Neurogenin1 and NeuroD1 induce cochlear non-sensory epithelial cells to develop as ectopic neurons
Previous studies have shown that Neurog1 and NeuroD1 are expressed in the spiral ganglion () and are required for formation of all (Neurog1) or most (NeuroD1) neurons within the vestibulocochlear ganglion (Ma et al., 1998
). This finding led us to investigate whether the expression of NeuroD1 or Neurog1 is sufficient to induce a neuronal identity in non-sensory inner ear epithelial cells that would not normally develop as neurons. To test this hypothesis, electroporation-mediated DNA transfection was used to induce ectopic expression of Neurog1
in non-sensory epithelial cells located within Kolliker’s organ (KO) or the lesser epithelial ridge (LER) of cochlear explant cultures. Cochleae were dissected from E13 embryos and electroporated. After 6 days in vitro
(DIV) development of neuronal phenotypes was assayed based on morphology and expression of the neuronal markers TuJ1 (β-tubulin III) (Lee et al., 1990
; Hallworth and Luduena, 2000
) and Microtubule-associated-protein 2 (Map2) (Hafidi et al., 1992
) both of which are expressed in spiral ganglion neurons but not in glial cells marked by Sox10 immunolabeling (). As previously reported, electroporation resulted in multiple transfected cells in both KO and the LER. In addition, a subset of epithelial cells transfected with either Neurog1.EGFP
was positive for TuJ1 ( and data not shown). Moreover, most of the TuJ1-positive cells, regardless of whether they expressed Neurog1.EGFP
, extended long processes (), many of which ended in growth cones (), a morphology that is consistent with developing neurites. Transfected cells with neuronal morphologies were also positive for Map2 (). To determine how rapidly expression of either NeuroD1 or Neurog1 induced a neuronal fate, explants were analyzed after only 1 DIV. A comparable number of TuJ1-positive transfected cells were detected in these explants (data not shown), suggesting a rapid induction of neuronal fate. In contrast, cells transfected with a Control.EGFP
vector were consistently negative for expression of either TuJ1 or Map2 ( and data not shown). Moreover, the morphology of these cells, which included a centrally located nucleus with basal and lumenal projections, was consistent with that of epithelial cells ().
Neurog1 and NeuroD1 are expressed in spiral ganglion neurons
NeuroD1 and Neurog1 induce neuronal phenotypes in non-sensory cells within the cochlea
To determine the efficiency of induction of a neuronal fate, the percentage of transfected cells that also expressed TuJ1 was determined for cells transfected with Control.EGFP, Neurog1.EGFP
. As shown in , 73% of cells (n = 156) transfected with NeuroD1.EGFP
and 26% of cells transfected with Neurog1.EGFP
(n = 111) were positive for TuJ1. Since a relatively large percentage of transfected cells, in particular cells transfected with Neurog1.EGFP
, failed to develop as neurons, we wanted to determine whether these cells might have been induced to develop with an alternative cell fate. Previous results have demonstrated that non-sensory cells within KO or the LER are competent to develop into hair cells or supporting cells (Zheng & Gao, 2000
; Woods et al., 2004
). Therefore expression of the hair cell markers Myosin6 (Myo6) and Myosin7a (Myo7a) and the support cell marker, p27Kip1
were determined in cells transfected with Neurog1.EGFP
. No induction of either hair cell or support cell markers was observed in any of these cells (, Fig. S1A,B
, and data not shown). Finally, to determine whether the ability to induce non-sensory cells to develop with a neuronal fate is specific to Neurog1 or NeuroD1, non-sensory cells were transfected with another bHLH transcription factor, Atoh1, and then assayed for expression of neuronal markers. Consistent with previous results (Zheng & Gao et al., 2000
; Woods et al., 2004
; Jones et al., 2006
), 98% of Atoh1.EGFP
transfected cells were positive for Myo7a (). In contrast, none of the transfected cells were positive for TuJ1 ().
