The expression of α7 exhibits distinct spatiotemporal patterning in developing cochlear structures. Previously, we demonstrated the earliest expression of α7 in the developing embryo to be in rhombomeres 3 and 5 of the E9.0 embryo (Rogers et al. 2012
). Thus, we initiated studies of α7GFP
staining at this time. From E9.5 through approximately E12.5, the otic and cochlear structures did not express detectable α7GFP
(A and not shown, see Rogers et al. 2012
). The earliest detected expression of α7GFP
in the cochlear structures was at E13.5 in cells of the spiral prominence (SP; B). The SP retains α7GFP
expression throughout embryonic and post-natal development (see below). By E14.5 (C and D), α7GFP
expression extends to cells in the sensory domain of the lesser epithelial ridge near the site of the presumptive outer hair cells (OHC) and Deiters' cells (Morsli et al. 1998
; Lanford et al. 1999
; Kiernan et al. 2005a
). Light staining of the greater epithelium ridge was also present from E14.5 and thereafter, although this staining is inconsistently observed (B and C and not shown). Coincident with this expression was strong staining of pioneering efferents that become separated into individually distinguished processes as they progress through the spiral ganglion (SG) to reach the external face of this sensory domain (C; see below). The staining of the epithelial cells of the lesser epithelial ridge intensifies thereafter (e.g., E15.5 in E). At this stage, expression of α7GFP
by cells of the SG was in general only weakly observed in scattered cells (E). By E16.5, α7GFP
expression continues to increase in cells of the lesser epithelial ridge of the prosensory domain where OHC and Deiters' cells can now be distinguished (F and G and insert). Cells throughout the SG were also revealed by expression of α7GFP
by this developmental stage. Pillar cells do not express α7GFP
and there were no identifiable efferent processes labeled by the expression of this receptor at this stage or thereafter (see the following sections).
Figure 2 The expression of α7GFP varies with cochlear development. The expression of α7GFP identified by immunohistochemical detection of GFP (Methods) is shown for the embryonic cochlear structures in the sagittal section plane. (A) E12.5 shows (more ...)
The pattern of α7GFP expression in the E18.5 cochlear structure undergoes significant remodeling as both sensory hair cells and the associated supporting cells complete their differentiation (H and I). This includes a decrease of α7GFP expression by OHCs and underlying Deiters' cells that progresses away from the inner hair cells and proceeds in a basal-to-apical direction (next section). This is coincident with the appearance of signal in Hensen's cells that are most proximal to the outer line of OHCs (returned to below). Ganglionic afferent fibers ending at the base of the inner hair cells are also detected (see subsequent sections). In the postnatal mouse, as shown in the P6 cochlear sensory structure (J and K), the expression of α7GFP becomes limited to Hensen's cells immediately adjacent to the most distal OHC. Cells of the spiral ligament also acquire α7GFP expression, while the spiral prominence remains unchanged. In the SG, the expression of α7GFP is well established and the projections from these labeled cells can be followed to the vicinity of the inner hair cells (IHC) where their terminals appear to surround the base of the inner hair cell (IHC; J and K). A summary diagram illustrating the expression of the α7GFP during these major developmental stages is shown in L.
Remodeling of α7GFP in the cochlear structure after E16.5 is in a basal-to-apical direction
The remodeling of the sensory cell region of the cochlear structure between E16.5 and E18.5 as suggested by the progression in changing α7GFP expression was examined further. Through E16.5, all otic structures exhibit a similar α7GFP expression pattern (A). This was not the case in the E18.5 cochlear structure where the loss of α7GFP expression by OHC and Deiters' cells and acquisition of staining by Hensen's cells was first observed in the most basal structures and it then appears in the more apical structures successive developmental stages (B and C and not shown). This generates a striking contrast in α7GFP expression between cochlear structures at the apex relative to the base with intermediary turns, exhibiting the progressive stages of this change in α7GFP expression (B). By P4, this gradient was not evident (not shown) and the mature α7GFP expression pattern first observed in the E18.5 basal cochlear structures was present across the entire structure. In D, a diagram depicts the remodeling of α7GFP expression seen in the E18.5 developing cochlear structure.
Figure 3 Remodeling of α7GFP Expression is from basal to apical. (A) A sagittal section showing the E16.5 cochlear structure and α7GFP expression (green). At this stage, all cochlear structures exhibit a similar pattern of α7GFP expression (more ...)
