Biology subjects

Doetzlhofer, A.

Publications and source records attributed to Doetzlhofer, A..

11 recordsLinked to original sources

bioRxiv2026

The RNA-binding protein TRIM71 Promotes Supporting Cell Reprogramming and Hair Cell Regeneration by Activating Sall4

Hair cell (HC) loss in the auditory organ (cochlea) is irreversible and represents the leading cause of deafness in humans. The absence of HC regeneration is primarily due to the limited plasticity of cochlear supporting cells (SCs). Using a novel doxycycline-inducible TRIM71 transgenic mouse model, this study demonstrates that overexpression of the RNA-binding protein TRIM71 promotes spontaneous cochlear HC regeneration in neonatal mice. Experiments in cochlear organoids indicate that TRIM71 reprograms SCs into progenitor-like cells and that this reprogramming is further enhanced by coactivation of follistatin (FST). FST augments TRIM71-induced increases in chromatin accessibility at progenitor and HC-specific gene loci, facilitating their transcriptional activation in SCs. Notably, FST also enhances TRIM71-induced reactivation of Sall4, which, like Trim71, is highly expressed in early otic progenitors. Using cochlear organoids and explants, we show that the early otic transcription factor SALL4 is required for TRIM71-induced HC formation. By contrast, SALL4 overexpression inhibits HC formation by maintaining reprogrammed SCs in an undifferentiated, proliferative state. These findings establish TRIM71 as an activator of HC regeneration in vivo, provide mechanistic insights into TRIM71's role in SC reprogramming, and identify SALL4 as its essential effector.

cell biology↗

bioRxiv2026

Notch1 and Notch2 receptors mediate the prosensory functions of the Notch ligand Jagged1 in the mammalian cochlea

Mechanosensory hair cells in the cochlea of the inner ear are essential for sound detection. Previous studies have shown that loss of the Notch ligand Jagged1 (JAG1) disrupts maintenance of prosensory progenitors, from which hair cells and their surrounding supporting cells derive. However, the molecular and cellular mechanisms by which JAG1 regulates prosensory progenitor maintenance are poorly understood, and the Notch receptors that mediate this function in the developing cochlea remain unknown. To address this, we generated Jag1 conditional knockout mice and characterized their prosensory deficits using transcriptomic and phenotypic analyses. We show that JAG1 loss abolishes prosensory expression of CDKN1B (p27Kip1) and reduces expression of essential neurotrophic factors, thereby disrupting the stereotyped pattern of cell-cycle exit and innervation within the prosensory domain. Furthermore, conditional knockout of Notch1 and Notch2 (Notch1/2) receptors in supporting cells phenocopied the Jag1 deletion phenotype, showing both a severe reduction in outer hair cell formation and a disrupted pattern of cell-cycle exit and innervation, thereby confirming their essential roles in mediating the prosensory functions of JAG1.

developmental biology↗

bioRxiv2026

Loss of Zbtb20 disrupts cochlear supporting cell differentiation and maturation and extends the postnatal hair cell regenerative window in mice.

Cochlear hair cell (HC) loss is a leading cause of hearing loss in humans. HCs can be generated from adjacent supporting cells (SCs); however, this regenerative capacity is lost after the onset of hearing. Using Emx2Cre Zbtb20 knockout mice, we show that ZBTB20 deficiency delays cell-cycle exit, differentiation, and maturation of cochlear SCs. Transcriptomic analysis of postnatal cochlear sensory epithelia indicates that ZBTB20 loss postpones the downregulation of progenitor genes, including Sox11 and Hmga2, and delays activation of a maturation-specific gene program. Additionally, experiments with cochlear organoid and organotypic explant models, reveal that prolonged, and to a lesser extent acute, ZBTB20 loss increases the mitotic and HC-regenerative potential of cochlear SCs. Transcriptomic profiling shows that acute ZBTB20 loss upregulates the midkine receptor Ptprz1, and further studies show that exogenous midkine, similar to ZBTB20 loss, promotes cell-cycle reentry and proliferation in cochlear organoid cultures.

