the cristae, we found that this axis provided a better hair cells at each age. This is due to the fact that the horizontal cristae spatial representation of the central and peripheral regions than have fewer hair cells in the adult than the other two cristae types, and an analysis of the y-axis for all three cristae types (Fig. 3A and B). the percentages of BrdUþ hair cells are similar across the types of Because this analysis is used to digitally “unfold” or flatten the cristae, with 21.1% in the posterior, 29.1% in the anterior, and 25.2% in cristae, we refer to the top of the cristae as being central and the thehorizontalcristae.Nonetheless,atE12.5,themajorityofBrdU- bottom as being peripheral. For this analysis, we divided the positive hair cells were still found within the more central bins

Table 2 A summary of the numerical data for the analysis of the spatial pattern of hair cell birth along the medial to lateral axis (z-axis). BrdU-positive hair cells were grouped into four bins according to their position along the z-axis. These bins are identified in order of most central to most peripheral by color as blue, green, orange, and red.

Numerical summary of the number and percentage of BrdUþ hair cells along the z-axis

POSTERIORS

E12.5 (n¼9) E15.5 (n¼6) E17.5 (n¼4)

Mean [# (%)] SEM [# (%)] Mean [# (%)] SEM [# (%)] Mean [# (%)] SEM [# (%)]

Bin colora Red 0.44 (0.3) 7 0.24 (0.2) 18.83 (16.47) 7 3.89 (2.6) 12.25 (31.45) 7 1.44 (2.7) Orange 12.67 (9.56) 7 3.03 (2.2) 43.67 (39.99) 7 3.99 (3.9) 12.50 (29.87) 7 3.52 (5.8) Green 64.11 (49.43) 7 6.28 (3.0) 38.00 (34.26) 7 5.47 (4.3) 11.00 (26.32) 7 3.39 (6.2) Blue 51.11 (40.7) 7 4.85 (3.9) 10.33 (9.28) 7 3.00 (2.8) 4.25 (12.36) 7 0.85 (4.4)

ANTERIORS E12.5 (n¼4) E15.5 (n¼5) E17.5 (n¼3)

Mean [# (%)] SEM [# (%)] Mean [# (%)] SEM [# (%)] Mean [# (%)] SEM [# (%)]

Bin colora Red 0.00 (0.00) 7 0.00 (0.0) 20.00 (13.41) 7 5.48 (3.2) 34.00 (46.09) 7 6.66 (6.1) Orange 10.50 (5.94) 7 2.02 (1.0) 54.00 (36.05) 7 10.90 (5.9) 23.00 (30.88) 7 8.51 (10.3) Green 86.25 (49.95) 7 8.66 (5.3) 64.00 (44.05) 7 8.57 (5.6) 13.33 (19.56) 7 4.98 (7.8) Blue 76.75 (44.12) 7 10.05 (5.2) 9.20 (6.50) 7 6.48 (4.6) 2.33 (3.48) 7 1.45 (2.1)

HORIZONTALS E12.5 (n¼4) E15.5 (n¼4) E17.5 (n¼3)

Mean [# (%)] SEM [# (%)] Mean [# (%)] SEM [# (%)] Mean [# (%)] SEM [# (%)]

Bin colora Red 0.50 (0.48) 7 0.29 (0.3) 23.25 (15.07) 7 4.19 (2.8) 22.67 (31.65) 7 9.06 (11.7) Orange 14.75 (15.74) 7 5.11 (5.1) 56.25 (36.69) 7 10.05 (7.2) 24.00 (34.55) 7 5.00 (5.8) Green 51.50 (56.25) 7 9.51 (9.1) 61.50 (38.80) 7 12.06 (6.3) 18.00 (27.81) 7 6.51 (11.9) Blue 27.25 (27.53) 7 6.91 (5.5) 15.50 (9.45) 7 6.96 (4.0) 3.67 (5.99) 7 3.18 (5.4)

# – Number, % – Percentage. a The red bin represents the most peripheral region and the blue bin represents the most central region. A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 7

