Neural patterning involves regionalised cell specification. Recent studies indicate that cell dynamics play instrumental roles in neural pattern refinement and progression, but the impact of cell behaviour and morphogenesis on neural specification is not understood. Here we combine 4D analysis of cell behaviours with dynamic quantification of proneural expression to uncover the construction of the zebrafish otic neurogenic domain. We identify pioneer cells expressing neurog1 outside the otic epithelium that migrate and ingress into the epithelialising placode to become the first otic neuronal progenitors. Subsequently, neighbouring cells express neurog1 inside the placode, and apical symmetric divisions amplify the specified pool. Interestingly, pioneer cells delaminate shortly after ingression. Ablation experiments reveal that pioneer cells promote neurog1 expression in other otic cells. Finally, ingression relies on the epithelialisation timing controlled by FGF activity. We propose a novel view for otic neurogenesis integrating cell dynamics whereby ingression of pioneer cells instructs neuronal specification.https://doi.org/10.7554/eLife.25543.001
The inner ear is responsible for our senses of hearing and balance, and is made up of a series of fluid-filled cavities. Sounds, and movements of the head, cause the fluid within these cavities to move. This activates neurons that line the cavities, causing them to increase their firing rates and pass on information about the sounds or head movements to the brain. Damage to these neurons can result in deafness or vertigo. But where do the neurons themselves come from?
It is generally assumed that all inner ear neurons develop inside an area of the embryo called the inner ear epithelium. Cells in this region are thought to switch on a gene called neurog1, triggering a series of changes that turn them into inner ear neurons. However, using advanced microscopy techniques in zebrafish embryos, Hoijman, Fargas et al. now show that this is not the whole story.
While zebrafish do not have external ears, they do possess fluid-filled structures for balance and hearing that are similar to those of other vertebrates. Zebrafish embryos are also transparent, which means that activation of genes can be visualized directly. By imaging zebrafish embryos in real time, Hoijman, Fargas et al. show that the first cells to switch on neurog1 do so outside the inner ear epithelium. These pioneer cells then migrate into the inner ear epithelium and switch on neurog1 in their new neighbors. A substance called fibroblast growth factor tells the inner ear epithelium to let the pioneers enter, and thereby controls the final number of inner ear neurons.
The work of Hoijman, Fargas et al. reveals how coordinated activation of genes and movement of cells gives rise to inner ear neurons. This should provide insights into the mechanisms that generate other types of sensory tissue. In the long term, the advances made in this study may lead to new strategies for repairing damaged sensory nerves.https://doi.org/10.7554/eLife.25543.002
Neural specification relies on proneural genes, which are expressed in specific patterns and underlie the genesis, organisation and the function of the neurons that will subsequently differentiate (Bertrand et al., 2002; Huang et al., 2014). Many signals that pattern the nervous system have been identified. For example, gradients of Shh, BMP and Wnt establish thirteen different domains of neural progenitors in the mouse neural tube (Ulloa and Briscoe, 2007); FGF8 and FGF3 control the site of retinogenesis initiation in chick and fish through regulation of ath5 expression (Martinez-Morales et al., 2005); and EGFR signalling determines the expression of a wave of l(1)sc in the Drosophila optic lobe (Yasugi et al., 2010).
Concomitant with cell specification, neural tissues undergo phases of morphogenesis and/or growth. Thus, the cells within a given domain are not static but perform complex cell behaviours. Recently, the contribution of such cell dynamics to neural patterning has been identified. In the neural tube, for instance, sharply bordered specification domains involve the sorting of cells along a rough Shh-dependent pattern (Xiong et al., 2013). Additionally, differences in the rate of differentiation of cells (which migrate out of the tissue) between distinct domains of the neural tube help to establish the overall pattern during tissue growth (Kicheva et al., 2014). Thus, dynamic spatial rearrangements of cells within a field that is being specified are integrated with patterning mechanisms of positional information by morphogens.
In the inner ear, developmental defects in neurogenesis could result in congenital sensorineural hearing loss (Manchaiah et al., 2011). Neurogenesis begins when an anterior neurogenic domain appears at the placode stage by the expression of the proneural gene neurog1, which specifies neuronal precursors. The rest of the otic placode is non-neurogenic and generates non-neuronal cell types (Ma et al., 1998; Andermann et al., 2002; Abello and Alsina, 2007; Radosevic et al., 2011). In the neurogenic domain, neurog1 induces neurod1 (Ma et al., 1996, 1998) expression, which is required for delamination of neuroblasts from the epithelium (Liu et al., 2000). Delaminated neuroblasts subsequently coalesce to form the statoacoustic ganglion (SAG) and differentiate into mature bipolar neurons (Hemond and Morest, 1991; Haddon and Lewis, 1996). The spatial restriction of the otic neurogenic domain relies on the integration of diffusible signals such as FGFs, SHH, Retinoic acid and Wnt (reviewed in Raft and Groves, 20142015) as well as the function of transcription factors such as Tbx1 (Radosevic et al., 2011; Raft et al., 2004), Sox3 (Abelló et al., 2010), Otx1 (Maier and Whitfield, 2014), Eya1 (Friedman et al., 2005) and Six1 (Zou et al., 2004). In the inner ear, several FGFs (Adamska et al., 2001; Mansour et al., 1993; Léger et al., 2002; Alsina et al., 2004; Vemaraju et al., 2012; Alvarez et al., 2003), regulate the sequential steps of neurogenesis starting from the expression of neurog1 (Vemaraju et al., 2012; Léger et al., 2002; Alsina et al., 2004) and continuing to later events involving neuroblast expansion (Vemaraju et al., 2012). Together with the regulation of spatial regionalisation, the number of neuronal progenitors produced depends on local cell–cell interactions mediated by the Notch pathway (Adam et al., 1998). Remarkably, to date no studies have addressed how morphogenesis, cell behaviour and proneural dynamics impact otic neuronal specification.
Here we use the zebrafish inner ear as a model to analyse the role of cell dynamics on neuronal specification. We identify pioneer cells that are specified outside the otic epithelium, ingress into the placode during epithelialisation and control local neuronal specification, suggesting an instructive role of these cells. Furthermore, we show that FGF signalling affects otic neurogenesis through the regulation of otic placode morphogenesis, influencing pioneer cell ingression.
We have previously identified cell behaviours contributing to otic vesicle morphogenesis (Hoijman et al., 2015) and here we focused on the influence of cell dynamics in the establishment of the neurogenic domain. For this, we used a zebrafish BAC reporter line that expresses the fluorescent protein DsRed-Express (DsRedE, a faster maturation version of DsRed [Bevis and Glick, 2002]) under control of the neurog1 regulatory elements (Drerup and Nechiporuk, 2013). We imaged in 4D the otic development from stages of otic placode morphogenesis (15 hpf) until neuroblast delamination is abundant and the central lumen is expanding (20.5 hpf, Figure 1A and B; Videos 1 and 2). The overall pattern of DsRedE expression is highly consistent between embryos, being restricted to the most ventroanterolateral region of the placode until 19 hpf and expanding posteromedially at around 20.5 hpf (Figure 1A and B; Videos 1 and 2). This DsRedE expression pattern recapitulates the endogenous spatiotemporal pattern of neurog1 as analysed by in situ hybridisation (ISH) (Radosevic et al., 2014; Vemaraju et al., 2012; Andermann et al., 2002). Moreover, DsRedE expressing cells delaminate (Figure 3H; Videos 1 and 11) and are incorporated into the SAG (Figure 1A and B; Video 3), supporting the use of this line to analyse single cell dynamics of neuronal specification.
We also analysed the cellular organisation of the neurogenic domain by performing a 3D morphometric analysis of this region. During the stages of neuronal specification, the shape of the otic vesicle is asymmetric, exhibiting a protuberance in the anterolateral region (Figure 1C). To compare the properties of the neurogenic region with the rest of the otic vesicle, we built a rectangular cuboid with the vertices of the vesicle and divided it in eight regions of equal volume (Figure 1D), in which we quantified the number of cells and the volume of tissue. By 19 hpf, the neurogenic domain region accumulated more cells (15.4 ± 0.4% of the total number of cells in the vesicle, 49 ± 3 cells of 311 ± 16 cells respectively) than other regions (mean non-neurogenic region: 12.0 ± 0.1%, 36 ± 2 cells, Figure 1E) and presented higher cellular density (Figure 1F; neurogenic region: 2.16 ± 0.03 nuclei/1 × 103 μm3, mean non-neurogenic region: 1.60 ± 0.03 nuclei/1 × 103 μm3). Quantification of all the mitotic events inside the vesicle between 14 and 18.5 hpf revealed that cell proliferation is also highly enriched in this region (Figure 1G). While the increase in cell number in the neurogenic domain was moderate (about 3% more cells than other regions), the enrichment in mitotic events led to about 41% of the total number of divisions to occur in this domain. Thus, in addition to a phase of transit-amplification of neuroblasts after delamination (Vemaraju et al., 2012), neuronal progenitors also appear to multiply inside the otic vesicle. This analysis indicates that the neurogenic domain presents high cell number, high cell density and an increased proliferative activity.