Nonsensory cells of the cochlear structure express α7GFP
As suggested by the preceding discussion, there was expression of α7GFP
by both neuronal and non-neuronal cells (). This is particularly clear in the postnatal mouse (e.g., P6–P12), where the predominant expression of α7GFP
in neuronal cells was by cells of the SG (A). The strongest labeling of cochlear structures was restricted to Hensen's cells and the spiral prominence (A–E). Evident at the P6 stage was α7GFP
signal in individual cells of the spiral ligament (C and D). Also evident were the extended branching that is characteristic of the morphology of type II fibrocytes located in this region (D; Spicer and Schulte 1991
; Sun et al. 2012
). In the P12 cochlear structure, the branches were more abundant and form a ‘feathered’ structure that emanates from cell bodies defined by α7GFP
expression (E). Cells of the stria vascularis or other members of the cell family composing the structures of the lateral wall and surrounding cochlear duct were not observed to express α7GFP
in these later stages of development ().
Figure 4 Postnatal expression of α7GFP in the cochlear structure. (A) An image of a sagittal section showing the P6 cochlear structure and the expression of α7GFP in afferents originating from spiral ganglion (SG) cells that terminate (arrow head) (more ...)
The expression of α7GFP during innervation of the developing cochlear structure
Innervation of cochlear sensory cells follows a series of distinct steps that were in part revealed by α7GFP visualization (). As noted, the first detection of α7 expression was in the prominently labeled efferent processes that appear to form bundles upon entering the SG and then disperse into small solitary fibers (E14.5; A and C,D). These solitary processes exhibit a beaded structure as they proceed to the base of the developing sensory cells (B).
Figure 5 The α7GFP expression during cochlear innervation. Innervation of the developing cochlear structure is revealed by α7GFP labeling. (A) An E13.5 sagittal section shows a group of efferent processes (arrow) that distribute to solitary fibers (more ...)
The origin of these efferent fibers was examined in serial sections of the E14.5 hind brain. These fibers appear to originate from a cell grouping in the basal brain stem caudal to trigeminal nucleus V that could be distinguished by their transient α7GFP
expression (C). These cells occur in clusters (C insert) and their prominently labeled processes can be followed using serial section sets to the cochlear structure where they give rise to the fiber bundles and the point of dissemination associated with the SG (C and insert). The anatomical location of these cells suggest that these cells are within the forming olive complex, which is consistent with the reports of pioneering fibers that originate from the developing olive complex and extend to the developing cochlea (Zuo et al. 1999
). These fibers were not detected after E15.5.
During the E15.5–16.5 period, there was essentially no labeling of neuronal processes by α7GFP
(D–F). However, ongoing innervation of cochlear sensory cells was identified using peripherin labeling (E; see Simmons et al. 1996
; Hafidi 1998
; Huang et al. 2007
) or for olivocochlear efferents that were identified by labeling for calcitonin gene-related protein (CGRP; F, Fritzsch 2003
). By E18.5, the SG α7GFP
signal was present in afferent processes that extend to the base or near vicinity of the IHCs (G).
At birth and thereafter (P0–P12 analyzed), the expression of α7GFP
was strongly detected in SG afferent fibers where they terminate near or at the base of IHC sensory cells (H and I). This basic pattern of α7GFP
expression was reinforced during the remaining postnatal period as fibers continue to form a dense plexus that appears to surround the base of the IHCs. The other efferent fibers not detected by α7GFP
continue to be trimmed and also associate with their final targets (Merchan-Perez and Liberman 1996
; Simmons et al. 1996
; Hafidi 1998
; Huang et al. 2007
). The outcome of this remodeling was evident by P12 when the SG1 afferent terminals surrounding the IHC were distinguished by strong α7GFP
staining of the terminal clusters (I and inset). This was approximately the same time hearing onset occurs in mice (~P10; Kros et al. 1998
). Processes originating from SG cells identified by peripherin expression that were not colabeled with α7GFP
form distinct efferent terminals on or very near OHCs cells and on the terminals that end on the IHC afferent terminals identified by α7GFP
labeling (I; Huang et al. 2007
). While not entirely evident from the images shown, not all SG cells at P12 expressed α7GFP
, suggesting this could identify a functionally distinct subpopulation (I; Happe and Morley 1998
). Again, no α7GFP
labeling of olivocochlear efferents was detected. A diagram summarizing these findings is shown in J.
