Single-Cell RNA Sequencing Analysis Reveals Greater Epithelial Ridge Cells Degeneration During Postnatal Development of Cochlea in Rats

Greater epithelial ridge cells, a transient neonatal cell group in the cochlear duct, which plays a crucial role in the functional maturation of hair cell, structural development of tectorial membrane, and refinement of audio localization before hearing. Greater epithelial ridge cells are methodologically homogeneous, while whether different cell subtypes are existence in this intriguing region and the degeneration mechanism during postnatal cochlear development are poorly understood. In the present study, single-cell RNA sequencing was performed on the cochlear duct of postnatal rats at day 1 (P1) and day 7 (P7) to identify subsets of greater epithelial ridge cell and progression. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to examine genes enriched biological processes in these clusters. We identified a total of 26 clusters at P1 and P7 rats and found that the cell number of five cell clusters decreased significantly, while four clusters had similar gene expression patterns and biological properties. The genes of these four cell populations were mainly enriched in Ribosome and P13K-Akt signal pathway. Among them, Rps16, Rpsa, Col4a2, Col6a2, Ctsk, and Jun are particularly interesting as their expression might contribute to the greater epithelial ridge cells degeneration. In conclusion, our study provides an important reference resource of greater epithelial ridge cells landscape and mechanism insights for further understanding greater epithelial ridge cells degeneration during postnatal rat cochlear development.

Gpr125 Marks Distinct Cochlear Cell Types and Is Dispensable for Cochlear Development and Hearing

The G protein-coupled receptor (GPR) family critically regulates development and homeostasis of multiple organs. As a member of the GPR adhesion family, Gpr125 (Adgra3) modulates Wnt/PCP signaling and convergent extension in developing zebrafish, but whether it is essential for cochlear development in mammals is unknown. Here, we examined the Gpr125lacZ/+ knock-in mice and show that Gpr125 is dynamically expressed in the developing and mature cochleae. From embryonic day (E) 15.5 to postnatal day (P) 30, Gpr125-β-Gal is consistently expressed in the lesser epithelial ridge and its presumed progenies, the supporting cell subtypes Claudius cells and Hensen’s cells. In contrast, Gpr125-β-Gal is expressed transiently in outer hair cells, epithelial cells in the lateral cochlear wall, interdental cells, and spiral ganglion neurons in the late embryonic and early postnatal cochlea. In situ hybridization for Gpr125 mRNA confirmed Gpr125 expression and validated loss of expression in Gpr125lacZ/lacZ cochleae. Lastly, Gpr125lacZ/+ and Gpr125lacZ/lacZ cochleae displayed no detectable loss or disorganization of either sensory or non-sensory cells in the embryonic and postnatal ages and exhibited normal auditory physiology. Together, our study reveals that Gpr125 is dynamically expressed in multiple cell types in the developing and mature cochlea and is dispensable for cochlear development and hearing.

The Notch Ligand Jagged1 is Required for the Formation, Maintenance, and Survival of Hensen Cells in the Mouse Cochlea

ABSTRACTDuring cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1’s expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen cells (HeCs), a highly specialized SC-subtype located at the edge of the auditory epithelium. Deletion of Jag1 at the onset of differentiation, at stage E14.5, disrupted HeC formation. Similar loss of HeCs was observed when Jag1 was deleted at P0/P1 and fate-mapping analysis revealed that in the absence of Jag1 some HeCs die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in P0 cochlea lacking Jag1.

Mesenchymal ETV Transcription Factors Regulate Cochlear Length

Mammalian cochlear development encompasses a series of morphological and molecular events that results in the formation of a highly intricate structure responsible for hearing. One remarkable event occurs during development is the cochlear lengthening that starts with cochlear outgrowth around E11 and continues throughout development. Different mechanisms contribute to this process including cochlear progenitor proliferation and convergent extension. We previously identified that FGF9 and FGF20 promote cochlear lengthening by regulating auditory sensory epithelial proliferation through FGFR1 and FGFR2 in the periotic mesenchyme. Here, we provide evidence that ETS-domain transcription factors ETV4 and ETV5 are downstream of mesenchymal FGF signaling to control cochlear lengthening. Next generation RNA sequencing identified that Etv1, Etv4 and Etv5 mRNAs are decreased in the Fgf9 and Fgf20 double mutant periotic mesenchyme. Deleting both Etv4 and Etv5 in periotic mesenchyme resulted in shortening of cochlear length but maintaining normal patterning of organ of Corti and density of hair cells and supporting cells. This recapitulates phenotype of mesenchymal-specific Fgfr1 and Fgfr2 deleted inner ear. Furthermore, analysis of Etv1/4/5 triple conditional mutants revealed that ETV1 does not contribute in this process. Our study reveals that ETV4 and ETV5 function downstream of mesenchymal FGF signaling to promote cochlear lengthening.

