Ptf1a expression is necessary for correct targeting of spiral ganglion neurons within the cochlear nuclei

Abstract

Two transcription factors, Atoh1 and Ptf1a, are essential for cochlear nuclei development. Atoh1 is needed to develop glutamatergic neurons, while Ptf1a is required to generate glycinergic and GABAergic neurons that migrate into the cochlear nucleus. While central projections of inner ear afferents are normal following loss of Atoh1, we wanted to know whether the loss of Ptf1a affects central projections. We found that in Ptf1a mutants, initially, afferents show a normal projection; however, a transient posterior expansion of projections to the dorsal cochlear nucleus occurs at a later stage.. In addition, in older (E18.5) Ptf1a mutant mice, excessive neuronal branches form beyond the normal projection to the anterior and posterior ventral cochlear nuclei. Our results on Ptf1a null mice are comparable to that observed in loss of function Prickel1, Npr2, or Fzd3 mouse mutants. The disorganized tonotopic projections that we report in Ptf1a mutant embryos might be functionally relevant, but testing this hypothesis requires Ptf1a KO mice at postnatal stages that unfortunately cannot be performed due to their early death.

1. Introduction:

The cochlear nuclei, located in the dorsal brainstem, receive incoming auditory stimuli via the spiral ganglion neurons (SGNs) and are the first site of auditory processing. The cochlear nuclei develop from rhombomeres (r) 2–5 to form the anteroventral (AVCN), the posteroventral (PVCN), and the dorsal (DCN) cochlear nuclei (Duncan et al., 2022). The cell fate determination of cochlear nuclei neurons is crucially dependent upon the expression of the basic helix–loop–helix (bHLH) genes, Atoh1 and Ptf1a. Atoh1 expression is needed for the development of glutamatergic neurons (Fritzsch et al., 2006; Maricich et al., 2009; Wang et al., 2005), whereas Ptf1a is required for the development of glycinergic and GABAergic positive neurons (Fujiyama et al., 2009) within the AVCN, PVCN, and DCN. Using Atoh1-LacZ we showed that the different cochlear nuclei develop with a different distribution of neurons in these three nuclei, such that they have a different neuronal density: AVCN>PVCN>DCN [Supplement Fig. 1A; (Elliott et al., 2021a)]. In contrast, neurons belonging to the Ptf1a-lineage show a different distribution, such that they have the highest density in the DCN and fewer neurons of this lineage is found in the ventral cochlear nucleus (VCN) [Supplement Fig.1B; DCN>>VCN, (Fujiyama et al., 2009)].

The gene Ptf1a encodes a bHLH transcription factor protein that is a sequence-specific DNA-binding subunit of the trimeric pancreas transcription factor-1 (Kawaguchi et al., 2002). Ptf1a was present in the earliest deuterostomes (Jin and Xiang, 2019) and plays a role for the exocrine lineage of the pancreas as well as specific neuronal development (Hoshino et al., 2021a; Hoshino et al., 2005; Jin and Xiang, 2019). Within the nervous system, Ptf1a is broadly expressed from the forebrain to the spinal cord. In particular, Ptf1a has been shown to contribute to the development of GABAergic neurons in the cerebellum (Haldipur and Millen, 2019; Hoshino et al., 2021a; Hoshino et al., 2005; Lowenstein et al., 2022; Millen et al., 2014; Pascual et al., 2007; Yamada et al., 2014) and glycinergic and GABAergic neurons in the cochlear nuclei in the dorsal brainstem (Fujiyama et al., 2009; Iskusnykh et al., 2016). In addition, single cell RNAseq studies revealed Ptf1a expression in a subset of SGNs (Petitpré et al., 2022). In the dorsal brainstem, Ptf1a is expressed in two neuronal precursor areas, dA4 and dB1. These neurons migrate to populate r2–5, contributing to the cochlear nuclei (Fujiyama et al., 2009; Iskusnykh et al., 2016). Loss of Ptf1a leads to the absence of the glycinergic and GABAergic neurons (Fujiyama et al., 2009) that normally migrate from dA4 and dB1 progenitor domains (Supplement Fig. 1D, E). Moreover, in Ptf1a KO mice, the number of dA3 and dB3 neurons in increased in the dorsal barainstem [Supplement Fig. 1E; (Iskusnykh et al., 2016)]. Most of the glycinergic and GABAergic cochlear nucleus neurons receive a direct input from the SGNs, while others do not [Supplement Fig. 1C; (Oertel and Cao, 2020; Trussell and Oertel, 2018)]. However, a role for Ptf1a in guiding/targeting SGN projections to these cochlear nuclei neurons has not yet been known.