To analyse how the neurogenic domain is built, we decided to evaluate when and where cells of the neurogenic domain start to express neurog1. We first aimed to capture the earliest specified cells. Epithelialisation of the otic placode progresses from 12.5 hpf until about 18 hpf (Hoijman et al., 2015). While it has been reported that neurog1 expression in the otic placode begins at 15 hpf (Radosevic et al., 2014), we found that already at 13 hpf there are rows of DsRedE expressing cells lateral to the neural tube and anterior to the epithelializing otic placode (Figure 2A; Video 4). These cells coincide with neurog1 expressing cells detected by ISH (Figure 2—figure supplement 1A), and previously assumed to belong to the anterior lateral line placode (Andermann et al., 2002). Unexpectedly, when we followed these cells we found that some of them migrate posteriorly and become incorporated into the anterolateral region of the otic epithelium, in a position corresponding to the neurogenic domain (red brackets in Figure 2B; Video 5). Therefore, these cells develop into otic and not lateral line cells. To confirm this cell ingression, we injected NLS-Eos mRNA at 1 cell stage to obtain a homogeneous nuclear staining with the photoconvertable protein throughout the embryo. At 13 hpf, we photoconverted Eos protein (from green to red fluorescence) in a group of nuclei anterior to the otic epithelium where the migrating cells are located. At 20 hpf, we detected photoconverted nuclei inside the vesicle (Figure 2—figure supplement 1B).
We also detected in the same anterior region a second pool of neurog1+ cells (expressing also neurod1; Figure 2—figure supplement 1C) that moves posteromedially without ingressing, remaining in the region of the SAG (blue brackets in Figure 2B; Video 5). The migrating cells are located laterally relative to a population of sparse cells from which they are segregated by an F-actin rich layer that runs anteroposteriorly until it reaches the placode (Figure 2—figure supplement 1F and Figure 2H). These observations suggest that neurog1 expression is not sufficient for cell ingression. Additionally, neurog1 expression was not required for cell ingression, as some neurog1- cells ingress. Consistently, we detected cell ingression events in neurog1 mutant embryos (neurog1hi1059, Figure 2—figure supplement 1G).
Interestingly, 3D tracking of individual cells of the ingressing pool revealed that some cells activate neurog1 expression while moving towards the epithelium and before their epithelialisation (Figure 2C; Video 6). Immediately after ingressing into the neurogenic domain, these cells divide and delaminate, thus undergoing a complete cycle of epithelialisation and de-epithelialisation in only a few hours. Analysis of the movement of these cells suggests that their migration is a directional process occurring in individual cells (Figure 2D,E and F; Video 7; some cells of the same region migrate in other directions). We also observed that the leading front of cells periodically protrudes, followed by a rapid forward translocation of the nucleus (Figure 2G; insets of Video 7), as has been described during fibroblast migration (Petrie and Yamada, 2015). When tracking three neighbouring cells, we observed that while two of them ingress (white and pink tracks), the third one (blue track), which is initially positioned closer to the otic placode, divides during migration and the daughters do not ingress (Figure 2D,E and F; Video 7). These observations highlight that ingressing cells are interspersed with other cells that do not join the otic placode, and factors other than anteroposterior positional cues within the migrating population determine whether a cell will ingress or not into the otic placode.
Particular morphogenetic features could facilitate the ingression of cells from the anterior region. As we previously reported, the otic placode is only epithelialised medially at these stages (Hoijman et al., 2015). As epithelialisation progresses, at 14 hpf the posterior part of the placode is segregated from the surrounding cells, while the anterior region of the placode is not (Figure 2—figure supplement 1D; Video 8). Thus, the posterior part folds approximately 3 hr before the anterior one (Figure 2—figure supplement 1D; Video 8). During this period, and by the anterior unfolded region, migrating cells ingress into the otic epithelium. Moreover, the basal lamina at these early stages is only rudimentary and not continuous (contrary to the one present at later stages surrounding the whole organ; Figure 2—figure supplement 1E). Therefore, the fact that the epithelium is still organizing could allow the migrating cells to ingress into the tissue before it is fully formed.
In summary, our results show cells that are being specified outside the otic epithelium, migrate and ingress into the prospective neurogenic domain, constituting the earliest neuronal specified cells of the organ.
We next evaluated if, in addition to ingressing cells, other cells start to express neurog1 within the neurogenic domain. We visualised the activation of neurog1 expression inside the otic vesicle in real-time (Figure 3A; Video 9), a process that we refer to as ‘local specification’. Dynamic quantification of DsRedE fluorescence levels in individual cells (cell) indicated that the rate of increase in the signal is variable among cells (Figure 3B, mean rate of increase ranging between 0.15 and 0.54 a.u./min, n = 11 cells). However, we found that when the signal reaches a critical level (between 45.5 and 52.5 a.u. in Figure 3B, gray region with red dots), cells begin to delaminate (visualised by the movement of the cell body to the basal domain of the epithelium). This suggests that cells delaminate relative to neurog1 levels and not to the time elapsed since they initiated neurog1 expression (Figure 3B and C).
As we mentioned above, higher mitotic events occur in the neurogenic domain. Therefore, division could also contribute to the domain by adding neurog1 expressing cells (neurog1+ cells) to the domain. To address this, we performed a 4D analysis of cell divisions and found that every cell divides only once in the 7 hr period analysed (n = 27/27). Mitotic cells are found either contacting the central lumen (Figure 3D) or not (peripheral divisions) (Figure 3E). Interestingly, these latter cells are apposed to an accumulation of the apical determinant Pard3 that forms a scaffold perpendicular to the central luminal surface of the vesicle, running from the lumen to the periphery (Figure 3F; Video 10). Thus, similar to the apical mitosis occurring in the central lumen, peripheral divisions are also in contact with an apical surface (Figure 3G and H).
In neurogenic tissues, either asymmetric (daughter cells become one progenitor and one neuron) or symmetric (both daughter cells with the same fate) divisions can occur (Taverna et al., 2014; Chenn and McConnell, 1995; Das and Storey, 2012). This depends on factors such as the apicobasal position of the dividing cell and the orientation of the mitotic spindle (Das and Storey, 2012). Our dynamic analysis of neurog1 activation allowed us to assess the modes of divisions within the otic neurogenic domain. We observed that all divisions in the neurogenic domain have the cleavage plane perpendicular to the apical surface regardless of their position in the epithelium or their neurog1 expression (Figure 3G and H). When analysing the fate of the daughter cells after division, we found all were symmetric (27/27): both daughter cells delaminate after division (20/27 delaminate during the timeframe analysed, 7/27 are positioned to delaminate at the end of the acquisition). However, division can occur either before (13/25) or after (12/25) the induction of neurog1 expression. Interestingly, daughter cells from mitoses of a neurog1+ cell with high levels of DsRedE expression (neurog1+Hi cell) rapidly delaminate, remaining in close contact as they move to the periphery of the tissue (Figure 3I and J; Video 11). On the other hand, daughter cells from mitosis of cells not expressing neurog1 (neurog1−), or only at low levels (neurog1+Low), remain in the epithelium after division, where they increase the DsRedE signal over a variable period of time (Figure 3—figure supplement 1).
In summary, divisions in the neurogenic domain are symmetric and apical. Furthermore, there is not a preferential sequence of events concerning neurog1 activation and division. Taken together, our analysis of the origin of neurog1+ cells revealed that they are added to the neurogenic domain by three different mechanisms: cell ingression, local expression and cell division.
The incorporation of the ingressing cells and their rapid exit from the otic vesicle led us to wonder about their role in the establishment of the neurogenic domain. These early-specified cells might contribute to the neurogenic domain by their inclusion as specified cells and/or play additional roles. To address this question, we decided to eliminate these cells during their migration, before they reach the otic epithelium. For this, we identified the stream of migrating cells by their DsRedE signal (Figure 4A), laser-ablated them unilaterally at 12.5 hpf (Figure 4B), and examined the effects on neuronal specification in 3D in the otic vesicle at 18.5 hpf (Figure 4C–H; Video 12), before delamination becomes significant. Neurog1 expression was analysed by quantification of the cell in all cells belonging to the neurogenic domain (Figure 4C and D). Ablation of a limited number of cells (2–3 cells per laser pulse; see Material and methods for more details) led to a decrease in the global level of DsRedE expression (calculated as the sum of the cell for all neurog1+ cells) in the vesicle of the ablated side as compared to the contralateral vesicle on the non-ablated side of the embryo (Figure 4C and E; non-ablated side: 1492 ± 58, ablated side: 454 ± 44 a.u). Applying an increased number of laser pulses ablated more cells, which seems to lead to a more severe specification phenotype (compare embryos 1 and 2 from Figure 4C, which received 1 and 3 laser pulses respectively), despite the overall morphology of the neurogenic domain being unaffected. Analysis of both neurog1 expression in the otic epithelium at 21 hpf and the phenotype of the SAG at 42 hpf confirms that the effect of ablation persists and, thus, does not appear to represent a delay in neuronal specification (Figure 4—figure supplement 2A,B and C; Video 12). The effect of ablation is specific to otic neurog1 expression, since DsRedE expression in the neural tube was not affected (Figure 4—figure supplement 2D). Moreover, we observed a phenotype only after ablating anterior future ingressing cells: ablation of neurog1+ cells in another location (posterior to the placode at 13 hpf, Figure 4—figure supplement 1B) or developmental stage (anterior to the vesicle at 19 hpf, Figure 4—figure supplement 1C) did not affect neurog1 expression in the otic vesicle.
