NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both ...pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.
Mammalian hearing requires the development of the organ of Corti, a sensory epithelium comprising unique cell types. The limited number of each of these cell types, combined with their close ...proximity, has prevented characterization of individual cell types and/or their developmental progression. To examine cochlear development more closely, we transcriptionally profile approximately 30,000 isolated mouse cochlear cells collected at four developmental time points. Here we report on the analysis of those cells including the identification of both known and unknown cell types. Trajectory analysis for OHCs indicates four phases of gene expression while fate mapping of progenitor cells suggests that OHCs and their surrounding supporting cells arise from a distinct (lateral) progenitor pool. Tgfβr1 is identified as being expressed in lateral progenitor cells and a Tgfβr1 antagonist inhibits OHC development. These results provide insights regarding cochlear development and demonstrate the potential value and application of this data set.
Mitochondrial form and function in hair cells O'Sullivan, James D.B.; Bullen, Anwen; Mann, Zoë F.
Hearing research,
February 2023, 2023-02-00, 20230201, Letnik:
428
Journal Article
Recenzirano
Odprti dostop
•Mitochondria play a significant role in function and death of auditory hair cells.•Advances in imaging have revealed new aspects of mitochondrial biology.•Spatially and functionally discrete ...mitochondrial populations support sensory function.•The relationship between mitochondrial structure and function is poorly understood.•Future work should focus on the role of mitochondria-organelle interactions.
Hair cells (HCs) are specialised sensory receptors residing in the neurosensory epithelia of inner ear sense organs. The precise morphological and physiological properties of HCs allow us to perceive sound and interact with the world around us. Mitochondria play a significant role in normal HC function and are also intricately involved in HC death. They generate ATP essential for sustaining the activity of ion pumps, Ca2+ transporters and the integrity of the stereociliary bundle during transduction as well as regulating cytosolic calcium homoeostasis during synaptic transmission. Advances in imaging techniques have allowed us to study mitochondrial populations throughout the HC, and how they interact with other organelles. These analyses have identified distinct mitochondrial populations between the apical and basolateral portions of the HC, in which mitochondrial morphology appears determined by the physiological processes in the different cellular compartments. Studies in HCs across species show that ototoxic agents, ageing and noise damage directly impact mitochondrial structure and function resulting in HC death. Deciphering the molecular mechanisms underlying this mitochondrial sensitivity, and how their morphology relates to their function during HC death, requires that we first understand this relationship in the context of normal HC function.
In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long ...(tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla (BP), requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging (FLIM) we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in hair cell morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.
Acoustic frequency analysis plays an essential role in sound perception, communication and behavior. The auditory systems of most vertebrates that perceive sounds in air are organized based on the ...separation of complex sounds into component frequencies. This process begins at the level of the auditory sensory epithelium where specific frequencies are distributed along the tonotopic axis of the mammalian cochlea or the avian/reptilian basilar papilla (BP). Mechanical and electrical mechanisms mediate this process, but the relative contribution of each mechanism differs between species. Developmentally, structural and physiological specializations related to the formation of a tonotopic axis form gradually over an extended period of time. While some aspects of tonotopy are evident at early stages of auditory development, mature frequency discrimination is typically not achieved until after the onset of hearing. Despite the importance of tonotopic organization, the factors that specify unique positional identities along the cochlea or basilar papilla are unknown. However, recent studies of developing systems, including the inner ear provide some clues regarding the signalling pathways that may be instructive for the formation of a tonotopic axis.
► The review discusses recent work on tonotopy in the mammalian, avian, and reptilian hearing organs. ► The functional specializations that mediate tonotopy in both mammals and birds/reptiles are discussed. ► The review links what is currently known about tonotopy in mammals, birds and amphibia with existing studies describing how these systems develop. ► The review also highlights important experiments that could be done to provide greater insight into how tonotopy is established during development.
