Macroscopic Changes and the Timeline of Neurosensory Loss
Foxg1-cre-mediated conditional knockout (CKO) of Dicer1 produced no obvious overall changes before E12.5 (Fig. 1a,a′). After E14.5, notable differences in skull morphology were the reduced growth of the eyes, snout, and forebrain area, leading to a depression of the frontal bone anlage and a shortened distance between the eye and the ear (Fig. 1b′,c′). The snout was markedly shortened and in older stages showed a reduced height. Overall the length of the head measured from the tip of the snout to the tentorium cerebelli was approximately 15% shorter. Most obvious was the shortening of the snout when compared with the lower jaw, which was almost as long as the snout in Foxg1-cre::Dicer1 f/f mice (Fig. 1c,c′).
Figure 1. These images show the progressive change in the appearance of the head of wild-type (WT) and Foxg1-cre::Dicer1f/f (CKO) mice. Note the limited reduction in the forebrain (FB) in the CKO mice at E12.5 (a, a′, d), suggesting that the initial formation of the forebrain is normal. Marked changes are obvious by E14.5 with a severe reduction of the forebrain, shortening of the skull and a reduction in eye size (b, b′, e). By E18.5 neither the forebrain nor olfactory bulb are present and the eye is much smaller in CKO mice (c, c′, f). The skull develops a sunken-in appearance and the snout is reduced in all dimensions by approximately 10%. Bar indicates 1 cm (a–c) and 0.5 mm (d–f).
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To investigate more closely the brain changes we cut the head into halves and remove the brains. Comparing the brains in both lateral and medial perspective showed no obvious differences until E12.5 when the forebrain showed accumulation of highly reflective cells. Such cells also appeared near the cerebellum and in various other places (Figs. 1d and 2a). By E14.5 the forebrain of the Foxg1-cre CKO mice was drastically reduced in size, formed only a small patch of white opaque cells compared with wild-type littermates, and was completely gone by E18.5 (Figs. 1e,f and 2b,b′). At this late stage the forebrain consisted of only an empty space in the meninges anterior to the diencephalon (Figs. 1f and 2c). Further, no olfactory bulb remained (Fig. 2c). These data show that the entire forebrain (telencephalon) and the olfactory bulb start to develop normally but disappear within 6 days in Foxg1-cre::Dicer1 f/f conditional null mutants.
Figure 2. Detailed examination of the brain shows not only the dramatic reduction in size and loss of the forebrain (a–c) but also the complete loss of the cerebellum (d–f) and olfactory bulb (c, c′) in the Foxg1-cre::Dicer1f/f (CKO) mice. Note the increasing density of many opaque cells in the degenerating forebrain (a, b) and cerebellum (d, e). While the forebrain and cerebellum of the CKO are almost identical to the WT at E12.5 (a, a′ and d, d′) size reductions are apparent at E14.5 (b-b′, e-e′), and loss of both areas is obvious at E18.5 (c, c′ and f, f′). Note that the loss of the cerebellum shows normal early embryonic development (d, d′) followed by complete loss (f, f′). Anti-cre immunochemistry of the WT cerebellum (Foxg1-cre::Dicer1f/+) reveals high positivity at E12.5 (d′ insert). Anti-β tubulin immunochemistry on the E12.5 CKO brain reveals positive fibers in the area of the olfactory bulb indicating the normal initial formation of olfactory neuron projection (insert in a). By E18.5 only vacant meninges remain where the olfactory bulb started to develop (c, c′). FB, forebrain; IC, inferior colliculus; C, cerebellum; CP, choroid plexus; OB, olfactory bulb. Bar indicates 2 mm bars in c, c′ and f, f′, 100 μm a and d′ insert, and 1 mm other images.