When comparing the number of neurog1+ cells (Nneurog1+), we also found a reduction in the ablated side vesicle compared to the control vesicle (Figure 4F; non-ablated side: 23.8 ± 1.4 cells, ablated side: 10.0 ± 0.8 cells). This result could be partially explained by the failure of the ablated cells to ingress into the forming neurogenic domain. These results also indicate that when ablating the cells that will be part of the neurogenic domain, the cells now located in the same position do not change their fate and become neural specified, as expected if cell identity would be dictated by cell position. Interestingly, the number of cells eliminated by ablation (and the ones produced by their divisions) would be too small to account for the large decrease in the number of neurog1+ cells in the vesicles of the ablated side (Figure 4F). This suggests that ingressing cells play an instructive role on the specification of other cells of the neurogenic domain (i.e. local specification). To shed light on this possibility, we calculated the mean value for cell ( cell) in vesicles from each experimental condition. This parameter was also reduced by the ablation (Figure 4G; non-ablated side: 60.1 ± 2.5, ablated side: 43.6 ± 4.8 a.u.), suggesting that the global reduction in fluorescence was not only caused by a decrease in the number of neurog1+ cells (Figure 4Iii), but that the neurog1 transcriptional activity inside these cells was also reduced. Accordingly, the number of neurog1+Hi cells (Nneurog1+Hi) was also significantly lowered by ablation (Figure 4H; non-ablated side: 6.0 ± 0.6, ablated side: 1.0 ± 0.4 cells). However, it is possible that the neurog1+Hi cells at the time point analysed are mainly ingressed cells, and thus by eliminating them, we decreased the cell in each vesicle by a relative increase in neurog1+Low cells (Figure 4Iiii, see figure legend for detailed explanation of the scheme). We discarded this possibility by backtracking cells identified as neurog1+Hi at 19 hpf from non-ablated embryos, and observing that most of them are neurog1- cells at 13 hpf positioned inside the epithelising placode before ingression takes place, therefore belonging to the pool of cells specified locally (Figure 4I and J).
Given that both the number and expression levels of neurog1+ cells were reduced by ablation, it is possible that a cell community effect takes place, in which the presence of more neurog1+ cells favours higher expression levels in the pool of progenitors being specified. However, the effect of cell ablation was not recapitulated when proliferation was blocked by incubation with aphidicolin and hydroxyurea (AH) (Hoijman et al., 2015). This treatment decreased the number of neurog1+ cells at 20 hpf (fold change AH/DMSO: 49,6 ± 6.3%, Figure 4—figure supplement 2E) but the mean levels of neurog1 expression were not affected (fold change AH/DMSO: 110 ± 11%, Figure 4—figure supplement 2E). This result suggests that cell number and expression levels are not necessarily linked during otic neurog1 expression and highlights the specific relevance of the ingressing cells in promoting the transcription of the neurog1 gene.
Altogether, these results indicate that these cells act as pioneer neurogenic cells, contributing to the neurogenic domain both through their incorporation as neurog1+ cells and by promoting neurog1 expression non-autonomously in other cells of the domain.
To understand how the specification processes identified above are promoted, we decided to explore the role of FGF signalling, a pathway reported to control both neurog1 expression in the vesicle and the number of neurons in the SAG (Wang et al., 2015; Vemaraju et al., 2012). To this aim, neurog1:DsRedE embryos were incubated with the FGFRs inhibitor SU5402 from 11 hpf until 19 hpf, beginning the treatment after placode induction and before otic morphogenesis starts (Figure 5A and B). Analysis of neuronal specification indicated that SU5402 treatment reduced the global level of DsRedE expression (Figure 5C), in agreement with the previous ISH analysis of neurog1 expression (Vemaraju et al., 2012; Léger et al., 2002). This reduction was caused not only by a decreased mean level of neurog1 expression in each cell (Figure 5B and C), but also by a reduction in the number of neurog1+ cells (Figure 5C, and particularly in the neurog1+Hi cells). To confirm that the FGF pathway is mediating the mentioned phenotype, we crossed a transgenic line expressing a dominant negative isoform of the FGF receptor 1 fused to GFP under the control of a heat-shock (hs) promoter (hsp70:dnfgfr1-EGFP) (Norton et al., 2005) with the TgBAC(neurog1:DsRedE)nl6 line. Inducing transgene expression at 10 hpf phenocopied at 20 hpf the effect on otic neurog1 expression observed in SU5402 treated embryos (Figure 5D and E).
We realised that the phenotypes produced by blocking FGF signalling are similar to those resulting from cell ablation. Furthermore, given that FGF blockade strongly reduces the number of SAG neurons when it is performed early during otic development (Wang et al., 2015), we hypothesise that FGF signalling might control the early cell ingression event. We tested this idea by blocking the FGF signalling from 11 hpf onwards (both using SU5402 or the hsp70:dnfgfr1-EGFP transgene), photoconverting NLS-Eos in cells located anterior to the otic epithelium at 13 hpf (Figure 5F and H, left panels) and, subsequently, quantifying the number of photoconverted nuclei inside the otic vesicle at 18 hpf (Figure 5F and H (right panels), G and I). As shown in Figure 5G and I, SU5402 treatment or DNFGFR1-EGFP induction significantly reduce the number of ingressed cells (DMSO: 4.7 ± 1.1 cells, SU5402: 1.0 ± 0.4 cells; heat-shocked siblings 5,3 ± 0.4 cells; heat-shocked hsp70:dnfgfr1-EGFP/+: 0.2 ± 0.2 cells). These results suggest that the FGF pathway contributes to neuronal specification in the otic vesicle by promoting the ingression of the pioneer cells into the neurogenic domain.
To gain insights into how the FGF pathway influences cell ingression, we performed time-lapse imaging during otic placode morphogenesis in embryos expressing DNFGFR1-EGFP. Tracking of photoconverted cells in these embryos showed that they still move towards the otic epithelium but remain outside (Figure 5—figure supplement 1E). Interestingly, in these embryos the anterior region of the epithelium folds at an earlier stage in development than in control embryos (Figure 5J; Video 13), becoming synchronous with folding of the posterior region (and not asynchronously as in the wild type embryos, Figure 2—figure supplement 1D; Video 8). Additionally, the otic basal lamina also formed earlier in DNFGFR1-EGFP expressing embryos than in siblings (Figure 5K). Conversely, overexpression of FGF3 by heat-shocking a hsp70:fgf3 line did not affect the anterior events (folding and cell ingression, Figure 5—figure supplement 1F and G) suggesting that endogenous anterior FGF levels are sufficient to mediate these processes. However, this manipulation led to a delay in folding of the posterior part of the epithelium, (a region where endogenous FGFs are not acting), supporting the notion that FGFs regulate otic epithelialisation. Altogether, these results suggest that endogenous FGF activity delays the final steps of anterior otic placode morphogenesis, providing time for cell ingression before the epithelial barriers appear.
Although important in other contexts, the control of proliferation does not seem to play a central role in the FGF signalling effect on otic specification, as blocking FGF did not modify the number of otic cells positive for phospho-Histone 3 (pH3+ cells, Figure 5—figure supplement 1, A and B). Moreover, not only does the FGF pathway control the number of neurog1+ cells but also the mean levels of neurog1 expression (as we show above with the AH experiments, both parameters were not coupled).
We have identified a new group of cells that act as pioneers of the otic neurogenic domain. These cells have two essential roles: they constitute the first specified cells of the domain and they promote specification of resident cells of the vesicle, thus spreading commitment to a neural fate (Figure 5L). To our knowledge, this is the first example of neuronal progenitors instructing specification of other progenitors. In the mammalian developing brain, differentiated neurons of the cortical plate migrate to invade the dorsal telencephalon and are able to control the timing of progenitor neurogenesis (Teissier et al., 2012). Our analysis challenges the view that otic neuronal specification takes place in a static tissue. Indeed, the results presented here show that elaborate cell behaviours underlie development of the neurogenic domain, including intra-organ cell movements, delamination, cell divisions and importantly, cell ingression (Figure 5L).
