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.

Keywords: Auditory system; Cochlear projections; Ear central projections; Hindbrain; Ptf1a.

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.

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.

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.

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.