The role of Atoh1 on the formation of central projections has been previously investigated and when completely or conditionally deleted, little to no apparent effect on the tonotopic organization of the central projections of SGNs is observed (Elliott et al., 2017). However, unlike Atoh1, a more profound reorganization of SGN central projections occured following the loss other transcription factor genes, including the bHLH gene Neurod1 or the LIM homedomain gene Isl1 (Filova et al., 2022a; Filova et al., 2020b; Macova et al., 2019). Given the variations in the ability of genes expressed in the cochlear nucleus, such as Atoh1 or Neurod1, to affect central pathfinding of SGNs, we wanted to determine whether Ptf1a plays a role in the targeting of SGNs in the cochlear nuclei. Here, we document the central projections of SGN in the cochlear nuclei in embryonic mice in which the Ptf1a gene was deleted (Iskusnykh et al., 2016). Our data show that loss of Ptf1a results in aberrant central projections, somewhat comparable to effects of Prickle1 (Yang et al., 2017), Npr2 (Schmidt and Fritzsch, 2019), and Fzd3 deletions (Duncan et al., 2019; Stoner et al., 2022). Loss of Ptf1a results in an exuberant central projection that transiently expands posteriorly and has additional branching within the AVCN.

2. Material and Methods.

We used Ptf1a^cre/+ mice that were bred to generate Ptf1a null (Ptf1a^cre/cre, referred to as Ptf1a KO mice throughout the paper), heterozygotes and control mice as previously described (Iskusnykh et al., 2016; Kawaguchi et al., 2002). All mice used in this study were genotyped as previously described (Iskusnykh et al., 2016). Noon of the day of the plug was designated as E0.5. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center (IACUC #15-057 and 18-037).

A total of four E18.5, five E16.5, two E14.5 and two E12.5 of Ptf1a KO and a similar number of control and heterozygotes were fixed for 24 hours in 4% PFA with 300 mM sucrose after which the fixation was diluted to 0.4% PFA with 300 mM sucrose and stored at 4° C until use. After fixation, heads were removed.

To trace neuronal projections between the inner ear and the brain, we used NeuroVue dyes (NV Jade, NVJ; NV Red, NVR; NV Maroon, NVM) as previously described (Schmidt and Fritzsch, 2019). Lipophilic dyes were applied using dye-soaked filter strips, which provide a more precise and reliable way of a dye application compared to crystals or dye-injections. The heads were hemisected and the ears were exposed by removing the tympanic membrane. Different colored dye-soaked filter strip wedges were inserted into the apex and into the basal turn through the round window. A third dye color was inserted into the utricle, anterior, and horizontal canals. Dyes were allowed to diffuse for 24–72 hours at 60°C depending on the age of the embryos. Mutants, heterozygotes, and control embryos were always processed in parallel, with an identical dye placement and an identical dye diffusion time. Brainstems and inner ears were subsequently micro dissected and mounted in glycerol. Images were obtained using a Leica SP8 confocal microscope with focal lengths of 10x (NA 0.5) and 20x (NA 0.8). Z-series stacks were collapsed into single images using the Leica imaging system (LasX). Figures were assembled using CorelDraw software.