Ingression of progenitors to the otic epithelium could also be relevant for sequential stages of their own differentiation, in a similar way that migration is important for maturation of either immature neurons in the mouse cortex (Ayala et al., 2007), or progenitors of the Drosophila optic lobe (Apitz and Salecker, 2015). Thus, the sequential epithelialisation and de-epithelialisation could be a general and crucial step for differentiation, as it has been recently proposed (Zheng et al., 2014). Our data indicate that the SAG integrates neuronal cells from at least two different origins: the ingressing cells and the ones specified locally. Different neuronal populations have been already identified in the SAG, including vestibular and auditory neurons (Torres and Giráldez, 1998; Bell et al., 2008). It still needs to be addressed whether the different populations of progenitors contributing to the neurogenic domain will differentiate into different functionally subgroups of neurons inside the ganglion.
In chick, a transitory population of cells surrounding the invaginating otic placode was described and termed ‘otic crest cells’ (Hemond and Morest, 1991). This population of cells seem to migrate to the rostral part of the SAG. These cells could be similar to the second pool of neurog1+ cells described here migrating directly to the SAG, suggesting similarities between chick and zebrafish. Given that single cells were not followed over time, a putative ingression of ‘otic crest’ into the otic placode might have been missed. Moreover, ingression of cells from outside to the otic epithelium might be an evolutionarily conserved event, since it was also reported to occur during mouse otic development (Freyer et al., 2011). Some of these ingressing cells have been shown to ultimately reside in the SAG. Whether these cells also have a function in neuronal specification of other cells remains to be explored.
The otic neurogenic domain emerges in a defined ventroanterolateral position due to the dialogue of several signalling pathways that regionalise the otic placode (Maier et al., 2014; Fekete and Wu, 2002; Abello and Alsina, 2007; Raft and Groves, 20142015). In light of this, within the otic placode the fate of each cell would be dictated by its position in the tissue (Bok et al., 2007, 2005; Brigande et al., 2000; Whitfield and Hammond, 2007) upon the influence of the extrinsic signals. However, we observe that some ingressing cells are specified prior to their incorporation to the anterolateral domain of the otic epithelium. Moreover, when ingressing cells are laser ablated, the cells in the otic vesicle located in the position of the ingressed cells (i.e. receiving the same putative diffusing morphogens) do not seem to adopt a neurogenic fate. This suggests that secreted factors establish a region competent for neurogenic specification, to which the ingressing cells (and probably other mechanisms) provide instructive signals to induce neurog1 expression. In agreement with this possibility, Tbx1, the main transcription factor involved in otic neurogenic regionalisation, is a repressor of neurog1 expression. Tbx1 is excluded from the anterior part of the vesicle, making the region competent to be induced by neurogenic signals (Bok et al., 2011; Radosevic et al., 2011; Raft et al., 2004). Thus, in addition to the reported role of cell movements on the spatial delimitation of different domains of the neural tube (Xiong et al., 2013; Kicheva et al., 2014), we propose that coordination between cell movement and cell communication contributes to the neuronal pattern of the otic vesicle.
In embryos mutant for FGF3, FGF8 and FGF10, and embryos in which FGF signalling has been temporally blocked, distinct phases of otic neural development are impaired (Wright and Mansour, 2003; Zelarayan et al., 2007; Pirvola et al., 2000; Léger et al., 2002; Vemaraju et al., 2012; Alsina et al., 2004; Alvarez et al., 2003). Our work indicates that FGF signalling promotes ingression of pioneer cells into the neurogenic domain, suggesting that some of the previously reported effects on neurog1 expression could be due to this novel role. Additionally, FGF signalling is known to control cell behaviour in other organs, such as epithelialisation and cell migration during kidney tubulogenesis and lateral line development (Atsuta and Takahashi, 2015; Aman and Piotrowski, 2008). Particularly in the inner ear, FGF signalling controls epithelial invagination during otic morphogenesis in the chick (Sai and Ladher, 2008). We have identified a role of this pathway in zebrafish otic morphogenesis, delaying tissue folding during epithelialisation, and thus influencing neurogenesis. Additionally, it is possible that the FGF pathway also impinges on cell migration. The candidate ligands for the FGF effects on morphogenesis might be FGF8 and FGF3 coming from the hindbrain (Maves et al., 2002) and FGF3 from the endoderm and mesoderm (McCarroll and Nechiporuk, 2013). FGF10a is also expressed at these stages in the region where the pioneer cells are migrating (McCarroll and Nechiporuk, 2013). However, neurog1 expression is normal in otic vesicles of FGF10a mutant embryos (Figure 5—figure supplement 1C and D), indicating that this ligand is most probably not involved in these processes.
A question that emerges from our analysis is how ingressing cells regulate neurog1 expression in their neurogenic domain neighbours. The Notch pathway could participate in this process. However, since Notch activation reduces the number of specified neuronal cells via lateral inhibition (Haddon et al., 1998; Abelló et al., 2007) and ingression enhances it, the instructive signal should inhibit Notch activity in the resident cells of the vesicle. Given that inhibition of cell ingression reduced not only the number of neurog1+ cells but also the mean expression levels, the mechanism for instruction seems to rely on the activation of the neurog1 promoter more than in stimulation of proliferation. This hypothesis is supported by the fact that: (a) FGF pathway blockade reduced both the number of neurog1+ cells and the mean neurog1 expression levels without affecting proliferation, and (b) AH inhibition of proliferation did not affect the mean levels of neurog1 expression.
Our 4D analysis allowed us to address the mode of division in the otic neurogenic domain for first time. We found that in all cases including both neurog1− and neurog1+ cells, both daughter cells acquire a neuronal fate. During the time frame analysed, no divisions were found where one daughter cell remained as a neurog1- progenitor while the other activated the proneural expression, as has been described in the neural tube (Wilcock et al., 2007; Das and Storey, 2012; Taverna et al., 2014). We cannot exclude, however, that asymmetric divisions occur at later times or at very low frequency.
Studies of fixed chick otic vesicles described the presence of mitosis in the basal side of the epithelium in addition to the luminal ones (Alvarez et al., 1989). Such mitoses were termed ‘basal divisions’ similar to the ones taking place in the retina in which mitotic cells are no longer polarized apically and in contact with the ventricular membrane (Weber et al., 2014). In our study, we also observed non-luminal mitoses, but our data show that these divisions remain in contact with a Pard3 scaffold and therefore still keep their apical polarity.
Neural specification usually occurs in epithelialised tissues. However, we observed activation of neurog1 expression in pioneer cells before epithelialisation, suggesting that stable cell-cell contacts would be dispensable to initiate proneural expression. Similarly, in mouse neurog2 is expressed in migrating sensory neuron precursors (Marmigère and Ernfors, 2007), although its expression begins before exiting the epithelium and migration (Zirlinger et al., 2002). We were able to visualise the transit of an otic neuronal progenitor from neurog1 expression to delamination. Analysis of neurog1 expression levels suggests that delamination occurs once a given threshold of proneural expression is reached; probably associated to neurod1 induction.
The otic placode and other cranial placodes originate from a large common pre-placodal region (PPR) adjacent to the neural plate (Bailey and Streit, 20052006). Precursors from the PPR segregate and coalesce into individual cranial placodes, which progressively acquire specific identities (Breau and Schneider-Maunoury, 2014; Streit, 2002; Bhat and Riley, 2011; Saint-Jeannet and Moody, 2014; McCarroll et al., 2012). Our data revealed that otic neurog1 is expressed before of what it was conceived and outside the epithelium by a group of cells that ingress during morphogenesis. This suggests that neural specification might precede the acquisition of a defined placodal identity. Thus, we propose that some PPR precursors might already be committed to a neural fate and that their subsequent allocation into the placodes (by random or directed movements) provides them one or another placodal identity. Further work in this direction might shed light into this hypothesis.
In conclusion, our study reveals that cell movements underlie an instruction essential for otic neuronal specification, a crucial step in neurogenesis. Unravelling the complex mechanisms that determine the number of neurons incorporated in a forming ganglion may provide insights leading to a better understanding of the anomalies associated with auditory neuropathies.
The following zebrafish lines were used in this study: AB wild-type, TgBAC(neurog1:DsRedE)nl6 (Drerup and Nechiporuk, 2013), Tg(neurod:GFP) (Obholzer et al., 2008), Tg(actb1:Lifeact-GFP) (Behrndt et al., 2014) Tg(Xla.Eef1a1:H2B-Venus) (Recher et al., 2013), Tg(hsp70:dnfgfr1-EGFP)pd1 (Lee et al., 2005), Tg(elA:GFP) (Labalette et al., 2011), neurog1hi1059 (Golling et al., 2002), Tg(hsp70:fgf3) (Hammond and Whitfield, 2011), and a cross between the TgBAC(neurog1:DsRedE)nl6 and the mutant fgf10a+/− (Norton et al., 2005). They were maintained and bred according to standard procedures (Westerfield, 1993) at the aquatic facility of the Parc de Recerca Biomèdica de Barcelona (PRBB). All experiments conform to the guidelines from the European Community Directive and the Spanish legislation for the experimental use of animals.