Efficiency of neuronal induction by NeuroD1, Neurog1, and Sox2 at different ages
To further characterize the ectopic neurons induced by overexpression of either Neurog1.EGFP
, we examined the expression of several markers of mature neurons. Cells transfected with Neurog1.EGFP
failed to express markers for more mature neurons including islet1, neurofilament-200, neurofilament-L, 2H3, synaptophysin and GAP-43. Furthermore, preliminary experiments in which Neurog1.EGFP
transfected explants were treated with different factors known to enhance neuronal differentiation, including FGF2, retinoic acid, nerve growth factor or neurodazine – an inducer of neurogenesis (Williams et al., 2007
) failed to induce the expression of additional neuronal markers. However, treatment with neurodazine did lead to an increased percentage of induction of TuJ1-positive neurons from 26% (n=111) to 53% (n=116), suggesting that the environment within KO and the LER may not be conducive for neuronal formation.
To confirm that transfected that were positive for TuJ1 or Map2 were in fact neurons, whole-cell recordings were made from transfected cells within cochlear explant cultures. Transfected cells which are selected based on GFP expression, while non-transfected cells within the same explant were used as controls. Examples of Neurog1.EGFP-transfected cells showing neuronal characteristics are shown in . In the voltage clamp recording shown in , positive voltage steps activated a transient inward current and a persistent outward current, consistent with expression of voltage-gated sodium and potassium channels, respectively. In a current clamp recording from another cell shown in , depolarizing current injections evoked action potentials, as indicated by their abrupt activation threshold, inflection in their rising phase, and peak membrane potential that exceeded 0 mV. Responses exhibiting evidence of expression of voltage-gated sodium and potassium channels were observed in 3 out of 9 Neurog1.EGFP transfected cells from which recordings were made, demonstrating in vitro acquisition of electrophysiological properties characteristic of neurons. In the remaining 6 out of 9 cells, a purely passive membrane response was observed in both voltage and current clamp recordings (). Similar passive responses were observed in all non-transfected cells (n=3).
Ectopic neurons exhibit electrophysiological characteristiscs consistent with neurons
It has been suggested that NeuroD1 is a direct target of Neurog1 signaling since targeted deletion of Neurog1
results in a disruption of NeuroD1 expression in the developing inner ear (Ma et al., 1998
). To test this hypothesis directly, we examined the expression of NeuroD1 in Neurog1-transfected cells. No induction of NeuroD1 was observed in any Neurog1-transfected cell (data not shown). These results suggest that Neurog1 and NeuroD1 could function in different signaling cascades or that Neurog1 requires specific co-factors to induce NeuroD1 expression.
Sox2 is expressed in the developing spiral ganglion neurons
As discussed, members of the SoxB1 family play a significant role in neural development in the spinal cord and elsewhere. To investigate the potential role of SoxB1 genes in the development of spiral ganglion neurons, the expression of Sox1, Sox2 and Sox3 was compared with Neurog1 and NeuroD1 in the otocyst and early post-natal cochlea. Expression of Sox2 was observed along with Neurog1 and NeuroD1 in otocyst-derived neuroblasts as early as E10.5 (). To confirm that Sox2 is expressed in neurons within the spiral ganglion, localization was confirmed by double labeling with TuJ1 in P0 cochleae ().
SoxB1 gene expression correlates with spiral ganglion development
Expression of Sox1 was also detected in spiral ganglion neurons at P0 based on co-expression of β-galactosidase and TuJ1 in a Sox1LacZ/+ reporter mouse (). Finally, restriction of TuJ1 to neurons within the spiral ganglion was confirmed by doubling labeling with glial marker, Sox10, at P0. The nuclei of Sox10-positive glial cells were noticeably smaller than those of TuJ1-positive neuronal cells and the two populations of cells were non-overlapping (). Since both Sox1 and Sox2 co-localize with TuJ1 these results strongly suggest that Sox1 and Sox2 are only expressed in neurons within the spiral ganglion. In contrast, Sox3 was not observed in spiral ganglion neurons at P0, and instead was restricted to a subset of cells within the epithelial cell layer of Reissner’s membrane (data not shown).