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An additional area of brain tissue loss was the cerebellum which was almost completely absent at E18.5 (Fig. 2f). The first obvious macroscopic indication of cerebellar change was around E14.5 when the cerebellum was more epithelial compared with the solid plate-like cerebellum of wild-type littermates (Fig. 2e,e′). Closer examination of E12.5 showed opaque cells in high abundance in the cerebellum that was otherwise indistinguishable from the wild-types (Fig. 2d,d′). Some minor reductions in other areas of the brain were negligible compared to the forebrain and cerebellar loss, and were always characterized by an accumulation of opaque cells (data not shown).
We next wanted to investigate in more detail the formation of bone in the heads of the Foxg1-cre::Dicer1 f/f conditional null mutants using bone and cartilage staining. We expected that the absence of brain growth in the CKO mutant would result in premature closure of sutures. As predicted, the overall smaller skull of the CKO mice had narrower sutures between the forming bones of the skull (Fig. 3).
Figure 3. Staining with Alizarin red S for bone reveals that ossification has smaller sutures between the growing bony plates of the skull in Foxg1-cre::Dicer1f/f (CKO) mice (arrow and asterisk in a and b). The overall reduction of the frontal bone growth and smaller eye socket are obvious in the lateral view. Bar indicates 1 mm.
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A prominent early change visible in the head of Foxg1-cre::Dicer1 CKO mutant mice were changes in the eye. Around E12.5 the anterior part of the eye became depigmented and subsequently showed stunted growth (Figs. 1a and 4a) such that by E18.5 the Foxg1-cre CKO mutant eye was about the size of an E16.5 wild-type eye. Direct comparison showed that at E18.5 the mutant eye was only the size of the wild-type lens (Fig. 4c). In addition, the mutant eyes showed a pigment-free anterior streak, an asymmetric iris pointing toward the anterior half and had no lens beyond a few distributed cells inside the vitreous (Fig. 4c,e). The radius of the mutant eye was progressively lacking behind the wild-type (Table 1). Assuming a spherical shape, this would result in an approximate 85% reduction in volume of the mutant eye at E18.5 (Table 1). There was no lens in these eyes and a small protrusion extended from the neural retina to partially covering the lens space (Fig. 4e). While a retina was obviously present, the cellular layers so characteristic of the normal littermate retina could not be distinguished (Fig. 4d,d′,e,e”). We tentatively identified a ganglion cell layer with little indication of nerve fibers and a partially developed inner plexiform layer. The anterior half of the retina was even more disorganized than the posterior retina, showing rosettes and overgrowth of receptor anlage (Fig. 4e”).
Figure 4. Eyes of wild-type (WT) and Foxg1-cre::Dicer1f/f (CKO) mice start development almost identical but show differences in growth over the observed period. The eyes of CKO mice show almost no growth past E12.5 whereas there is doubling in the diameter and a 20× increase in the volume (Table 1) in the WT animal. The anterior (right) part of the CKO eye shows a reduced size and loss of pigment at E14.5 (b, arrow), suggesting a loss of the anterior, Foxg1-Cre expressing part of the eye. At E18.5 the reduced eye of the CKO is approximately the same size of the lens of the WT animal (c). In addition, histology does not reveal a lens in the E18.5 CKO eye but instead shows a protrusion of the neural retina to fill the gap (e). Beyond the size differences are histological differences. The control retina showed well-developed fiber (F), ganglion (G), and inner plexiform layers (IPL) but no such layers are as obvious in the CKO mice (d, e). Higher magnification shows the presence of apoptotic cells as single profiles or several of them engulfed by a macrophage-like cell (arrow in e). F, fiber layer; G, ganglion cell layer; IPL, inner plexiform layer; L, lens. Bar indicates 1 mm a–c and 100 μm d–e”.