Table 3 For the posterior cristae, at E12.5, hair cell birth was evenly dis- A summary of the statistical data for the analysis of the spatial pattern of hair cell tributed along the longitudinal axis (Fig. 4B, green). At E15.5, there birth along the central to peripheral axis (z-axis). BrdU-positive hair cells were was a significant decrease in the number of hair cells born in the grouped into four bins according to their position along the z-axis. These bins are identified in order of most central to most peripheral by color as blue, green, or- medial bin spanning the crux eminentia (Fig. 4B, yellow). At E17.5, ange, and red. Reported p-values come from two-way ANOVAs with Bonferroni there was a significant reduction in the number of hair cells born post-tests comparing both the number and the percentage of BrdU-positive hair in the remaining regions of the cristae, such that the levels along cells from E12.5 to E15.5 and E15.5 to E17.5. The symbol, ns, denotes that the the longitudinal axis were again evenly distributed (Fig. 4B, pur- comparison was not significant. ple). Overall, the spatial patterns of hair cell birth along the Statistical summary of the number and percentage of BrdUþ hair cells along longitudinal axis are not very strong during these ages of devel- the z-axis opment; however, in the posterior cristae the decrease in hair cell birth begins in the regions near the central crux eminentia at E15.5 POSTERIORS and progresses to the regions near the peripheral planum semi- # – Bins: F ¼17.69 po0.0001 Age: F ¼23.63 po0.0001 Interaction: 3,48 2,16 lunatum at E17.5. This “off wave” of hair cell birth along the F6,48 ¼23.46 po0.0001

% – Bins: F3,48¼13.51 po0.0001 Age: F2,16¼0.00 p¼1.00 Interaction: longitudinal axis is indicative of an earlier wave of hair cell birth ¼ o F6,48 20.98 p 0.0001 along this axis. E12.5 vs E12.5 vs E15.5 vs E15.5 vs A similar pattern of hair cell birth along the longitudinal axis E15.5 (#) E15.5 (%) E17.5 (#) E17.5 (%) was present in the anterior cristae. At E12.5, hair cell birth was Bin colora Red t¼3.198, t¼3.555, ns t¼2.690, fairly evenly distributed along the longitudinal axis, with a slight po0.01 po0.01 po0.05 reduction in the two medial bins that is likely due to the position Orange t¼5.392, t¼6.693, t¼4.426, ns of the crux eminentia, which is always present and complete in o o o p 0.001 p 0.001 p 0.001 anterior cristae (Fig. 4C, green). Similar to posterior cristae, the Green t¼4.542, t¼3.337, t¼3.834, ns po0.001 po0.01 po0.01 number of hair cells born in the anterior cristae decreased at E15.5 Blue t¼7.093, t¼6.911, ns ns and E17.5. In addition, the anterior cristae appeared to have a si- po0.001 po0.001 milar “off wave” pattern to the posterior cristae, where the medial ANTERIORS bins decreased at E15.5 and the lateral bins decreased at E17.5

# – Bins: F3,27¼9.84 p¼0.0001 Age: F2,9 ¼26.01 p¼0.0002 Interaction: (Fig. 4C, yellow and purple). ¼ o F6,27 14.70 p 0.0001 The horizontal cristae, which do not have a crux eminentia, – ¼ ¼ ¼ ¼ % Bins: F3,27 6.41 p 0.002 Age: F2,9 2.77 p 1.00 Interaction: showed a somewhat unique pattern of hair cell birth, in that there F ¼13.53 po0.0001 6,27 is a greater difference between E12.5 and E15.5 in the horizontal E12.5 vs E12.5 vs E15.5 vs E15.5 vs cristae than the other cristae. In addition, the distribution at each E15.5 (#) E15.5 (%) E17.5 (#) E17.5 (%) age showed a greater degree of asymmetry. At E12.5, hair cell birth a Bin color Red ns ns ns t¼4.308, was unevenly distributed towards one side of the cristae (dorsal), po0.001 shown as the right side (Fig. 4D, green). At E15.5, the bins re- Orange t¼4.268, t¼4.321, t¼2.794, ns po0.001 po0.001 po0.05 presenting the left side of the cristae increased such that hair cell Green ns ns t¼4.567, t¼3.228, birth was fairly evenly distributed at this age, and this is reflected po0.001 po0.05 in the results from the ANOVA that compares the two ages. Blue t¼6.628, t¼5.398, ns ns po0.001 po0.001 HORIZONTALS 6.4. Differentiation of hair cells in embryonic inner ears

