Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

MacDonald, Glen; Rubel, Edwin W.

doi:10.1016/j.heares.2008.05.009

Cited by 62 publications

(48 citation statements)

References 26 publications

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“…Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Whole-mount immunolabeling with myosin VIIA antibody, dehydration and clearing of inner ears was performed as published (MacDonald and Rubel, 2008). Images of embryos, inner ears, sections and whole mounts of inner ear sensory epithelia were captured with a DFC 490 camera (Leica) on a Labophot-2 (Nikon) or MZ16FA fluorescence stereomicroscope (Leica).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea

et al. 2015

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Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the respecification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirrorimage copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea.

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Section: Immunohistochemistrymentioning

confidence: 99%

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea

et al. 2015

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“…For Movies 1A–C, embryos were rendered optically transparent (cleared) prior to imaging using a methyl salicylate and benzyl benzoate (MSBB) method described previously (78). Briefly, embryos were dehydrated using a series of ethanol solutions in distilled water (70%, 95%, 100%), and placed in a methyl salicylate, benzyl benzoate (5:3) solution for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

In vivo time-lapse imaging reveals extensive neural crest and endothelial cell interactions during neural crest migration and formation of the dorsal root and sympathetic ganglia

George

Dunkel

Hunnicutt

et al. 2016

Developmental Biology

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During amniote embryogenesis the nervous and vascular systems interact in a process that significantly affects the respective morphogenesis of each network by forming a “neurovascular” link. The importance of neurovascular cross-talk in the central nervous system has recently come into focus with the growing awareness that these two systems interact extensively both during development, in the stem-cell niche, and in neurodegenerative conditions such as Alzheimer’s Disease and Amyotrophic Lateral Sclerosis. With respect to the peripheral nervous system, however, there have been no live, real-time investigations of the potential relationship between these two developing systems. To address this deficit, we used multispectral 4D time-lapse imaging in a transgenic quail model in which endothelial cells (ECs) express a yellow fluorescent marker, while neural crest cells (NCCs) express an electroporated red fluorescent marker. We monitored EC and NCC migration in real-time during formation of the peripheral nervous system. Our time-lapse recordings indicate that NCCs and ECs are physically juxtaposed and dynamically interact at multiple locations along their trajectories. These interactions are stereotypical and occur at precise anatomical locations along the NCC migratory pathway. NCCs migrate alongside the posterior surface of developing intersomitic vessels, but fail to cross these continuous streams of motile ECs. NCCs change their morphology and migration trajectory when they encounter gaps in the developing vasculature. Within the nascent dorsal root ganglion, proximity to ECs causes filopodial retraction which curtails forward persistence of NCC motility. Overall, our time-lapse recordings support the conclusion that primary vascular networks substantially influence the distribution and migratory behavior of NCCs and the patterned formation of dorsal root and sympathetic ganglia.

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“…After dehydration, samples were incubated in a 1:1 mixture of methyl salicylate (Fisher Scientific) and benzyl benzoate (Sigma-Aldrich)(MSBB) (MacDonald and Rubel, 2008) and pure ethanol for four hours. Samples were then cleared using three changes of MSBB for 2 hours, 4 hours, and overnight.…”

Section: Methodsmentioning

confidence: 99%

A central to peripheral progression of cell cycle exit and hair cell differentiation in the developing mouse cristae

Slowik

Bermingham‐McDonogh

2016

Developmental Biology

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The inner ear contains six distinct sensory organs that each maintains some ability to regenerate hair cells into adulthood. In the postnatal cochlea, there appears to be a relationship between the developmental maturity of a region and its ability to regenerate as postnatal regeneration largely occurs in the apical turn, which is the last region to differentiate and mature during development. In the mature cristae there are also regional differences in regenerative ability, which led us to hypothesize that there may be a general relationship between the relative maturity of a region and the regenerative competence of that region in all of the inner ear sensory organs. By analyzing adult mouse cristae labeled embryonically with BrdU, we found that hair cell birth starts in the central region and progresses to the periphery with age. Since the peripheral region of the adult cristae also maintains active Notch signaling and some regenerative competence, these results are consistent with the hypothesis that the last regions to develop retain some of their regenerative ability into adulthood. Further, by analyzing embryonic day 14.5 inner ears we provide evidence for a wave of hair cell birth along the longitudinal axis of the cristae from the central regions to the outer edges. Together with the data from the adult inner ears labeled with BrdU as embryos, these results suggest that hair cell differentiation closely follows cell cycle exit in the cristae, unlike in the cochlea where they are uncoupled.

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Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

Cited by 62 publications

References 26 publications

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea

In vivo time-lapse imaging reveals extensive neural crest and endothelial cell interactions during neural crest migration and formation of the dorsal root and sympathetic ganglia

A central to peripheral progression of cell cycle exit and hair cell differentiation in the developing mouse cristae

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