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Table 1. Reduction in Eye Size
| ||WT||Foxg1-cre::Dicer1 f/f||% Reduction|
|E18.5 radiusa||1197 ± 217||636.5 ± 150.5||46.8|
As with the eyes, the changes in the ear started around E12.5 with the appearance of opaque cells in the delaminated sensory neurons. A reduction in size of the ear was first apparent at E14.5. Formation of periotic cartilage in later stages indicated that the size of the otocyst of CKO mutant mice was reduced at E18.5, but also showed less ossification of the ear and a smaller tympanic ring (Fig. 5a,a′). Interestingly the inner ear had only a superior part left with a short ventral extension reminiscent of the cochlear duct (Fig. 5b). Small otoconia were usually present near the small posteroventral duct, as well as within this small duct (Fig. 5b). This indicates that possibly a gravistatic sensory epithelium might have formed in the ear. We investigated neurosensory development of the Foxg1-cre CKO mutant ear using anti-β tubulin and anti-Myo7a immunochemistry to label neurons and hair cells, respectively (Fig. 5h–j). Our data indicated the presence of very few nerve fibers to a small patch of hair cells resembling in shape and position the utricle. In addition, a few Myo7a-positive cells with a single fiber innervating them were found in the small posterior duct. We tentatively identify these cells as the “saccule” as they are reminiscent of the reduced saccule of Pax2-cre::Dicer1 f/f mice (Soukup et al.,2009). It is unclear if larger prosensory domains characterized by expression of specific genes form initially and degenerate or if they never develop, as pigment cells usually identified with such domains, manage to reach the reduced ear but remain in the otocyst wall. We next investigated the development of innervations. Some innervation to the ear developed initially but ultimately was reduced to a few remaining fibers to vestibular endorgans only (Fig. 5d–g).
Figure 5. The otic capsule of Foxg1-cre::Dicer1f/f (CKO) mice is modestly reduced at E18.5 (a) and shows a nearly complete absence of ossification of the otic capsule. Cartilage and bone staining (a, a′) indicates that ossification of the malleus (M) seems to proceed normally and a smaller annulus tympanicum (AT) forms that undergoes normal ossification. In contrast, the inner ear is reduced by 38.6% (note differences in magnification in b, c). The CKO inner ear shows loss of canal formation beyond the canal plate found at E12.5, and has a very short cochlear duct (CD) instead of a coiled cochlea (b, c). Labeling of afferents and efferents to the ear with lipophilic dyes (NVMaroon) inserted into the brainstem shows that fibers grow out nearly normal up to E14.5 (d, e) but are much reduced at E16.5 (f, g). Notably is the near complete loss of fiber growth to the cochlea (f) and the reduction to vestibular organs such as the posterior canal crista (PC). Fibers outside the ear such as the facial nerve (FN), geniculate ganglion (GG), and greater petrosal nerve (GPN) are near normal (f, g). Tracing the development of hair cells with Myo7a (h, j) and anti-β tubulin immunochemistry (i, j) reveals hair cells only in a single organ, tentatively identified as the utricle (h, j), and two to three cells at the base of the cochlear duct, possibly representing the saccule (S). Nerve fibers reach these hair cells and a few very thin, likely autonomic fibers are found around the cochlear duct. AC, anterior canal crista; CD, cochlear duct; CP, canal plate; PC, posterior canal crista; S, saccule; U, utricle; CoE, cochlear efferents; FN, facial nerve; GG, geniculate ganglion; GPN, greater petrosal nerve. Bar indicates 1 mm a–c and 100 μm d–j.
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Because of the apparent reduction in size of the snout by approximately 10% in all dimensions we also investigated the olfactory system. Olfactory receptors seem to develop initially and project to forebrain (Fig. 2a; insert). However, at E18.5 we found a reduction of conchae and loss presumably of all olfactory neurons (data not shown), defects that are more profound than those reported in Foxg1 null mice (Duggan et al.,2008; Kawauchi et al.,2009). No olfactory fibers could be detected passing through the cribrifom plate, which was solid bone. The inability to innervate the absent olfactory bulb (Figs. 1c and 2c) could certainly accelerate the demise of olfactory receptors, but it is unclear where and when loss started.