#-Bins: F3,24¼8.59 p¼0.0005 Age: F2,8¼14.95 p¼0.0020 Interaction: ¼ ¼ F6,24 4.01 p 0.0064 In the cochlea, the terminal mitoses of hair cells are uncoupled ¼ ¼ ¼ ¼ ¼ %-Bins: F3,24 7.98 p 0.0007 Age: F2,8 -2.00 p 1.00 Interaction: F6,24 4.05 from their differentiation. In the apical turn, hair cells exit the cell p¼0.0061 cycle around E12.5, but do not begin to differentiate until E16.5, E12.5 vs E12.5 vs E15.5 vs E15.5 vs four days later. To determine if the pattern of differentiation fol- E15.5 (#) E15.5 (%) E17.5 (#) E17.5 (%) lowed the pattern of cell cycle exit in the cristae, E14.5 inner ears Bin colora Red ns ns ns ns were examined for the expression of the early hair cell markers, ¼ ¼ Orange t 4.063, ns t 2.923, ns Gfi1 and Myo6 (Fig. 5). po0.01 po0.05 Green ns ns t¼3.943, ns For both the posterior and the anterior cristae, hair cell differ- po0.01 entiation appeared to begin in the region nearest to the crux Blue ns ns ns ns eminentia. The expression of both Myo6 (Fig. 5C and E) and Gfi1 (Fig. 5C″ and E″) occurred in two distinct patches for each anterior # – Number, % – Percentage, p-values come from a two-way ANOVA with Bonfer- and posterior crista. The non-expressing region between the two roni post-tests. patches appeared to be just the width of a few cell bodies, which is a The red bin represents the most peripheral region and the blue bin represents the most central region. approximately the size of the mature crux eminentia (Fig. 1C–F). In the horizontal crista, which does not have a crux eminentia and is instead one continuous sensory region, hair cell differ- (Fig. 3E). At E15.5, there was a significant increase in the number of entiation appeared to begin towards one side of the sensory re- BrdU-positive hair cells in the peripheral bin. At E17.5, the overall level gion, marked by Sox2 (Fig. 5D–D″). This side is furthest from the of hair cell birth was significantly reduced, specifically in the inter- developing cochlea towards the and the lat- mediate bins (Tables 2 and 3)(Fig. 3HandK). eral portion of the head, and is known as the superior half of the crista. This asymmetric pattern of differentiation is consistent with 6.3. Hair cell birth on the longitudinal axis the pattern of hair cell birth along the longitudinal axis for the horizontal cristae (Figs. 2C and 4D). To analyze the distribution of hair cell birth on the longitudinal Overall, the relative expression patterns and levels of Myo6 and axis, cristae were divided into ten evenly spaced bins using the list Gfi1 agreed with the known order of cell cycle exit and differ- of Myosin7a-positive hair cells and the number of BrdU-positive entiation for the inner ear organs, which from earliest to latest are hair cells in each bin was counted (Fig. 4A, summarized in Table 4). the , , anterior crista, posterior crista, horizontal 8 A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14