For axon reconstruction, Z-series images were loaded in ImageJ software and ten individual afferents per mutant and control within the AVCN were manually segmented using the Simple Neurite Tracer (SNT) plugin as previously described (Duncan et al., 2019; Stoner et al., 2022). Linear Sholl analysis (Duncan et al., 2019) was performed to determine the straightness of projections within the AVCN. Individual reconstructed axon tracings obtained from the ImageJ software were copied into CorelDraw. Each axon was manually rotated such that the terminal points fell along a horizontal line. The top and bottom boundary limit of the vertical (y-axis) trajectory of each control axon was determined. The mean y-axis width and standard deviation were calculated from twenty control axons. These values (mean, +1, and +2 standard deviations) were used in determining the number of times an axon crossed a value boundary. A linear Sholl crossing was counted when an axon trajectory crossed the boundary of the mean width or crossed either of the standard deviation boundaries. In addition, the number of times an individual reconstructed axon crossed over another axon was counted as previously described (Duncan et al., 2019; Stoner et al., 2022). Means and standard error of the means were calculated. Student’s t-test was performed, and significance was determined at p<0.05.

3. Results

Early observation of the central projections of SGNs to the cochlear nuclei in E12.5 and E14.5 mice was accomplished following insertion of lipophilic dye into the spiral ganglion. At these early stages, it was technically impossible to split the SGN labeling into the basal and apical SGNs. These early SGN central projections (green) of Ptf1a KO mice were nearly indistinguishable from that of control littermates (Fig. 1A–B). In addition, the trigeminal (red) and glossopharyngeal/vagal fibers (lilac) were normal, suggesting the change in the central organizations within the hindbrain due to the loss of Ptf1a and subsequent upregulation of Lmx1b (Supplement Fig.1D–E; (Iskusnykh et al., 2016) is not affecting the initial central projections of peripheral neurons, despite the massive reorganizations of brainstem neurons.

Fig. 1. Loss of Ptf1a does not affect central projections at E14.5.

The central projection of SGNs to the cochlear nuclei is shown in E14.5 mice (A, B). At this stage, there is little, if any, difference in central projection of the cochlea to the cochlear nucleus between Ptf1a KO and control littermate mice. Trigeminal (red) and glossopharyngeal (lilac) central projections are also shown (A, B). Note that the projections that extend from r2-r5 is identical between control and Ptf1a KO mice. AVCN and DCN indicate central projections to AVCN and DCN nuclei, respectively. Anterior is left, posterior is right, dorsal is up, ventral is down. Bar indicates 100 μm.

However, by E16.5, central projections of Ptf1a KO mice are atypical compared to that of control littermates (Fig. 2). In mice, the DCN occupies r5 and is not present in r6 (Di Bonito and Studer, 2017; Fritzsch et al., 2022). Thus, the central projections of control mice and heterozygotes (data not shown) end up in r5 (arrow in Fig 2A). In E16.5 Ptf1a KO mice, the apex labeled fibers appeared to be expanded posteriorly (Fig. 2B, arrow). A marker between r5 and r6 that is identified as the anterior inferior cerebellar artery (AICA) that passes the glossopharyngeal (NIX) nerves to define the transition between r5 and r6. Compared to the basal (red) fibers, the apex (green) fibers were found at more posterior positions and showed additional branching in Ptf1a KO mice (Fig. 2C–E, white arrows).

Fig. 2. Central projection abnormalities in Ptf1a KO mice at E16.5.

The central projection of E16.5 Ptf1a KO mice shows more side branches in the AVCN/PVCN than in control littermates (A, B). In control littermates, apex (green) fibers end up in the posterior DCN (known to occupy r5) (arrow in A). In the mutants, the caudal fibers appear extended posteriorly beyond r5 (arrow in B). (D, E) In the mutants, the apex (green) fibers extend to more posterior positions than base (red) fibers and show additional branching to reach r6 (arrows in D, E).. All Ptf1a KO central projections had a branch that loops around the vestibular ganglion neurons (VGN) to eventually project to the cochlear nuclei (C). Efferent fibers (not indicated) overlap with basal fibers (base, C) and apical fibers (green, C). Anterior is left, posterior is right, dorsal is up, ventral is down. Bar indicates 100 μm.