In summary, loss of Foxg1-cre-mediated Dicer1 results in complete loss of certain areas of the brain (forebrain, olfactory bulb, and cerebellum), near complete loss of the inner ear, reduction of the eye and of conchae as well as olfactory epithelium. We next wanted to investigate how this loss was orchestrated at the molecular level in the absence of Dicer1 enzyme and likely all miRNAs and other small RNAs.
Loss of cells follows Cre protein expression which is followed by loss of miRNA, upregulation of anti-cleaved caspase 3, and digestion of dying cells by macrophages.
We first investigated the upregulation of cre using immunocytochemistry and found cre expression in the forebrain, cerebellum and ear as early as E12.5 (Fig. 2d′; insert) consistent with the known expression of Foxg1. We next investigated the expression of one miRNA as a proxy for other neuronal miRNAs, miR-124. Expression of this miRNA was detected in the forebrain (data not shown) and the ear at E11.5 (Fig. 6a,b), but disappeared in the ear, the forebrain, and the cerebellum around E12.5 (Fig. 6c,d). Areas without miR-124 were abundant throughout the brain around E14.5 and no miR-124 was visible in the olfactory epithelium past E14.5 (data not shown). These data suggest that it takes approximately 6 days after the cre-mediated deletion of Dicer1 before the miRs are almost completely depleted.
Figure 6. In situ hybridization using a LNA probe for miR-124 shows little difference at E11.5 (a, b), indicating that sensory neurons form a ganglion (G) and produce functional miRNA. In contrast, by E12.5 there is no miR-124 left in the ganglion of the Foxg1-cre::Dicer1f/f CKO (c) whereas the control littermate reacted with the CKO ear shows prominent staining (d). Staining for anticleaved caspase 3 at E12.5 shows prominent expression in the inner ear ganglion but a much reduced presence of cleaved caspase 3 in the adjacent trigeminal (V) and glossopharyngeal/vagal (IX, X) ganglia (e). Higher power shows the nearly ubiquitous presence of anti-cleaved caspase 3 expressing cells that nevertheless show some anti-β tubulin staining (blue, e, e′), indicating some neuronal development. Staining for PSVue (f, f′) shows many cells staining positive throughout the ear at E16.5, indicating continued cell degeneration during later development. Bar indicates 100 μm.
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Following our previous data (Soukup et al.,2009) and those of others (Huang et al.,2010; Shi et al.,2010; Zehir et al., 2010), we expected that miRNA depletion will result in apoptosis of proliferating and differentiating neuronal precursors. We therefore next investigated the distribution of apoptotic cells using anti-cleaved caspase 3 antibodies and a PSVue stain that targets phosphatidylserine (PS) exposed on the membranes of dying cells (MTTI). Phosphatidylserine is normally found only in the inner plasma membrane leaflet of membranes and is inaccessible to this stain. Flippases normally transfer any PS that diffuses into the outer leaflet back into the inner leaflet. However, these flippases are the target of caspase 7 which in turn is activated by caspase 3. Flippases are inactivated when caspase 3/7 are activated in apoptotic cells. Without flippases, PS accumulates in the outer leaflet (Krijnen et al.,2010). Exposure of phosphatidylserine in the outer leaflet is a signal to macrophages to engulf apoptotic cells. The PSVue dye was designed for labeling these late-stage dying cells. In addition we used anti-β tubulin to stain developed neurons to understand if neurons start to differentiate and subsequently die or die shortly after cell cycle exit without any differentiation.
Eyes and Ears
While loss of the forebrain, olfactory bulb and cerebellum was complete at E18.5 (Figs. 1 and 2), loss of eyes and ears was incomplete but possibly for different reasons. Foxg1 is only expressed in the anterior half of the retina whereas the posterior half expresses a different forkhead gene, Foxd1 (Herrera et al.,2004). Consequently there is no Foxg1-cre expression in the posterior retina and we find little defects (Fig. 4). In contrast, the anterior retina seems to degenerate completely. Near equatorial sections through the retina show that the posterior retina begins differentiation at E18.5 whereas the anterior retina shows numerous condensed nuclei (Fig. 4e). It remains unclear how and why the lens disintegrates in these eyes but it is definitely absent by E18.5 (Fig. 4e). These data suggest that the anterior half of the eye may completely disappear by Dicer 1 CKO induced cell death. The remaining posterior half reforms the eye cup but is unable to restore the full growth of the eye, leading to a markedly smaller and deformed eye (Figs. 1 and 4; Table 1).