SAT-448 Limited Adaptive Response by Deiodinase Enzymes in the Perinatal Hypothyroid Cochlea

Thyroid hormone (TH) is essential for cochlear development and normal auditory function. Considering the importance of TH in mediating cochlear development, understanding the degree to which developing tissues can adapt to perturbations in thyroid hormone signaling is extremely important. The deiodinases (Dio2 and Dio3) are enzymes that tightly control TH availability at the tissue level and have been proposed to function as adaptive mechanisms that maintain tissue TH homeostasis. Dio2 converts thyroxine (T4) to a biologically active ligand triiodothyronine (T3); locally amplifying a T3 signal. Conversely, Dio3 inactivates T3 and T4 by converting these iodothyronines to the inactive metabolites diiodothyronine (T2) and reverse T3 (rT3), respectively. During cochlear development, Dio3 expression is high prenatally while Dio2 expression is low. During the first postnatal week, the expression levels of these enzymes invert, resulting in high expression of Dio2 and low expression of Dio3. Together, the deiodinases control the timing of postnatal cochlear remodeling; suggesting a genetic developmental clock controls TH-mediated cochlear development. Considering the role deiodinases as adaptive mechanisms, it is important to understand whether such a developmental clock can be negated during times of developmental thyroid hormone insufficiency. We hypothesize that the perinatal change in deiodinase expression is controlled by a developmental clock rather than environmental clues and therefore have limited capacity function as compensatory mechanisms in response to low TH during development. To test this, timed-pregnant mice were treated with thyroid gland inhibitors to induced hypothyroidism from gestational day 12.5 until pup sacrifice. A parallel set of untreated timed-pregnant mice served as controls. Cochlea were harvest from control and hypothyroid mice at postnatal (P) ages P1, P5, P10 and P15 for qRT-PCR, and P7 for in situ hybridization. In situ hybridization for Dio2 mRNA demonstrated a striking reduction in Dio2 mRNA in hypothyroid animals compared to euthyroid animals. Additionally, we observed a significant effect of age (p<0.001) and an interaction between age and treatment (p=0.0035) on Dio2 mRNA levels analyzed by qRT-PCR. Specifically, Dio2 mRNA levels were similar or reduced in hypothyroid animals compared to controls at P1, P5, and P10. This finding indicates that Dio2 mRNA at these ages did not respond in manner consistent with compensation. However, at P15, Dio2 mRNA levels were increased in hypothyroid animals compared to controls; a finding that is consistent with a compensatory mechanism. Taken together, our results suggest that developmental programs in tissues may be dominant over potential compensatory mechanisms and that developing tissues may be more susceptible to perturbations in tissue TH levels due to a reduced capacity to compensate.

CDK5RAP2 primary microcephaly is associated with hypothalamic, retinal and cochlear developmental defects

BackgroundPrimary hereditary microcephaly (MCPH) comprises a large group of autosomal recessive disorders mainly affecting cortical development and resulting in a congenital impairment of brain growth. Despite the identification of >25 causal genes so far, it remains a challenge to distinguish between different MCPH forms at the clinical level.Methods7 patients with newly identified mutations in CDK5RAP2 (MCPH3) were investigated by performing prospective, extensive and systematic clinical, MRI, psychomotor, neurosensory and cognitive examinations under similar conditions.ResultsAll patients displayed neurosensory defects in addition to microcephaly. Small cochlea with incomplete partition type II was found in all cases and was associated with progressive deafness in 4 of them. Furthermore, the CDK5RAP2 protein was specifically identified in the developing cochlea from human fetal tissues. Microphthalmia was also present in all patients along with retinal pigmentation changes and lipofuscin deposits. Finally, hypothalamic anomalies consisting of interhypothalamic adhesions, a congenital midline defect usually associated with holoprosencephaly, was detected in 5 cases.ConclusionThis is the first report indicating that CDK5RAP2 not only governs brain size but also plays a role in ocular and cochlear development and is necessary for hypothalamic nuclear separation at the midline. Our data indicate that CDK5RAP2 should be considered as a potential gene associated with deafness and forme fruste of holoprosencephaly. These children should be given neurosensory follow-up to prevent additional comorbidities and allow them reaching their full educational potential.Trial registration numberNCT01565005.