Live embryos were embedded in low melting point agarose at 1% in embryo medium including tricaine (150 mg l−1) for dorsal confocal imaging using a 20x (0.8 NA) glycerol-immersion lens. Imaging was done using a SP5 Leica confocal microscope in a chamber heated at 28.5°C. 20 to 80 µm thick z-stacks spanning a portion or the entire otic vesicle (a z-plane imaged every 0.5–2 µm) were taken every 1 to 3 min for 2–12 hr. Raw data were processed, analysed and quantified with FIJI software (Schindelin et al., 2012). For visualisation purposes, the images were despeckled. For quantifications of neurog1 expression, images were not modified. Videos were assembled selecting a plane from every z-stack at every time point to better visualise the phenotype (or track a cell) or shown as 3D reconstructions. A representative video from at least three different embryos is shown. Images in figures are either shown as confocal coronal sections, 3D reconstructions or average z-projections. To track the trajectory of individual cells, 3D videos were analysed using the MtrackJ, Manual tracking plugins of ImageJ (Meijering et al., 2012), and temporal colour code applied to generate a single image of the tracks.
To perform quantifications in different regions of the otic vesicle, we live imaged a z-stack and built a rectangular cuboid defined by external vertices of the otic vesicle. The cuboid was divided in eight equally sized regions, and quantifications were performed inside each region. Before quantification, the z-stacks were aligned in 3D to correct for variability in orientations during mounting to guarantee the coronal sectioning of the vesicle. For volume calculation, the x-y area of the tissue in each plane of the z-stack was measured and then multiplied by the z spacing every plane (the volume of the lumen was subtracted). The number of cells in each region was determined manually by counting H2B-mCherry stained nuclei on z-stacks, using the Cell counter plugging of ImageJ. 3D visualisation of Lyn-GFP plasma membrane staining helped the identification of each single cell. To quantify the number of cell divisions in the otic epithelium in a period of time, high temporal resolution videos (1 min frequency) in 3D of H2B-GFP stained nuclei were analysed manually to detect every chromosome segregation event. The number of divisions in each region of the vesicle was determined building a cuboid as described above for each time point.
To ablate a group of cells, a two-photon laser beam (890 nm) from a Leica SP5 microscope was applied over one side of the embryos mounted in agarose (the contralateral side was maintained intact as a control). We used embryos with mosaic H2B-mCherry nuclear staining (mRNA injected at 16 cell stage) to calibrate the settings of the microscope required to ablate 2–3 cells in each ablation pulse (Figure 4—figure supplement 1A; Video 14). Each pulse consisted in approximately 5 s of 30% laser power applied in a ROI of about 70 µm2 imaged with a 20x air objective and a digital zoom of 64x. In neurog1-DsRedE embryos, the cells to ablate were identified by single photon confocal imaging recognizing the DsRedE fluorescence in cells anterior (or posterior) to the otic placode/vesicle. Right after ablation, imaging of the vesicle was performed to confirm the damage caused (dead cells were clearly visualised). Sequential pulses at different locations were applied to ablate an increased number of cells. No damage outside the ablated region was observed. Ablated embryos were maintained mounted at 28°C until the moment in which specification analysis was performed (see below).
To detect ingression of cells into the epithelium, photoconversion of NLS-Eos expressing nuclei was performed with UV light (λ = 405 nm, using a 20x objective in a Leica SP5 system) on 13 hpf mounted embryos. A 3D ROI of about 1 × 105 μm3 located 25 µm apart from the anterior limit of the epithelialising placode was photoconverted. Photoconversion was checked by confocal imaging right after UV illumination. The number of photoconverted cells was quantified using the Cell counter plugin from FIJI (DMSO = 58 ± 9 cells; SU5402 = 59 ± 7 cells, n = 8). The embryos were then removed from the agarose and incubated in embryo medium until 20 hpf to check for cell ingression by 3D imaging. When blockade of FGFR was performed, the embryos were dechorionated at 11 hpf, incubated with SU5402 or DMSO in embryo medium until 13 hpf, mounted in agarose including SU5402 or DMSO, photoconverted, imaged, unmounted, and incubated in presence of the drugs in solution until 19 hpf. In some cases, the TgBAC(neurog1:DsRedE)nl6, the neurog1hi1059 (embryos genotyped by PCR after imaging), Tg(hsp70:fgf3), or Tg(hsp70:dnfgfr1-EGFP) lines were used. In the latter case, time-lapses at 5 min resolution time were performed to track photoconverted nuclei over time.
To analyse specification phenotypes z-stacks were acquired with fixed settings (laser power and detector gain) between different experimental groups (or vesicles in the case of ablations). The settings were adjusted to detect a range of increased or decreased fluorescence levels without saturation or lack of signal. DsRedE fluorescence was quantified in single slices using imageJ. A small region of a few pixels was created and a mean fluorescence level in each cell (cell) was calculated by averaging three quantifications in different x, y and z positions of the cytosol (the background was deducted from each measurement). To consider a cell positive for DsRedE expression, a threshold was defined empirically for each set of experiments, as the minimum level at which DsRedE expression in different z slices is unambigously detected (to avoid mistakes produced by fluorescence coming from cells located at other z positions). We then calculated the mean cell in each vesicle ( cell), the number of neurog1+ positive cells, and the global level of DsRed expression as the sum of the cell for all the neurog1+ cells in a vesicle. neurog1+Hi cells were defined as the ones that have fluorescent level higher than 1.5x cell of the control (DMSO or non-ablated side) vesicles. Dynamic quantifications were performed by sequentially measuring fluorescence at consecutive times of a video in the same cell. The mean rate of increase in fluorescence was calculated as . The same single cell fluorescence quantifications were performed in the neuroepithelial cells of the hindbrain, in a region adjacent to the otic vesicle.
To label cellular and subcellular structures, mRNA encoding for the following fusion proteins were injected at 1 cell stage after being synthesised with the SP6 mMessenger mMachine kit (Ambion): H2B-mCherry, H2B-GFP or NLS-Eos (100–150 pg) (Sapede et al., 2012), Pard3-GFP (50–75 pg) (Buckley et al., 2013), Lyn-EGFP (memb-GFP 100–150 pg), membrane-mCherry (100–150 pg). For the specification analysis, TgBAC(neurog1:DsRedE)nl6 dechorionated embryos were treated with SU5402 25 µm (Merk Millipore 572630), aphidicolin 300 µM (Merck) in combination with hydroxyurea 100 mM (Sigma), or DMSO (Sigma) added to the embryo medium. For determination of the number of pH3+ cells, DMSO or SU5402 treated embryos from 13 to 16 hpf were fixed and processed for the immunostainings.
The heat shock was performed by incubating 10 hpf embryos in preheated water at 39° during 30 min. Fluorescence from DNFGFR1-EGFP was detectable from about one hour after initiation of the shock. Induced embryos were selected at 12 hpf. For photoconversion or laminin immunostaining, EGFP- embryos were used as controls. For DsRedE expression analysis in which a membrane staining is relevant, neurog1:DsRedE embryos injected with memb-GFP at 1 cell-stage were heat shocked and used as controls.
For experiments using the fgf10a+/-; neurog1:DsRedE line, the embryos were mounted and imaged at 20 hpf for DsRedE expression analysis, recovered from the agarose, and incubated until 5 dpf, when the fgf10a-/- mutants embryos were identified by the absence of pectoral fins.
For immunostaining, dechorionated zebrafish embryos were fixed in 4% PFA overnight at 4°C and immunostaining was performed either on whole-mount or cryostat sections. Embryos for sections were cryoprotected in 15% sucrose and embedded in 7.5% gelatine/15% sucrose. Blocks were frozen in 2-Methylbutane (Sigma) for tissue preservation and cryosectioned at 14 µm on a Leica CM 1950 cryostat. After washing in 0.1% PBT, and blocking in 0.1% PBT, 2% Bovine Serum Albumin (BSA), and 10% normal goat serum (NGS) for 1 hr at RT, embryos were incubated overnight at 4°C in blocking solution with the appropriate primary antibodies: rabbit anti-Laminin (Sigma, 1:200), rabbit anti-pH3 (Abcam, 1:200). After extensive washing in 0.1% PBT, donkey anti-rabbit Alexa-488 (Thermo fisher scientific A21206; 1:400) was incubated overnight at 4°C in blocking solution. Sections were counterstained with 1 µg/ml DAPI, mounted in Mowiol (Sigma-Aldrich) and imaged in a Leica SP5 confocal microscope.