Fig. 4. Uniform pattern of hair cell birth along the longitudinal axis. A) Cristae were divided into ten evenly distributed bins based on the x-values of the Myosin7a-positive hair cells shown in gray. The number of BrdU-positive hair cells (colored points) were then counted for each bin. B–D) Graphs of the mean number of BrdU-positive hair cells in each bin show the pattern of hair cell birth with age in the posterior (B), anterior (C), and horizontal (D) cristae. Error bars depict SEM and asterisks indicate significance from two-way ANOVAs with Bonferroni post-tests comparing the bins of E12.5 to E15.5 and of E15.5 to E17.5 (po0.05 – *, po0.01 – **, po0.001 – ***). crista, and cochlea (Ruben, 1967; Sans and Chat, 1982; Sher, 1971). support cells was counted for each bin. The data for the support The saccule had the most Gfi1-positive cells, followed by the cells (Fig. 6B,D,F, and H) is shown alongside the corresponding utricle, the anterior crista, and the posterior and horizontal cristae data for the hair cells in the E12.5 posterior cristae (Fig. 6A,C,E, and (Fig. 5C″,D″,E″,F″, and F‴). In addition, the Myo6 staining was most G) shown in previous figures. distinct in the saccule where the distinctive lamination of hair cells In E12.5 posterior cristae, the spatial pattern of support cell was apparent (Fig. 5F,F′, green). In the utricle, Myo6 appeared to development was very similar to that seen for hair cells (Fig. 6A be localized to distinct cells, but these cells did not exhibit the and B). At this age, both cell types were born in the central region typical hair cell lamination (Fig. 5F,F′, green). In the cristae, the of the cristae and have a fairly even distribution along the long- Myo6 expression was diffuse but located within a portion of the itudinal (medial-lateral) axis (Fig. 6C and D). The exception to this Sox2-positive sensory region (Fig. 5C–E′). The cochlear duct, as was a slight enrichment in the most medial bin for the support expected, was not immunolabeled by either Gfi1 or Myo6 at this cells, which reflects the large number of Sox2-positive non-sen- age. sory cells born in the crux eminentia at this age (Fig. 6B and D). An analysis of the z-axis confirmed that the majority of support cells 6.5. Support cell birth follows a similar pattern were born in central and intermediate regions of the cristae (Fig. 6F and H). This is different from the hair cells, where a higher In the above analyses, we demonstrated that hair cell birth and percentage of the cells born at this age were located in the central differentiation proceed from the central to peripheral regions of (blue) bin (Fig. 6E and G). However, this difference may be partly the developing cristae; however, since we hypothesized that the due to the fact that support cell nuclei are located beneath the hair timing of differentiation underlies regenerative competence and cell layer, and thus fall into the lower (ie. more peripheral) bin. hair cell regeneration is driven by the transdifferentiation of sup- Overall, these data indicate that hair cells and support cells in the port cells, we also analyzed the spatial pattern of development of same region exit the cell cycle at similar times. support cells for E12.5 posterior cristae using immunolabeling for Sox2. In order to birthdate support cells we used the same methods 7. Discussion described above to birthdate hair cells with the exception that BrdU-positive support cells were identified as being positive for 7.1. Spatial patterns of development in the cristae BrdU and Sox2 and negative for Myosin7a. Bins were created for both the x-axis and the z-axis as described previously using the list By labeling dividing cells in mouse embryos with BrdU, we de- of Myosin7a-positive hair cells, and the number of BrdU-positive termined the spatial pattern of hair cell development throughout the A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 9

Table 4 A summary of the results and the statistical data for the analysis of the spatial pattern of hair cell birth along the longitudinal axis. BrdU-positive hair cells were grouped into ten bins according to their position along the x-axis. These bins are identified in numerical order from left to right. Means for each bin and age (E12.5, E15.5, and E17.5) are reported along with the standard error of the mean. Reported p-values come from two-way ANOVAs with Bonferroni post-tests comparing the number of BrdU-positive hair cells from E12.5 to E15.5 and E15.5 to E17.5. The symbol, ns, denotes that the comparison was not significant.