developmental biology↗

bioRxiv2025

CASZ1 regulates the rate at which outer hair cells mature and is required for hearing

The transcriptional activator ATOH1 is a master regulator of the development of mechanosensory hair-cells (HCs) in the ear. We report that the ATOH1 target gene Casz1 encodes a transcription factor that regulates the rate of outer HC (OHC) maturation by gene repression. Genetic deletion of Casz1 during (but not after) development of the mouse cochlea caused: hearing loss; abnormal organization of mechanosensory stereocilia bundles in OHCs; abnormally low F-actin density in OHC cuticular plates; progressive loss of OHCs; and mild morphological alterations in inner HCs. RNA sequencing revealed that Casz1 deletion delayed downregulation of genes expressed in immature OHCs, including the actin regulator-encoding gene Coro2a, and accelerated upregulation of genes expressed in mature OHCs. Coro2a knockdown restored the density of cuticular plate F-actin in Casz1 mutant OHCs. Our data indicate that CASZ1 regulates transcriptional and morphological maturation of OHCs, and that CASZ1 in maturing HCs is necessary for hearing.

developmental biology↗

bioRxiv2025

Title: The RNA-binding protein TRIM71 is essential for hearing in humans and mice and regulates the timing of auditory sensory organ development

The RNA-binding protein TRIM71 is essential for brain development, and recent genetic studies in humans have identified TRIM71 as a risk gene for congenital hydrocephaly (CH). Here, we show that mono-allelic missense mutations in TRIM71 are associated with hearing loss (HL) and inner ear aplasia in humans. Utilizing conditional Trim71 knockout mice carrying a CH and HL-associated mutation, we demonstrate that loss of TRIM71 function during early otic development (embryonic day 9-10) causes severe hearing loss. While inner ear morphogenesis occurs normally in Trim71 knockout mice, we find that early otic loss of TRIM71 function disrupts the highly stereotyped timing of cell cycle exit and differentiation within the inner ear auditory sensory organ (cochlea), resulting in the premature formation and innervation of mechano-sensory hair cells. Transcriptomic profiling of Trim71 deficient cochlear progenitor cells identifies Inhba and Tgfbr2 as targets of TRIM71 repression, and our analysis of InhbaTgfbr1 double knockout mice indicates that TRIM71 maintains hair cell progenitors in a proliferative and undifferentiated state by restricting TGF-{beta}-type signaling. Characterization of hair cells and their associated neurons in adult Trim71 knockout mice revealed abnormally short inner hair cell stereocilia, reduced pre-synaptic terminals, and neuronal degeneration in the outer hair cell region, providing a basis for the observed hearing deficits in Trim71 knockout mice.

neuroscience↗

bioRxiv2025

The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration.

Hair cells (HCs) within the inner ear cochlea are specialized mechanoreceptors required for hearing. Cochlear HCs are not regenerated in mammals, and their loss is a leading cause of deafness in humans. Cochlear supporting cells (SCs) in newborn mice have the capacity to regenerate HCs, but persistent Notch signaling, presumably activated by SC-specific Notch ligand Jagged1 (JAG1), prevents SCs from converting into HCs. Here, employing an organoid platform, we show that while JAG1 participates in HC-fate repression, JAG1s primary function is to preserve the "progenitor-like characteristics" of cochlear SCs. Transcriptomic and mechanistic studies reveal that JAG1/Notch signaling maintains the expression of progenitor and metabolic genes in cochlear SCs and sustains pro-growth pathways, including PI3K-Akt-mTOR signaling, a function that is mediated by Notch1 and Notch2. Finally, we show that JAG1/Notch signaling stimulation with JAG1-Fc peptide enhances the HC-forming capacity of cochlear SCs undergoing maturation in cochlear explants and in vivo.

developmental biology↗

bioRxiv2023

Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming, however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 reactivation increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71s RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.

neuroscience↗

bioRxiv2020

LIN28B controls the regenerative capacity of neonatal murine auditory supporting cells through activation of mTOR signaling