Synthesis of antisense RNA and whole-mount in situ hybridisation were performed as previously described (Thisse et al., 2004) to generate a probe against neurog1 (Itoh and Chitnis, 2001). Dechorionated Tg(elA:GFP) (which express GFP in rhombomeres 3 and 5) zebrafish embryos were fixed in 4% paraformaldehyde (PFA) overnight at 4°C and dehydrated in methanol series, rehydrated again and permeabilized with 10 mg/ml proteinase K (Sigma) at RT for 5–10 min depending on their stage. Digoxigenin-labeled probe was hybridised overnight at 70°C, detected using anti-digoxigenin-AP antibody at 1∶2000 dilution (Roche) and developed with NBT/BCIP (Roche). After the ISH, an immunostaining for the GFP expressed from the transgene was performed (primary antibody: rabbit anti-GFP (Torrey Pinnes; 1:400), secondary antibody: anti-rabbit Alexa-488 (Thermo fisher scientific A21206; 1:400)). Embryos were post-fixed overnight in 4% PFA and used for imaging mounted in 100% glycerol.
All statistical comparisons are indicated in figure legends including one sample and unpaired t-test performed using GraphPad. The box plot was generated in excel.
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Marianne BronnerReviewing Editor; California Institute of Technology, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]
Thank you for submitting your work entitled "Pioneer neurog1 expressing cells ingress in the otic primordium and instruct neuronal specification" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
Altogether, there was a great deal of enthusiasm about the paper but equally extensive discussion about the need for additional quantitation throughout. In particular, the reviewers request better characterization of the cells that ingress (and do not ingress), better characterization of the ablation experiments, and better characterization of the role of FGF. Given that eLife only allows 2 months for revision, the reviewers and editor did not feel you could realistically make the required changes in that window of time. Therefore we are returning the manuscript to you. However, if you should be able to address their comments in the future, we would welcome seeing a revised version that addresses these major concerns and would make every effort to return the paper to the original reviewers.
In this interesting and beautifully illustrated paper, Alsina and colleagues identify a population of cells, which they believe are derived from the lateral line placodes, that express ngn1 and invade the otocyst by insertion into the otic epithelium. They present evidence that these pioneer ngn1+ cells are at least partly necessary to induce further ngn1-expressing cells from the otic epithelium. If these pioneer cells are ablated, the authors suggest this leads to a reduction in the number of induced neurogenic cells in the otocyst.
The images are beautiful and well presented and I think the message of the paper is potentially interesting and challenging to our current ideas of otic neurogenesis. My main concern with the paper is that I feel the evidence for induction of ngn1 cells by the ingressing pioneer cells is less convincing than the rest of the paper. This is due in part to a lack of absolute numbers presented in the paper, which I expand on below.
1) In Figure 1, the authors provide a detailed temporal and spatial reconstruction of ngn1-expressing cells in the developing otocyst. It would be extremely helpful to the reader to give absolute numbers of cells in this part of the paper in addition to percentages. At 19hpf, how many cells are in the otocyst, and how many of them express the ngn1 reporter? This will be important for the rest of the paper (see below).
2) In the Results section the authors write: "The modest increase in the number of cells in this region cannot account for the large enrichment in cell proliferation, suggesting a higher proliferation rate in the NgD region." This sentence does not make sense to me. Can the authors rephrase it?
3) The Eos technique needs to be described better in the main text to make clear that a NLS-Eos construct was injected into the embryos at the 1 cell stage.
4) The authors suggest that the ingressing cells they observe are derived from the lateral line based on ngn1 expression. Are there other more specific lateral line markers or lineage tracers they can use to confirm this? Some posterior lateral line mutants such as cxcr4 and 7 and Kremen1 have migration defects. Are these expressed in cells invading the otocyst, and do mutants in these genes affect the development of ngn1+ cells in the otocyst? Such mutants might support the ablation studies presented in the paper.
5) I would like a better description or estimation of how many lateral line cells the authors believe ingress and contribute to the NgD. Is it possible to use the NLS-Eos labeling to estimate their contribution in the mature ganglion? Is it possible to evaluate whether they divide more than their otocyst counterparts, either in the otocyst itself or as transit amplifying cells once they have delaminated again?
6) The description of the lateral line cell ablation could be more detailed. The authors should indicate exactly how many lateral line cells were ablated per embryo and how many laser pulses were given per cell and per embryo. In addition to the contralateral control, they should also ablate cells outside the otocyst that are not lateral line cells and see if this affects neurogenesis in the NgD. It is possible that simply killing cells in the vicinity of the otocyst affects NgD development (for example, perhaps by altering FGF release or signaling).
7) The reason why points 1 and 6 are so important is that it is not clear whether the deficit in cells seen in the NgD domain after ablation is due to a loss of inductive signals from the ingressing cells (which is what the authors suggest) or is simply the result of depleting cells that would otherwise contribute to the NgD. At the moment, it is not clear to the reader which is more likely. As mentioned above, a better description and discussion of the quantitative data would be in order here – how many lateral line cells do the authors think ingress into an otocyst (1? 10? 50?) versus how many NgD ngn1+ cells are generated within the otocyst. The authors offer their reverse tracing of their movies as evidence to suggest that most cells expressing high levels of the ngn1 reporter come from inside the otocyst and thus the contribution of the ingressing cells is small, but it is not completely clear if this experiment was performed in normal or ablated embryos.
8) The authors suggest that FGF may be responsible for helping the lateral line-associated cells ingress into the otocyst. In support of this, they show a reduction in ngn1-reporter cells in the otocyst after SU5402 treatment, and a reduction in NLS-Eos labeled lateral line cells in the otocyst. I have two questions – have the authors made time lapse films of the SU5402 treated embryos directly showing a failure of ngn1-reporter cells to ingress, and in their NLS-Eos experiments, is the TOTAL number of photoconverted cells (inside the otocyst + outside the otocyst) the same, but it is just their distribution in the two compartments that differs?
9) In their Discussion, the authors mention the Notch signaling pathway in relation to zebrafish otic neurogenesis. Do ngn1+ neurons express Δ ligands, and if so, wouldn't these inhibit rather than promote neurogenesis if they ingressed into a field of Notch-expressing progenitors?
Hoijman and colleagues have provided a detailed description of neurogenesis in the zebrafish otic primordium. They provide a number of interesting findings. Using reporter constructs expressing dsred under regulation of neurog1 genomic elements, they identify cells that express dsred that subsequently ingress into the otic vesicle, while others begin expression within the vesicle before dividing. Additional cells express dsred after division, with both daughters expressing the reporter. Ablating ingressing cells reduces the overall number of dsred+ cells suggesting that the ingressing cells have instructive roles in promoting neurog1+ expression in cells within the otic vesicle. Together the work describes a potentially novel mechanism for neurogenesis within the otic placode that may have more general implications.
1) It is not clear that only dsred+ cells ingress into the developing otic vesicle. It looks like in some videos that dsred- cells also ingress. Moreover it is not clear that only anterior cells ingress – in Video 6 there appear to be cells in the ventral posterior that also do so. These potentially alternate observations might suggest that there is a substantial addition to the otic vesicle after its initial formation but would change the interpretation of dsred+ cells are undergoing a characteristic pioneer behavior. It appears that the authors have the ability to track all cells contributing to the otic vesicle to resolve this issue. It's important to do so, for other results such as the effects of FGF would be interpreted in a different light.
2) It is not clear how specific the ablations are for the dsred+ pioneer cells. Are other dsred- neighbors damaged or spared? If dsred- cells are also ingressing (see comment 1) then the specificity of ablation may be an issue.
3) Is the reduction of dsred+ precursors due to the specific ablation of ingressing cells or is it due to the reduction of the overall number of dsred+ cells? That is, could there be a 'community effect' where dsred+ cells induce others irrespective of their initial origin outside the otic vesicle?
4) Does ablation of dsred+ ingressing cells alter proliferation of cells within the otic vesicle?
In this manuscript the authors study otic neurogenesis. They describe a population of pioneer cells in zebrafish, which arises outside of the otic placode, invades the placode and generates neurons. Their experiments suggest that these cells play a role in promoting neurogenesis in neighbouring otic epithelial cells, since their ablation reduces neurog1 expression (both levels and cell numbers). They also perform morphometric and proliferation analysis of the otic vesicle, and finally some experiments to suggest that FGF signalling somehow controls the integration of the pioneer cells into the vesicle.
The idea of pioneer cells is an interesting and novel finding that warrants further investigation and provides a novel view on how neuroblasts are determined in the ear. However, throughout the manuscript the authors make quite forceful conclusions that are not always supported by their data (e.g. for most experiments numbers are extremely low, or not given). The morphometric analysis does not add much to the paper, and I wonder if it should be removed. The FGF results are very preliminary and in their current form are not conclusive. The authors do not provide a clear model for FGF function and thus do not provide any mechanistic insight.