Summary of the number of BrdUþ hair cells along the longitudinal axis

POSTERIORS

Bins: F9144 ¼2.74 p¼0.0055 Age: F2,16 ¼24.08 po0.0001 Interaction: F18,144 ¼1.81 p¼0.0296

E12.5 (n¼9) E15.5 (n¼6) E17.5 (n¼4)

Mean SEM Mean SEM E12.5 vs E15.5 Mean SEM E15.5 vs E17.5 Bin number1 1 11.56 7 2.66 13.17 7 2.54 ns 1.75 7 0.63 t¼3.548, po0.01 2 16.00 7 1.63 13.67 7 1.31 ns 3.00 7 0.71 t¼3.315, po0.05 3 13.44 7 1.68 9.67 7 1.67 ns 4.00 7 0.71 ns 4 13.00 7 1.25 8.33 7 1.15 ns 6.75 7 1.03 ns 5 11.78 7 2.18 3.67 7 1.17 t¼3.087, po0.05 4.00 7 0.91 ns 6 10.67 7 1.56 4.33 7 1.56 ns 2.50 7 0.29 ns 712.11 7 1.80 13.50 7 1.95 ns 4.50 7 0.65 ns 813.11 7 0.70 12.33 7 2.60 ns 4.50 7 2.02 ns 915.11 7 1.72 15.33 7 2.64 ns 4.00 7 1.68 t¼3.522, po0.01 10 11.67 7 2.07 16.83 7 3.78 ns 5.50 7 2.72 t¼3.522, po0.01

ANTERIORS

Bins: F9,81 ¼3.04 p¼0.0035 Age: F2,9 ¼25.52 p ¼0.0002 Interaction: F18,81¼2.22 p¼0.0082

E12.5 (n¼4) E15.5 (n¼5) E17.5 (n¼3)

Mean SEM Mean SEM E12.5 vs E15.5 Mean SEM E15.5 vs E17.5

Bin number1 1 11.75 7 3.50 15.00 7 3.49 ns 5.67 7 3.48 ns 2 20.25 7 1.89 20.80 7 2.18 ns 7.67 7 2.19 t¼3.585, po0.01 3 17.75 7 3.66 11.00 7 0.71 ns 10.67 7 2.40 ns 4 19.50 7 1.44 13.60 7 2.25 ns 8.00 7 1.00 ns 5 13.00 7 2.38 10.80 7 1.24 ns 9.33 7 1.20 ns 6 14.50 7 3.23 8.40 7 1.40 ns 4.00 7 0.58 ns 7 23.75 7 4.17 10.00 7 1.90 t¼4.086, po0.001 5.00 7 0.58 ns 8 22.25 7 3.61 18.00 7 1.10 ns 10.33 7 1.76 ns 9 18.00 7 3.39 20.20 7 2.27 ns 8.33 7 1.20 t¼3.239, po0.05 10 12.50 7 2.96 19.40 7 3.04 ns 5.00 7 0.58 t¼3.931, po0.01

HORIZONTALS

Bins: F9,72 ¼5.92 po0.0001 Age: F2,8 ¼14.38 p ¼0.0022 Interaction: F18,72 ¼2.5 p¼0.0033

E12.5 (n¼4) E15.5 (n¼4) E17.5 (n¼3)

Mean SEM Mean SEM E12.5 vs E15.5 Mean SEM E15.5 vs E17.5 Bin number1 1 1.50 7 0.96 12.50 7 1.19 t¼3.552, po0.01 1.67 7 0.88 t¼3.239, po0.05 2 4.75 7 2.84 16.50 7 2.53 t¼3.794, po0.01 10.33 7 1.20 ns 3 9.25 7 3.09 18.75 7 2.84 t¼3.068, po0.05 7.00 7 2.52 t¼3.513, po0.01 4 10.25 7 3.07 20.75 7 1.55 t¼3.391, po0.05 7.33 7 1.86 t¼4.011, po0.01 5 14.00 7 3.85 18.75 7 2.96 ns 9.33 7 1.33 ns 6 13.75 7 3.88 11.50 7 0.87 ns 12.67 7 2.60 ns 7 7.75 7 1.89 20.25 7 1.93 t¼4.037, po0.01 6.67 7 1.86 t¼4.061, po0.01 8 13.00 7 1.78 15.75 7 1.25 ns 8.00 7 1.53 ns 9 12.25 7 1.80 13.75 7 2.78 ns 4.33 7 1.86 ns 10 7.50 7 1.44 8.50 7 1.44 ns 1.67 7 0.67 ns