The mechanisms of formation of the distinct sensory organs of the inner ear and the non-sensory domains that separate them are still unclear. Here, we show that several sensory patches arise by ...progressive segregation from a common prosensory domain in the embryonic chicken and mouse otocyst. This process is regulated by mutually antagonistic signals: Notch signalling and Lmx1a. Notch-mediated lateral induction promotes prosensory fate. Some of the early Notch-active cells, however, are normally diverted from this fate and increasing lateral induction produces misshapen or fused sensory organs in the chick. Conversely Lmx1a (or cLmx1b in the chick) allows sensory organ segregation by antagonizing lateral induction and promoting commitment to the non-sensory fate. Our findings highlight the dynamic nature of sensory patch formation and the labile character of the sensory-competent progenitors, which could have facilitated the emergence of new inner ear organs and their functional diversification in the course of evolution.
Precise frequency discrimination is a hallmark of auditory function in birds and mammals and is required for distinguishing similar sounding words, like 'bat,' 'cat' and 'hat.' In the cochlea, tuning ...and spectral separation result from longitudinal differences in basilar membrane stiffness and numerous individual gradations in sensory hair cell phenotypes, but it is unknown what patterns the phenotypes. Here we used RNA-seq to compare transcriptomes from proximal, middle and distal regions of the embryonic chicken cochlea, and found opposing longitudinal gradients of expression for retinoic acid (RA)-synthesizing and degrading enzymes. In vitro experiments showed that RA is necessary and sufficient to induce the development of distal-like hair cell phenotypes and promotes expression of the actin-crosslinking proteins, Espin and Fscn2. These and other findings highlight a role for RA signalling in patterning the development of a longitudinal gradient of frequency-tuned hair cell phenotypes in the cochlea.
The auditory systems of animals that perceive sounds in air are organized to separate sound stimuli into their component frequencies. Individual tones then stimulate mechanosensory hair cells located ...at different positions on an elongated frequency (tonotopic) axis. During development, immature hair cells located along the axis must determine their tonotopic position in order to generate frequency-specific characteristics. Expression profiling along the developing tonotopic axis of the chick basilar papilla (BP) identified a gradient of Bmp7. Disruption of that gradient in vitro or in ovo induces changes in hair cell morphologies consistent with a loss of tonotopic organization and the formation of an organ with uniform frequency characteristics. Further, the effects of Bmp7 in determination of positional identity are shown to be mediated through activation of the Mapk, Tak1. These results indicate that graded, Bmp7-dependent, activation of Tak1 signalling controls the determination of frequency-specific hair cell characteristics along the tonotopic axis.
A study of genes expressed in the developing inner ear identified the bHLH transcription factor Scleraxis (Scx) in the developing cochlea. Previous work has demonstrated an essential role for Scx in ...the differentiation and development of tendons, ligaments and cells of chondrogenic lineage. Expression in the cochlea has been shown previously, however the functional role for Scx in the cochlea is unknown. Using a Scx-GFP reporter mouse line we examined the spatial and temporal patterns of Scx expression in the developing cochlea between embryonic day 13.5 and postnatal day 25. Embryonically, Scx is expressed broadly throughout the cochlear duct and surrounding mesenchyme and at postnatal ages becomes restricted to the inner hair cells and the interdental cells of the spiral limbus. Deletion of Scx results in hearing impairment indicated by elevated auditory brainstem response (ABR) thresholds and diminished distortion product otoacoustic emission (DPOAE) amplitudes, across a range of frequencies. No changes in either gross cochlear morphology or expression of the Scx target genes Col2A, Bmp4 or Sox9 were observed in Scx(-/-) mutants, suggesting that the auditory defects observed in these animals may be a result of unidentified Scx-dependent processes within the cochlea.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract In the cochlea, cell damage triggers intercellular Ca2+ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca2+ waves are thought to convey ...information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca2+ concentration (Ca2+ cyt ), and yet little is known about their role during the propagation of such intercellular Ca2+ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell Ca2+ cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in Ca2+ cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca2+ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, enhanced the peak amplitude and duration of ATP-induced Ca2+ cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca2+ waves was significantly increased. Thus, mitochondria function as spatial Ca2+ buffers during agonist-evoked Ca2+ cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca2+ waves.
PDF