Ablation of the α7Cre-expressing cell lineage confirms α7GFP expression during cochlear development
expression was not detected in the developing cochlear structures until E13.5 (B), as reported previously the earliest α7 expression we have defined is at P9.0 in rhombomeres 3 and 5 (Rogers et al. 2012
). Because cochlear morphogenesis includes signaling from rhombomere 5 (Liang et al. 2010
), the possibility of α7GFP
contributing to the development of this structure was examined. This was done using embryos from α7Cre
mice crossed with mice harboring the conditional ROSA26-loxp (diphtheria-A toxin (DTA; termed α7Cre:DTA
; Rogers and Gahring 2012
). In these embryos, α7Cre:DTA
-expressing cells and their direct lineages were ablated, thus revealing expression that could have been be missed by α7GFP
measurements (Rogers and Gahring 2012
). An example of the cochlear structure at E16.5 taken from α7Cre:DTA
crosses is shown in . Because there is only occasional overlap with α7GFP
(see E), we used peripherin expression to aid in examining the fate of non-α7-expressing cells (A and B). The overall patterning of the cochlear structure and the formation of major boney structures of the cochlea inclusive of the otic capsule and modiolus were intact, albeit somewhat distorted. The cochlear ducts were collapsed (B), probably due to the absence or severe thinning of the distal lateral wall. Also absent was the sensory cell domain containing presumptive OHCs and Deiters' cells (C and D), as expected from results of α7GFP
expression (, ).
Figure 6 The ablation of α7 cell lineages is consistent with α7GFP expression. Comparison of a cochlear structure labeled for expression of the filament marker peripherin from an E16.5 α7GFP mouse (A) and similarly timed α7Cre: (more ...)
The SG of α7Cre:DTA embryos is reduced in size and the majority of cells remaining give rise to mostly peripherin-labeled efferents (see E). These fibers also appear to be more densely aggregated relative to the α7GFP control mouse (A and B). While peripherin-identified processes still project to the presumptive sensory cells (both IHC and OHC), they were less branched and those that did project to the former OHC target fields often turn and proceed backwards towards the vicinity of IHCs (C and D). These results are consistent with the earliest expression of α7 being after major cochlear structures are determined, and there was the expected selective ablation of OHCs and Deiters's cells. The necessity of the presence of the target sensory cell to coordinate the innervation process is also suggested by these findings.
Auditory pathways in the postnatal central nervous system are identified by α7GFP expression
The results of studies examining α7 expression using in situ hybridization and functional measurements using electrophysiology have shown that this receptor is an important contributor to various nuclei of the central auditory system (Happe and Morley 1998
; Vetter et al. 1999
; Morley and Happe 2000
; Morley 2005
). The α7GFP
mouse system offers an excellent opportunity to view these central systems and their connections as shown in . The connections between the SG and the cochlear nuclei were strongly identified at E18.5, presumably due to the dense projections from SG cells expressing α7GFP
that extend processes both to the IHC () and the developing cochlear nuclei of the brainstem (A).
Figure 7 Central auditory systems express α7GFP. Central auditory nuclei identified by α7GFP expression. (A) At E18.5 in this sagittal image of the entire otic complex and the adjacent basal brainstem is included. The cochlear nucleus (C) and the (more ...)
The expression of α7GFP
appears to intensify after P10, and by P12 signal is consolidated almost exclusively in the ventral-posterior cochlear nucleus (B). This is in agreement with reports from in situ hybridization studies reporting the strong expression of α7 in this nucleus, whereas other major cochlear nuclear divisions exhibited only weak or sporadic labeling (Yao and Godfrey 1999
; Morley and Happe 2000
). Also consistent with those studies was that the cells identified by α7GFP
expression resemble octopus cells (B, insert). Essentially, no expression of α7GFP
was detected in the dorsal cochlear nucleus, although some dispersed and weakly stained cells were present in the granular aspect. Also evident was the strong staining of neuropil, presumably in part due to terminals of SG cells associated with the eighth cranial nerve (B, inset). This strong labeling of the P12 SG and OHC afferents is consistent with other reports (Morley and Happe 2000
The expression of α7GFP
also persists into the adult animal. This is apparent in the ascending central auditory system nuclei and their fibers (C). After the cochlear nucleus, α7GFP
is present in the ventral lateral lemniscus, on through the dorsal lateral lemniscus, and to the inferior colliculus where dense staining of α7GFP
is present (C; Morley and Happe 2000
; Yao and Godfrey 1999
). The commissural fibers of the inferior colliculus are also identified by α7GFP
expression (D). Thereafter, efferents follow the brachium of the inferior colliculus to the medial geniculate nucleus where scattered cells expressing α7GFP
were seen. Not shown is that the expression of α7GFP
in the adult auditory cortex appears restricted to cells of layer 1. Labeling of olivocochlear fibers was not detected.