SEM – Standard error of the mean, p-values from a two-way ANOVA with Bonferroni post-tests. 1 Bin 1 is the left-most bin and bin 10 is the right-most bin. period of hair cell birth in the cristae. Our data confirms the previous Marovitz et al., 1976). As the cochlea elongates, the earliest born results from the rat horizontal cristae (Sans and Chat, 1982)demon- cells form the apical turns, whereas later born cells become the strating that hair cell birth largely changes along the central to per- more basal turns (Lee et al., 2006; Ruben, 1967). In contrast, the ipheral axis, occurring early in the central regions and later in the onset of hair cell differentiation begins in the midbasal turn and more peripheral edges in all three cristae types. In addition, we found extends both basally and apically between E14.5 and E16.5. that hair cell differentiation is coupled to cell cycle exit in the cristae Therefore, even though the cells in the apical turn are born first, and identified previously unknown patterns of hair cell development they are actually the last to differentiate (Chen et al., 2002; Lanford in the cristae along the longitudinal axis. et al., 2000; Lim and Anniko, 1985; Sher, 1971; Woods et al., 2004). In the cochlea, where the developmental process has been This extended period between terminal mitosis and differentiation more extensively characterized, cell cycle exit is uncoupled from is fairly unique to the cochlea, as in both the utricle (Zheng and cellular differentiation. Cells of the cochlear duct are born between Gao, 1997) and the central nervous system (reviewed in Gotz and E12 and E16 (Ruben, 1967) in a mitotic zone found at the junction Huttner (2005) and Nguyen et al. (2006)) most cells begin to dif- of the cochlea and the saccule (Marovitz and Shugar, 1976; ferentiate almost immediately after cell cycle exit. Our data from 10 A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14

Fig. 5. Expression of the hair cell markers Myo6 and Gfi1 in the E14.5 inner ear. A–B) Maximum intensity projections of E14.5 inner ears labeled in A for hair cells with Myo6 (green) and sensory regions with Sox2 (red) and in B for Gfi1 (white). AC – anterior crista, HC – horizontal crista, PC – posterior crista, U – utricle, S – saccule. C–F‴) Zoomed views of the sensory organs stained with Myo6 (green, C,D,E,F), Sox2 (purple, C′,D′,E′,F′), and Gfi1 (white, C″,D″,E″,F″,F‴) show the developing anterior crista (C–C″), horizontal crista (D–D″), posterior crista (E–E″), utricle (upper F–F′,F″), and saccule (lower F–F′,F‴). C–C″) In anterior cristae, the hair cells begin differentiating adjacent to the non-sensory crux eminentia. D-D”) In horizontal cristae, the hair cells develop as one continuous patch starting on the superior or dorsal half of the cristae (towards the left), consistent with the lack of a crux eminentia in the horizontal cristae. E–E″) In posterior cristae, the hair cells also begin differentiating adjacent to the crux eminentia. F–F‴) At E14.5, the utricle and saccule have more differentiated hair cells than the cristae. In the saccule, hair cells stained with Myo6 are beginning to show a characteristic hair cell lamination (F–F′). Scale bars – 10 μm. A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14 11

Fig. 6. Support cells in E12.5 posterior cristae show a similar pattern as hair cells. A–B) Support cells that double-labeled for BrdU and Sox2 were analyzed and plotted identically to those shown in the hair cell plot from Fig. 2A depicting the group data for E12.5 posterior cristae (A). C–D) Similar to the hair cells, the BrdU-positive support cells show a fairly even distribution along the x-axis at E12.5. Error bars depict SEM. E–F) The same plots as shown in panels A and B here color coded for z-bin in the color order from top to bottom of blue, green, orange, red. G–H) Similar to the hair cells, the BrdU-positive support cells are located in the higher, or more medial, z-bins, as seen in graphs of both the number and the percentage of BrdU-positive cells in each z-bin. The greater distribution of support cells in the green bin may be due partly to the support cell nuclei sitting below the hair cells.