Previous work has demonstrated that only very few areas of the developing ear are free of Foxg1 such as the stria vascularis (Pauley et al.,2006), an area of the lateral wall of the cochlea specialized in the secretion of the potassium-rich endolymph. In particular all neurosensory cells seem to be highly positive for Foxg1, suggesting that perhaps all neurosensory precursors express Foxg1 (Hwang et al.,2009; Pauley et al.,2006). Consistent with this expectation, we found at E18.5 a near complete loss of all neurosensory cells and a near complete loss of the entire ear with an elongated vesicle remaining (Fig. 5b). Only one patch of hair cells remained that expressed myosin Myo7a, a marker for hair cells. Based on its position near the undifferentiated canal growth plate (Fig. 5b,h–j) and due to its kidney shape, it is likely the utricle but could be a fusion of some epithelia (Nichols et al.,2008). Interestingly, a short ventral duct was always present in these mutants that had a few Myo7a-positive hair cells near the single primary epithelium in the vestibular sac. This extension appears in shape like a shortened cochlear duct. Innervation to this truncated ear was by several fibers to the major sensory epithelium whereas a single fiber extended to the few cells in the base of the cochlear duct.
This nearly complete loss of the ear could come about through spatial sparing of some cells or through delayed loss. We therefore next investigated the state of differentiation of the neurosensory system in earlier embryos. Interestingly, many more nerve fibers were detected radiating to multiple areas of the ear consistent with posterior canal crista, cochlea, etc (Fig. 5d–g). However, this early extension of nerve fibers soon disappeared resulting in the limited innervation that remained at E18.5 (Fig. 5f,g,i,j). We therefore suggest that many neurons are lost with a delay because of delayed loss of Dicer1 and miRNAs. Once both have disappeared, the partially differentiated neurons that extend processes will die. We directly investigated this hypothesis using anti-caspase 3 staining in combination with PSVue staining and found degenerating neurons in the ganglion (Fig. 6e,e′,f,f′). These data suggest that neurosensory cells in the ear are as dependent for their differentiation on miRNAs as are neurons in the CNS and undergo a very similar cell death of partially differentiated neurons followed by phagocytosis through macrophages/activated microglia. In situ hybridization using miR-124 showed absence of this miR as early as E12.5 (Fig. 6c,d), but it cannot be excluded that other miRNAs might remain longer in some neurons, thus enabling some limited development. Whether the very few neurons and Myo7a-positive hair cells we found at E18.5 will remain after birth could not be tested due to perinatal lethality of the mutant embryos. Based on the uniform later fate of all cells in the forebrain (see below), we assume that differentiating cells that go on to survive the longest have for whatever reason managed to retain enough miRNAs to initiate that process but eventually are depleted like other cells and undergo apoptosis.
Forebrain and Cerebellum
Consistent with this scenario was that numerous anti-cleaved caspase 3 and PSVue-positive cells were present in the forebrain at E12.5 (see Fig. 7), and by E14.5 the forebrain was reduced to a small vesicle attached to the lateral wall of the diencephalon with very high levels of reflective cells (Figs. 1e and 2b). By E18.5 not even a trace of a forebrain remained (Figs. 1f and 2c), clearly indicating that all developing neurons critically depend on Dicer1-mediated replenishment of small RNAs for survival. Within the forebrain, cell death started in the future cortical areas, consistent with the upregulation of Foxg1 (Tao and Lai,1992). At E12.5 the entire forebrain was filled with macrophages containing multiple lumps of cellular debris that stain positive for PSVue. Counterstaining with anti-β tubulin showed multiple neuronal processes associated with anti-caspase 3 positive cells but not with PSVue-stained cells inside macrophages. These data show that at least some neurons start their differentiation. Notably, olfactory axons reach into the forebrain at this stage but interact only with anti-caspase 3-positive cells (Fig. 7). Later stages show the near complete loss of tubulin and caspase 3 with most cells forming debris inside macrophages that still stain positive for phosphatidylserine (data not shown). It remains unclear for a given neuron how onset of differentiation relates to complete depletion of all miRNA.