Below, some specific comments that the authors should address.
1) Throughout the manuscript the figures need to be improved. To help the reader figures need better labelling: label SAG in Figure 1 (and other figures), label delaminating neurons to distinguish them from neuroblasts in the vesicle. Outline the otic placode/vesicle in figures and movies and add arrows for orientation (anterior-posterior, medial-lateral) into the movies. In some videos it is difficult to see which neurog1-dsRedE cells are in the vesicle, remain outside or delaminate. It needs to be clear which are the SAG cells and the placode cells.
2) Morphometric analysis in the Results section: these measurements do not add very much to the paper and do not really allow any particular conclusions and distract from the main part of the paper. Maybe this section should be removed.
If this section remains in the manuscript, the authors need to provide numbers: how many placodes were analysed? How representative are the results? This sentence is unclear: "The modest increase in the number of cells in this region cannot account for the large enrichment in cell proliferation, suggesting a higher proliferation rate in the NgD region." The authors conclude that the neurogenic domain has a higher proliferation rate, but have not assessed proliferation rate and therefore this conclusion is not valid.
3) The authors say that neurog1 cells become integrated into the vesicle during placode formation; can they specify the timing and explain this better for readers unfamiliar with the fish and with the otic development. This is also picked up in the Discussion, however the timing is not very clearly described in the paper.
4) In the Results section; Figure 2: The authors describe that neurog1 + cells from outside the otic vesicle become integrated into the otic vesicle, and generate neurons of the SAG. In Figure 2, Video 6 they show that some cells exhibit this behaviour, while others do not. It is not clear how this behaviour is determined; are those cells that do invade the vesicle always neurog1 positive? The authors need to compare the behaviour of neurog1 + cells; it is quite possible that only neurog1 + cells exhibit this behaviour, and that this is the reason that position of cells is irrelevant.
5) What are the cells in the "second pool of neurog1+ cells"? do they contribute to the SAG, other cranial ganglia?
6) What do the authors mean? "Additionally, other morphological features particular of these stages could contribute to cell ingression"
7) In the Results section: the authors suggest that levels of neurog1 determines when cells delaminate from the vesicle. Can they provide numbers: how many cells were measured? What is the threshold? Please provide statistics to support this claim.
The final conclusion from this section is: "This suggests that cells delaminate relative to neurog1 levels and not to the time elapsed from the beginning of neurog1 expression". The authors do not measure the time a cell spends in the vesicle; how do they come to this conclusion?
8) In the Results section the authors re-visit cell division within the neurogenic domain, which is already described in the first section of the paper. It is not clear what this section adds and why Pard3 staining is relevant in this context, and what this adds to the main message of the paper. The whole section seems a bit out of place. The conclusion is that "our analysis of the origin of neurog1+ cells revealed that they are added to the NgD by three different mechanisms: cell ingression, local expression and cell division. They are not really different mechanisms, neuroblasts are known to proliferate and expand before becoming terminally determined and this observation is therefore not surprising.
9) The authors show that ablation of neurog1+ cells (pioneer cells) leads to a reduction of neurog1 expression in the otic vesicle. They suggest that the number of ablated cells determines the how many neurog1+ cells later appear in the vesicle as well as the level of neurog1 expression in each cell. The authors only show 2 embryos; this is not sufficient to reach this conclusion. Are there more specimen; please provide numbers.
The authors state that otic neurogenesis does not recover after ablation, but do not provide much evidence (2 embryos in Figure 4—figure supplement 2). I would like to see more examples to reach such a firm conclusion. What happens to the SAG? Is there other compensation? Embryo 2 in Figure 4—figure supplement 2 appears to form some neurog1 cells eventually; have the authors looked later?
Do the authors suggest that the majority of otic neuroblasts is induced by the pioneer cells? If so, this is interesting and novel, but this requires more data with solid statistics and longer observations.
The authors measure the level of fluorescence in individual cells in pioneer ablated and non-ablated embryos: how many embryos were analysed, how many cells?
10) In zebrafish the otic vesicle does not delaminate as an epithelium but rather coalesces from cells that then form a lumen. As such it's quite possible that addition of cells to the vesicle continues after initial lumen formation. Therefore the issue in my mind was whether there was specificity for dsred+ cells invading. If nonspecific then the FGF requirement is just a continuation of the requirement for vesicle formation. If nonspecific then the specificity of the ablation needs to be determined – ablation of bystander dsred- cells will also contribute to any observed phenotype.
11) The FGF results are not convincing; more experiments are needed to provide a clear view of what exactly FGF is supposed to do in this context. The authors should provide a clear model of how they suggest that FGF acts: what is the source of FGF? Do pioneer cells have FGF receptors? Do they suggest that FGF is an attractant? Why do not all neurog1 cells invade the placode if that is the hypothesis? How can the effect on pioneer neuron invasion and proliferation be unravelled? Does FGF have multiple effects, not only affecting pioneer invasion, but also local induction of neuroblasts and their expansion?
Simply blocking signaling by SU5402 does not provide definitive proof that FGF is involved since other receptors are also affected by this drug. The authors need to use different ways to show that FGF is involved; these could be dominant negative receptors in pioneer cells, cell type specific knock-down of FGF response or secretion. Without further experiments this part of the manuscript opens up too many unanswered questions.
12) Discussion section. The authors need to be more careful with their choice of words (there are many examples throughout the text); they suggest that the pioneer neurons are the first "specified cells" and "promote neural commitment" – we do not know if these cells are truly specified; this can only be assessed by culturing them in isolation. What do the authors mean by promoting commitment? That pioneer neurons induce neighbouring cells to become neuroblasts?
13) The Discussion section is a bit convoluted and the arguments are not clearly structured, and often the authors seem to over-interpret their data. For example, the authors argue strongly that positional information does not determine whether or not a cell adopts pioneer identity, but it may equally be possible that by the time they evaluate this cells are not competent to do so.
14) It is surprising that the authors do not refer to an older paper by Hemon & Morest 1991 describing the 'otic crest', as well as a more recent paper describing neural crest contribution to the otic vesicle in mouse. This may be very relevant to the current study.
In this context, the authors should discuss whether their model could also be true in higher vertebrates like chick and mouse, where development of the placode is a much longer process than in amphibians and fish. For example, in fish and frog neurogenic markers are expressed much earlier than in mouse and chick; could their observation be species specific?
'Ingression' is not the right word to describe that individual cells from outside the otic vesicle are integrated into it. They invade, are incorporated, inserted or similar.
'Local specification': in developmental biology 'specification' is used to describe along which path cells or tissues can differentiate when cultured in isolation. Therefore in this context the term 'local specification' is somewhat misleading. The authors should find a better term.
16) A native speaker should read the manuscript; it contains errors, and peculiar constructions and expressions making the text at times a bit cumbersome to read. E.g. "Pioneer cells specify outside the otic primordium and ingress during otic placode formation". Should read: 'are specified'.
17) The authors should avoid too many abbreviations; they make the text more difficult to read in particular for readers outside the field. There is no reason to abbreviate terms like 'neurogenic domain', 'global level of dsRedE expression' (GLE) or similar.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for submitting your article "Pioneer neurog1 expressing cells ingress in the otic primordium and instruct neuronal specification" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Reviewing and Senior Editor.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
This manuscript provides live imaging data to demonstrate that neuronal specification in the inner ear of zebrafish is dependent on a number of neurogenin 1 (ngn1) expressing pioneer cells that enter the otic primordium transiently, which cause induction of otic epithelial cells to upregulate ngn1 and these cells then delaminate to form the statoacoustic ganglion (SAG). Furthermore, the authors demonstrate that this ingression process is dependent on FGF signaling using dominant negative FGFR transgenic fish and an Fgfr inhibitor, SU5402.
1) If their neurog- neighbors are also joining the placode, then ingression is not a distinct behavior of 'pioneer' cells. While this observation does not detract from authors' main conclusions the specific roles of the 'pioneer' cells in promoting the formation of later neurons, it does speak to whether the 'pioneer' cells are actually outside the otic primordium (defined as the group of cells that will form the otic placode). The authors make a convincing argument that global tracking would be time-consuming and not directly applicable to answering the central questions of the work presented. I therefore suggest that they drop claims that these 'pioneer' cells are outside the otic primordium as stated in the Abstract and elsewhere.
2) The study begs the question of whether ngn1 is required for the pioneer cells, the ingression process, the otic epithelial cells to respond to the pioneer cells, or all of the above. It may be difficult to tease out all requirements of ngn1 but whether cell ingression proceed normally in the ngn1 knockout mutants should be investigated.