By E18.5, control mice (Fig. 3A,C) show a large central projection to the AVCN that tapers toward the DCN and terminates in r5. The beginning of side branches to innervate the granule cells in the adult mice (Muniak et al., 2016; Nayagam et al., 2011) is shown to be coming from the apex, not the base (Fig. 3A). Parallel branches project to the bushy cells and T- and D-stellate cells of the AVCN (Oertel and Cao, 2020; Trussell and Oertel, 2018) that will eventually form the large Held terminals (Glover and Fritzsch, 2022). All other cochlear nuclei neurons will be innervated with puncta (Supplement Fig.1C), except for two neuron types in the DCN, the cartwheel cells and the superficial stellate cells. The notable parallel fibers are visible in the AVCN (Fig. 3A, B) that will innervate mostly spherical and globular bushy neurons in r2/3. Like with the control mice, at E18.5, the Ptf1a KO mice have no posterior expansion of central projections to DCN (Fig. 3B). However, there are side branches labeled with lipophilic dye implanted into the apex that go beyond the normal apical projection and terminate close to the fibers from the base (Fig. 3D). In addition, we find occasional neurons labeled from the basal dye injection that have topologically unusual central projections. Normally, SGN fibers will innervate bushy cells and T-stellate cells in the AVCN that lack the D-stellate cells in the mutant (Supplement Fig. 1C). It is possible that the branches are predominantly from the absence of D-stellate cells. Coronal sections of the cochlear nuclei show a normal central projection with a clear segregation of the base (red) and green in E18.5 control mice (apex, Fig. 4A). In contrast, in Ptf1a KO littermates, there is much less space between apical and basal fibers (Fig. 4B) and some apical fibers overlap with basal fibers.

Fig. 3. Central projection abnormalities in Ptf1a KO mice at E18.5.

By E18.5, the projection to cochlear nuclei show more side branches in the AVCN/PVCN of Ptf1a KO mice compared to littermate controls (A-D, arrow in D). Many more side branches are observed that emanate from the AVCN and PVCN in the Ptf1a mutants that reaches beyond the parallel fibers when compared with control littermates (C-D). However, central projection of SGNs to the DCN is similar between controls and Ptf1a KO mice (A-D). Thus, it appears that the projections beyond the DCN are transient and show no such expansion at E18.5 that had been as shown at E16.5 in Fig. 2. Note that occasionally the apex can be split, one anterior and one posterior to the basal projection (B). Anterior is left, posterior is right, dorsal is up, ventral is down. Bar indicates 100 μm.

Fig. 4. Reduced segregation of apical and basal fibers in Ptf1a KO mice.

Coronal sections after labeling from the base (red) and apex (green) show the segregation of the AVCN in control mice (A). In contrast, while there is still a segregation of the base (red) and the apex in the Ptf1a KO mice, the fibers are in close approximation to each other. Bar indicates 100 μm.

To further examine some of these aberrations in mutant mice as compared with controls, ten individual SGN axons per animal were traced within the cochlear nucleus at E18.5 (Fig. 5A–B’). Using a linear Sholl analysis, reconstructed SGN axons from Ptf1a KO mice projected with a broader path compared with axons from control littermates (t-test, p<0.05; Fig. 5C). Moreover, reconstructed axons from Ptf1a KO mice were shown to cross over adjacent axons, whereas this was not observed in the control littermates (t-test, p<0.001; Fig. 5D).

Fig. 5. Aberrant axonal crossing in Ptf1a KO mice.