Figure 7. Immunochemistry of a Foxg1-cre::Dicer1f/f CKO reveals numerous anti-cleaved caspase 3-positive cells in the forebrain at E12.5 (a, a′). These anti-cleaved caspase 3-positive cells form a gradient with the highest concentration near the rostral (top) pole of the forebrain. Higher power images (a′) indicate that nearly every cell in the most rostral area shows anti-caspase 3 staining. PSVue staining (b, b′) shows prominently stained aggregates of cells in the more caudal and medial parts of the forebrain (b). Higher magnifications reveal surprisingly large aggregates of PSVue-positive cells that match the distribution of opaque cells seen at the macroscopic level (b′). Staining for the neuronal marker anti-acetylated β-tubulin shows fibers most prominent in areas of anticleaved c-cleaved caspase 3 staining (c, c′, d, d′) whereas areas of prominent PSVue staining reveal little nerve fiber staining (b, b′, d, d′). This suggests that loss of tubulin correlates with the appearance of phosphatidylserine on the surface of dying neurons. Bar indicates 100 μm.
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A similar sequence of events was found for the cerebellum but with a slight delay in onset and a faster completion of loss. At E12.5 the cerebellar anlage already showed many anti-cleaved caspase3-positive degenerating cells but comparatively fewer antiphosphatidylserine positive cells inside macrophages (Fig. 8a,b). By E14.5 the cerebellum was already massively reduced with the future vermis being completely eliminated and the future hemispheres being filled with numerous macrophages filled with cellular debris (Fig. 2e). By E18.5 there was no cerebellum, and the choroid plexus (CP) of the IVth ventricle was attached directly onto the midbrain (Fig. 2f). These data show a fast upregulation of critical steps in apoptosis followed by macrophage-mediated resorption of the debris (Fig. 8a,b”) in all brain areas with uniform expression of Foxg1-cre.
Figure 8. Staining with anti-cleaved caspase 3 and PSVue of the cerebellum of an E12.5 Foxg1-cre:: Dicer1 f/f CKO mouse shows widespread caspase 3 upregulation throughout the cerebellum (a). Staining for phosphatidylserine with PSVue shows an overlapping distribution of cells in possibly different stages of degeneration (a′, a”). Higher power images show that anti-cleaved caspase 3-positive cells are mostly individually distributed (b) whereas the PSVue labeled cells are mostly aggregated (b′). Combined imaging shows that either labeling is rarely overlapping with the other, or if they do, both stain weakly (b”). This suggests that either stain reveals cells at different stages of degeneration with the PSVue dye presumably showing cells already ingested by activated microglia cells that turned into macrophages. Bar indicates 100 μm a, a” and 5 μm b, b”.
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In summary, eyes and ears follow in principle the same rapid degeneration process in the absence of Dicer1 as do neurons in the CNS. However, there is incomplete loss of the retina due to expression of Foxg1-cre only in the anterior half of the eye. The limited number of hair cells and the few neurons remaining in the E18.5 ear could reflect a transient retention or might indicate that at least some hair cells can form in the absence of miRNAs, possibly correlated with less Foxg1-cre expression or very early development and thus more advanced stages in development before Dicer1 and miRNAs were depleted. Birthdating these cells using BrdU could test this hypothesis. Clearly, brain areas with uniform, early expression of Foxg1 disappear entirely (forebrain), but loss is also surprisingly profound in areas with little known Foxg1 expression (cerebellum).