3) The authors concluded that FGF normally delays epithelial barrier formation in the anterior otic primordium. As a result, less pioneer cells get into the primordium to induce subsequent ngn1+ cells. The authors demonstrated a reduction of ngn1+ cells within the otic epithelium when Fgfr function was knockdown. If the authors are correct, one should expect a pileup of pioneer cells outside the placode. A video and quantification of the pioneer cells demonstrating this fact is lacking and warranted. The premature basement membrane formation of the otic epithelium using anti-laminin staining is not convincing. While the dotted lines outline the border of the otic primordium, it also negated judgement of the data by readers. What happens with gain of FGF function models? Would there be an increase in cell ingression compare to wildtype? If so, would one conclude the rate-limiting step is the barrier formation of the anterior otic primordium. That is if FGF only functions in the otic epithelial cells and not in the pioneer cells.
4) In the Results section the argument claiming that small differences in the cell number but large differences in the mitotic event found in the neurogenic domain suggests an increase in proliferative activity is unclear. A high mitotic event should result in an increase in cell number at a later time point, at the least, or the moderate increase in cell number is due to neuroblast delamination.
5) The authors cited a reference by Raible's lab in regards to ngn1 gene expression in the pioneer cells. The ngn1 expression in Figure 2 of Raible's paper is later than the onset of pioneer cells ingressing the otic epithelium described here. It may be a good idea to determine whether ngn1 transcripts are detected in the pioneer cells. It will complement the functional study of ngn1 suggested above.
6) The illustration in Figure 4I is not helpful to describe all the scenarios that the authors are considering. At least, the authors should describe/cite Figure 4 Iii in the text. The text in the legend also needs clarification.
7) The summary diagram in Figure 5L is misleading and does not serve the manuscript. The summary diagram implies that FGF signaling is required for cell ingression, but according to the authors, FGF signaling mediates cell ingression indirectly by delaying epithelial barrier formation. At the minimum, the summary diagram should be flipped so that the medial region is towards the top to be consistent with the videos and images shown in the figures. The authors should modify the diagram; as it is it somehow suggests that it affects the incorporation of cells into the vesicle.
[Editors' note: further revisions were requested prior to acceptance, as described below.]
Thank you for resubmitting your work entitled "Pioneer neurog1 expressing cells ingress in the otic primordium and instruct neuronal specification" for further consideration at eLife. Your revised article has been favorably evaluated by Marianne Bronner (Senior and Reviewing editor) and three reviewers.
The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:
In general, the reviewers remain largely enthusiastic about your work. However, they all felt that you argued many of their points rather than making relatively easy changes to the manuscript. I'm afraid I am unable to accept your paper without better attending to the criticisms of the reviewers. I would like to highlight that it is highly unusual for eLife to allow more than one round of review. I am willing to do so in this case because I think you can make the needed changes with alterations to the text and figures. I stress that it is essential that you make every attempt to address the reviewers comments rather than arguing them. Otherwise, I will have no choice but to decline the paper. I have included the detailed comments of the individual reviewers so that you can better understand their requests and I ask you also to better address the concerns raised in the original review.
While it seems we are in a silly semantic argument I think the point I made in the original review still stands.
I would argue that the otic primordium/placode is the field of all cells that subsequently form the otic vesicle. I interpret the authors results to show that not all otic primoprdium/placode cells join the otic vesicle at once, as some cells ingress into the otic vesicle epithelium after it forms. I originally pointed out that it looked like cells other than the ones designated as neuronal pioneer cells were also ingressing. As the zebrafish otic vesicle forms by coalescence rather than the epithelial infolding and delamination described for amniote embryos, this type of MET would perhaps be expected.
If a whole bunch of cells are ingressing into the otic vesicle then the behavior is not specific to the neuronal pioneer cells. Indeed I would argue that if neuronal pioneer cells and their neighbors are all joining the otic vesicle, then these cells and their neighbors are part of the otic primordium/placode even if they join the vesicle after it has formed an epithelium. However if they are interspersed with other cells that do not join the otic vesicle, that would be evidence for a distinct behavior more along the lines of what the authors are implying. The authors were unwilling to perform the timelapse analysis and in toto lineage tracing to address this point. I agreed that in toto imaging is not necessary for the main conclusions of the current work. But I do not believe that the authors should state that ingression is a special behavior unique to pioneer cells, which is what is implied as the text currently stands.
The authors have addressed most points raised in previous review.
They did not change the summary diagram in Figure 5L; if I remember correctly all three reviewers raised the point that the summary does not accurately reflect the findings of the authors indepdently. THis suggests that the message the authors wish to convey is not clear. In its current form the diagram suggests that FGF affects the behavior of ingressing cells directly, but the authors show that it affectes the formation of the epithelial barries – this is how arrows are interpreted even if this is not the intention of the authors. It should not be very difficult to change this to make the figure accurately reflect their findings.
Ngn1 not required for the ingression process: The lack of cell ingression in the ngn1 null mutants is interesting and is relevant information. I don't see why it should not be incorporated into the manuscript with some additional quantification.
Knockdown of FGF signaling reduced ingression of ngn1-positive cells: Although there is no strong evidence for the predicted ngn1 positive cells piling up outside the FGF lof placode, the authors proposed several alternate explanations for this observation, which are acceptable. The authors provided additional FGF gain-of-function results, which further supported a role of FGF in regulating barrier formation in the otic placode. These results, together with the lof experiments, argue against a direct role of FGF in the ingression process.
Anti-laminin staining: A higher magnification of the control in a comparable region as the treated sample would help to convince readers. The anti-laminin staining in the mutants, though seems stronger than controls, is blotchy and discontinuous and raises the question whether there is indeed a basement membrane barrier. However, the additional gain-of-FGF function experiments supported a normal role of FGF in delaying barrier formation.
I appreciate the authors' attempt to explain their point of view. In Results paragraph two, it states "The large difference in the change of the two parameters (meaning low cell number increase but high percentage of mitotic events) suggests that the increased number of mitotic events is not a consequence of having more cell dividing at the same rate, but due to a specific increase in the proliferative activity of these cells". I interpreted this sentence to mean that there were not more cells going into cell division but the same cells are dividing faster, which contributed to the higher percentages of mitotic counts. If so, I am confused because I thought the authors measured mitotic events rather than measured how fast each cell go through the cell cycle. Proliferate activity and proliferate rate are not interchangeable terms in my mind. Increased proliferate activity (mitotic events) could be caused by either increased proliferate rate or increased number of cells in division. Either scenario should result in an increased in cell number. The moderate increase in cell number observed at the time of measurement could be simply due to timing. The authors measured mitotic events between 14 to 18.5 hpf. On average, each cell cycle takes about 8-12 hrs. Considering the cells have just reached the placode at 13-14 hpf, peak increase in cell number may not be apparent until after 18.5 hpf. By this time, some neuroblasts will start to delaminate from the epithelium, which will also reduce the total cell number in the region.
I do not find the option 1 and 2 described in the rebuttal letter particularly helpful, partly due of the interchangeable terms of proliferative rate and proliferative activity.
Summary diagram: I might have over-interpreted those videos. Based on the examples shown in the videos, it appears that ingression of cells is occurring at the lateral edge of the placode and delamination is slightly medial to where ingression takes place. This pattern is consistent with the position of the statoacoustic ganglion being located medial to the otocyst. If this is correct, the summary diagram should reflect the spatial relationship between ingression and delamination.
I still think the arrow of the FGF in the summary diagram is misleading, but it is up to the authors to decide the best way to summarize their data.
In summary, this work describes for the first time the cell ingression phenomenon in the otic placode of zebrafish. It also demonstrated that after ingression, the ngn1-positive cells instruct more neighboring cells to turn on ngn-1 within the placode. The mechanisms underlying these cellular events, however, are not known. With the additional data provided, neither ngn-1 nor FGF appears to be directly involved in the cell ingression process. In fact, the data from FGF become a distraction. This ingression process could very well be a general mechanism of how cells populate the otic placode and may not be specific for the neuronal specification, as suggested by reviewer 1. Despite the nice live imaging results, there are not much mechanism to grapple with.https://doi.org/10.7554/eLife.25543.035
- Berta Alsina
- Berta Alsina
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank A Nechiporuk for the neurog1:DsRedE fish line, S Schneider-Maunoury and M Breau for the hsp70:dnfgfr1-EGFP line, D Gilmour for the fgf10a mutant line, C Pujades for the eA:GFP line, E Marti, I Gutierrez Vallejo and E Gonzalez Gobart for helping with the fishes, P Bovolenta for critically reading the manuscript, R Aguillon and J Batut for their help and the fishes, and the members of the Advanced Light Microscopy Unit of the UPF/CRG.
Animal experimentation: All experiments conform to the guidelines from the European Community Directive and the Spanish legislation for the experimental use of animals. The protocols (AR081096P4 and AR081098P3) were approved by the Committee on the Ethics of Animal Experiments of the Parc de Recerca Biomèdica de Barcelona (PRBB), Spain.
- Marianne Bronner, Reviewing Editor, California Institute of Technology, United States
© 2017, Hoijman et al
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.