Reconstructed SGN axons within the AVCN from control (A) mice are ordered tonotopically and represented as rainbow colors from more basal to more apical SGN projections. In contrast, reconstructed axons within the AVCN from Ptf1a KO mice show more aberrant projections (B-B’). The orange neuron depicted below each image in A-B’ is an individual axon tracing overlaid onto boxes representing the mean dorsoventral limit of all control axons (dark gray), plus or minus one standard deviation (1 SD, medium gray), and plus or minus two standard deviations (2 SD, light gray). Grayscale circles represent mean/SD level crossings. The linear Sholl analysis of 20 axons from E18.5 control and Ptf1a KO mice shows a significant difference (*,p<0.05) for crossings of the mean and 1 SD boundaries (C). The mean number of times a single axon crossed over another axon from an analysis of 20 axons each from controls and Ptf1a KO mice (D) was significantly different (***, p<0.001). Error bars represent standard error of the means.

In summary, our data suggest a near normal central projection in the absence of Ptf1a in early embryonic mice. At E16.5, a transient posterior expansion of projections to the DCN was observed in Ptf1a mutants. Finally, we detected aberrant central projections to the AVCN/PVCN in Ptf1a KO mice.

4. Discussion

Our results presented here implicate Ptf1a in axon guidance within the cochlear nuclei. Thus, adding Ptf1a to growing list of genes involved in central pathfinding of inner ear afferents that include Neurod1, Lmx1a/b, Isl1, Prickle1, Fzd3, and Npr2 (Duncan et al., 2015; Filova et al., 2020a; Filova et al., 2022b; Jahan et al., 2010; Macova et al., 2019; Schmidt and Fritzsch, 2019; Stoner et al., 2022; Yang et al., 2017). In fact, the aberrant central projections to the cochlear nuclei in Ptf1a KO mice, are reminiscent of those in Prickle1, Fzd3, or Npr2 mutants. For instance, these mice have extra branching, especially in apical SGNs, as well as occasional overlapping projections of adjacent neurons.

The precise mechanism of Ptf1a is playing in SGN axon guidance is not yet known. Recent single cell RNAseq studies revealed Ptf1a expression in a subset of SGNs during embryonic development (Petitpré et al., 2022). For example, it has been shown that Fzd3 expression within the inner ear neurons themselves is necessary for their guidance (Stoner et al., 2022). Thus, it is also possible that Ptf1a acts intrinsically in SGNs to regulate their axon guidance. Alternatively, or in addition, Ptf1a expression in cochlear nuclei progenitors/neurons may be necessary for correct targeting of axons of SGNs to cochlear nuclei. Aberrant projections observed in the Ptf1a mutant mice could be due to lost pathfinding signals, or instead, since GABAergic and glycinergic cochlear nucleus neurons are absent in Ptf1a deletion mutants, abnormal SGNs projections to cochlear nuclei may simply be due to the impaired DCN neuronal composition.

Ptf1a expression in the cochlear nuclei could be interacting with ephrin-A3 (Hoshino et al., 2021b) or Fmr1 (Yu and Wang, 2022). There could additionally be something exclusive with the physical interaction of SGNs with these glycinergic and GABAergic neurons expressing Ptf1a, given that loss of Atoh1 expressed within the glutamatergic neurons has no obvious effect on central pathfinding (Elliott et al., 2017; Elliott et al., 2021b; Filova et al., 2020b; Maricich et al., 2009). Determining the mechanism will require conditional deletion of Ptf1a.

It remains to be shown whether the transient caudal expansion of central projections at E16.5 or the aberrant projections observed in the AVCN at E18.5 translate into a functional hearing defect in Ptf1a knockout mice. Whether or to what extent these tonotopic defects functionally impair hearing would require studying the conditional deletion of Ptf1a, allowing mice to grow past birth.

Highlights:

A transient posterior expansion of inner ear projections to the DCN was observed in Ptf1a mutants.

Broader central projections to the AVCN/PVCN were observed in Ptf1a KO mice.

Aberrant crossing of individual axons of SGNs was observed in Ptf1a KO mice.