Sox2 promotes neurogenesis
The expression of Sox1 and Sox2 in the spiral ganglion neurons suggested a role for one or both in neuronal development and/or maintenance. To ascertain whether Sox1 plays a role in any of these events, expression of TuJ1 was examined in Sox1LacZ/LacZ mutant cochlea at P0. As shown in , TuJ1 expression and ganglion cell morphology are unaffected in the absence of Sox1 suggesting that Sox1 is not required for ganglion cell formation or maintenance. Moreover, labeling of spiral ganglion neurites with anti-NF-200 in cochlear whole-mounts from Sox1+/LacZ and Sox1LacZ/LacZ mice indicated no effects on the pattern of innervation in the absence of Sox1 ( (inset)). In addition, no glial abnormalities were apparent based on immunolabeling with Sox10. Consistent with these results, non-sensory cells transfected with Sox1.EGFP did not develop a neuronal phenotype () suggesting that overexpression of Sox1 alone is not sufficient to induce a neuronal fate in the cochlea.
Sox1 is neither necessary nor sufficient for spiral ganglion neuron formation
In contrast with Sox1LacZ/LacZ mutant cochleae, expression of TuJ1 and Sox10 were absent in the spiral ganglion region of Sox2Lcc/Lcc mutant cochleae at E15.5 (). In fact, labeling with DAPI demonstrated a complete absence of cell nuclei in the spiral ganglion region (), suggesting a defect in neurogenesis. In order to confirm that the absence of cells within the spiral ganglion in these mutant cochleae is not due to delay in development, expression of TuJ1 and Sox10 were also analyzed at postnatal day 0 (). As was the case at E15.5, Tuj1 and Sox10 labeling were completely absent in cochleae from Sox2Lcc/Lcc mutants. These results demonstrate that Sox2 is necessary for spiral ganglion formation.
Sox2 is required for formation of the spiral ganglion
To determine whether Sox2 is also sufficient to induce neurogenesis, non-sensory cells in cochlear explants were transfected with Sox2.nucEGFP
and development of neuronal identity was analyzed as described for NeuroD1 and Neurog1. Approximately 39% of Sox2.nucEGFP
-positive cells (n = 505) were positive for TuJ1 ( and ). Moreover, as was observed in response to the over-expression of Neurog1 or NeuroD1, these cells also extended processes that appeared consistent with developing neurites (). Sox2-transfected cells that were positive for Map2 were also observed (). These ectopic neurons were able to survive for at least 15 days after transfection (data not shown). In addition, as was observed for Neurog1 and NeuroD1 transfections, expression of Sox2 did not induce hair cell, support cell or glial cell phenotypes (Fig S1C,D
and data not shown).
Sox2 induces neuronal phenotypes in cochlear non-sensory cells
The observation that Sox2 failed to induce expression of a hair cell phenotype is consistent with recent results demonstrating that Sox2 acts to antagonize Atoh1 expression in the cochlea (Dabdoub et al., 2008
). Furthermore, forced expression of Sox2 induces Prox1 expression in non-sensory epithelial cells in the cochlea (Dabdoub et al., 2008
). While Prox1, a homeobox transcription factor, is expressed in the spiral ganglion neurons (Bermingham-McDonogh et al., 2006
); forced expression of Prox1 in non-sensory cochlear cells did not induce a neuronal fate (data not shown). To determine whether the ability of Sox2, Neurog1 and NeuroD1 to induce a neuronal fate in cochlear non-sensory cells is dependent on the age of the cells at the time of transfection, non-sensory cells were transfected with each construct in cochleae isolated at E16 or P1. A progressive decrease in the percentage of cells that develop as neurons was observed ().
To determine whether Sox2 acts upstream, downstream or in parallel with NeuroD1 and Neurog1, expression of all three molecules was assessed in cells transfected with each construct. Sox2.nucEGFP-transfected cells did not express either NeuroD1 or Neurog1 nor were Neurog1.EGFP transfected cells positive for Sox2 (data not shown). Similar results were obtained for cells transfected with NeuroD1.EGFP. These results suggest that despite overlapping expression within the otocyst and developing spiral ganglion, Neurog1 and Sox2 act through independent pathways within the developing inner ear.