Fig. 7. Summary of the pattern of cell cycle exit in the cristae. A) A cartoon diagram summarizing the changes in the spatial distribution of hair cell birth with age. The colored dots are similar to the colors in Fig. 3 to indicate the position in the crista where the BrdUþ cells are found. B) A cartoon diagram of the regions in the adult cristae that maintain active Notch signaling and express the downstream Notch effector Hes5. This region correlates with the areas at E17.5 where some of the last hair cells exited the cell cycle. the E14.5 embryonic ears and the embryonic BrdU injections eminentia, even though at E12.5 hair cells exited the cell cycle suggest that the cristae adhere to the more common mechanism throughout the entire central region of the cristae. This suggests where cellular differentiation occurs shortly after cell cycle exit. that the cells nearest to the crux eminentia are born earlier than In the posterior and anterior cristae, hair cell birth begins near those closer to the lateral planum semilunatum. This is further the crux eminentia in two distinct patches, extends along the supported by the “off-wave” of hair cell birth found along the entire length of the crista in the central regions, and then finally longitudinal axis using embryonic BrdU injections. Although hair shifts to the more peripheral edges of the crista between E15.5 and cell birth was evenly distributed along the longitudinal axis at E17.5. Differentiation, as indicated by the expression of Gfi1 and E12.5, it decreased first in the regions near the crux eminentia at Myo6 at E14.5, begins in the regions nearest to the medial crux E15.5 and later in regions near the planum semilunatum at E17.5. 12 A.D. Slowik, O. Bermingham-McDonogh / Developmental Biology 411 (2016) 1–14

The horizontal cristae, which do not have a crux eminentia, ap- Unlike the cochlea, where the potential for regeneration is lost peared to develop less symmetrically along the longitudinal axis, with with age, in the vestibular sensory organs, later born regions ap- hair cell birth beginning in the more dorsal half of the cristae. This pear to maintain a low level of regenerative ability into adulthood. asymmetric development was seen to a lesser extent in the em- This suggests that the link between maturity and regenerative bryonic posterior and anterior cristae, where sometimes one hemi- ability is not a developmental phenomenon. Identifying the factors crista had slightly more Gfi1-expressing hair cells or more Myo6-la- that limit regeneration may come from observations of these re- beling than the other. This finding is interesting as the superior half of gional differences. By further studying these organs, we can better the horizontal cristae is specifically innervated by vestibulocerebellar understand the mechanism through which a small subset of afferents (Maklad et al., 2010) and the horizontal crista lies along the support cells is maintaining regenerative competence and develop dorsoventral axis, making it particularly susceptible to the dorso- methods to induce more robust regeneration in the larger quies- ventral gradients present in the developing inner ear (reviewed in cent population of support cells and in other non-regenerative Groves and Fekete (2012) and Wu and Kelley (2012)). neural systems.

7.2. Regional differences in hair cell regeneration Significance statement Many studies have shown that the mature mammalian inner ear has a limited capacity for hair cell regeneration through the Neural regeneration is rare in mammals and occurs at a very transdifferentiation of support cells, both spontaneously (Berggren low level. Each of the adult inner ear sensory organs maintains et al., 2003; Forge et al., 1993; Forge et al., 1998; Golub et al., 2012; some regenerative ability, but the determinants for this re- Kawamoto et al., 2009; Kirkegaard and Jorgensen, 2000; Li and generation are poorly understood. Here we provide insight into Forge, 1997; Lin et al., 2011b; Lopez et al., 2003, 1998, 1997; Meza the regenerative process by analyzing the spatial patterns of hair et al., 1996; Oesterle et al., 2003; Ogata et al., 1999; Rubel et al., cell development in the cristae of the inner ear. We find that hair 1995; Tanyeri et al., 1995; Taura et al., 2006; Walsh et al., 2000; cell differentiation is coupled to terminal mitosis in the cristae and Warchol et al., 1993; Yamane et al., 1995; Zheng and Gao, 1997) that there is a link between the relative maturity of different re- and through methods such as inhibition of Notch signaling (Hori gions and their regenerative potential in the adult. Understanding et al., 2007; Jung et al., 2013; Lin et al., 2011b; Mizutari et al., 2013; why these regions maintain developmental plasticity could help Slowik and Bermingham-McDonogh, 2013). However, it remains us to induce robust regeneration in the larger quiescent popula- unclear what is limiting regeneration in the adult and why the tion of support cells and in other non-regenerative neural systems. majority of support cells do not transdifferentiate, as occurs earlier in all of the developing organs in response to damage and mod- ulations such as Notch inhibition (Burns et al., 2012a; Collado Conflict of interest et al., 2011; Hayashi et al., 2008; Lanford et al., 1999; Munnamalai et al., 2012 ; Slowik and Bermingham-McDonogh, 2013; White The authors declare no competing financial interests. et al., 2006; Yamamoto et al., 2006; Zhao et al., 2011; Zine et al., 2001). Our data demonstrating that hair cell development in the Acknowledgments cristae begins in the central regions and shifts to the peripheral edges with age is consistent with the hypothesis that the last re- This work was supported by the following grants: PHS R21 gions of the sensory organ to differentiate retain regenerative DC010862 from NIDCD/NIH, PHS R01 DC009991 from NIDCD/NIH, potential the longest (Fig. 7). In the cristae, support cells in the PHS NRSA T32 GM07270 from NIGMS/NIH, PHS NRSA T32 peripheral region transdifferentiate in response to inhibition of GM007108 from NIGMS/NIH and P30DC004661 from NIDCD/NIH. Notch signaling as shown using lineage tracing with the PLP/CreER We thank Linda Robinson for time mating mice, Catherine Ray for mice (Slowik and Bermingham-McDonogh, 2013). The peripheral technical support, Dr. Hugo J. Bellen for the gift of the Gfi1 anti- region is also the only region that maintains expression of the body, past and present members of the Bermingham-McDonogh, Notch effector, Hes5, in the adult (Hartman et al., 2009). Reh, and Chao labs for helpful discussions, Drs. Paul Nakamura, This relationship between relative maturity and regenerative Thomas Reh, and Rachel Wong for critical comments on the potential is consistent with the other inner ear organs. In the manuscript, and Dr. Ronald Seifert and the Lynn and Mike Garvey utricle, hair cell development begins in the striolar region with Cell Imaging Lab for help with microscopy. later addition occurring along the lateral edges (Burns et al., 2012b; Sans and Chat, 1982; Zheng and Gao, 1997). Hair cell re- generation in the utricle has been found spontaneously after da- mage in the lateral extrastriolar region (Golub et al., 2012) and in References the posteromedial extrastriolar region using Hes5 siRNA (Jung et al., 2013). 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Stem Cell Rep. 2015; Shi et al., 2013; Walters et al., 2014; Yamamoto et al., 2006; 2; , pp. 311–322. Zheng and Gao, 2000). In other studies of vestibular organs mainly Burns, J.C., Cox, B.C., Thiede, B.R., Zuo, J., Corwin, J.T., 2012a. In vivo proliferative regeneration of balance hair cells in newborn mice. J. Neurosci. 32, 6570–6577. the utricle there does not appear to be a link between exit from Burns, J.C., On, D., Baker, W., Collado, M.S., Corwin, J.T., 2012b. Over half the hair the cell cycle and regenerative ability as the striolar region exits cells in the mouse utricle first appear after birth, with significant numbers the cell cycle early and yet appears to be the region of the greatest originating from early postnatal mitotic production in peripheral and striolar growth zones. J. Assoc. Res. Otolaryngol. 13, 609–627. regenerative capacity (Golub et al., 2012; Lin et al., 2011a; Wang Chen, P., Johnson, J.E., Zoghbi, H.Y., Segil, N., 2002. 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