Mechano-sensory hair cells within the inner ear cochlea are essential for the detection of sound. In mammals, cochlear hair cells are only produced during development and their loss, due to disease or trauma, is a leading cause of deafness. In the immature cochlea, prior to the onset of hearing, hair cell loss stimulates neighboring supporting cells to act as hair cell progenitors and produce new hair cells. However, for reasons unknown, such regenerative capacity (plasticity) is lost once supporting cells undergo maturation. Here, we demonstrate that the RNA binding protein LIN28B plays an important role in the production of hair cells by supporting cells and provide evidence that the developmental drop in supporting cell plasticity in the mammalian cochlea is, at least in part, a product of declining LIN28B-mTOR activity. Employing murine cochlear organoid and explant cultures to model mitotic and non-mitotic mechanisms of hair cell generation, we show that loss of Lin28b function, due to its conditional deletion, or due to overexpression of the antagonistic miRNA let-7g, suppressed Akt-mTORC1 activity and renders young, immature supporting cells incapable of generating hair cells. Conversely, we found that LIN28B overexpression increased Akt-mTORC1 activity and allowed supporting cells that were undergoing maturation to de-differentiate into progenitor-like cells and to produce hair cells via mitotic and non-mitotic mechanisms. Finally, using the mTORC1 inhibitor rapamycin, we demonstrate that LIN28B promotes supporting cell plasticity in an mTORC1-dependent manner. SIGNIFICANCE STATEMENTCochlear hair cell loss is a leading cause of deafness in humans and other mammals. In the immature cochlea lost hair cells are regenerated by neighboring glia-like supporting cells. However, for reasons unknown, such regenerative capacity is rapidly lost as supporting cells undergo maturation. Here we identify a direct link between LIN28B-mTOR activity and supporting cell plasticity. Mimicking later developmental stages, we found that loss of the RNA binding protein LIN28B attenuated mTOR signaling and rendered young, immature supporting cells incapable of producing hair cells. Conversely, we found that re-expression of LIN28B reinstated the ability of maturing supporting cells to revert to a progenitor-like state and generate hair cells via activation of mTOR signaling.

developmental biology↗

bioRxiv2020

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

During 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 JAG1s 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.

developmental biology↗

bioRxiv2019

Let-7 miRNAs control auditory sensory progenitor behavior in the vertebrate inner ear

The evolutionary conserved lethal-7 (let-7) family of microRNAs (miRNAs) is a well-known activator of terminal mitosis and differentiation. Surprisingly, we previously found that overexpression of let-7 miRNAs in the murine auditory organ accelerated the terminal mitosis of auditory sensory progenitors (pro-sensory cells) but failed to stimulate their differentiation into mechano-sensory hair cells (HCs). To further address the role of let-7 miRNAs in auditory sensory differentiation, we conducted gain and loss of function experiments in the developing chicken auditory organ, the basilar papilla (BP). Using a sponge approach, we show that the disruption of let-7 miRNA function in the developing BP delays pro-sensory cell exit and delays differentiation of auditory HCs, revealing that endogenous let-7 miRNAs limit pro-sensory cell self-renewal in the developing BP. However, consistent with the role of let-7 miRNAs in the murine auditory organ, let-7b overexpression in the developing BP delayed HC differentiation, suggesting that too low or too high let-7 miRNA levels disrupt HC differentiation. Furthermore, we provide evidence that the repressive role of let-7 miRNAs in HC differentiation may be due to its targeting of the chromatin remodeler CHD7. Mutation in the human CHD7 gene causes CHARGE syndrome, which amongst others is characterized by inner ear and hearing deficits. Using target prediction algorithms, we uncovered a highly predictive and evolutionary conserved let-7 binding site within the Chd7 transcript. Consistent with being a target of let-7 repression, we demonstrate that let-7b overexpression significantly reduced CHD7 protein expression in to the developing BP. Furthermore, utilizing an inducible let-7g transgenic mouse model, we show that let-7 miRNAs negatively regulate CHD7 protein expression in developing murine cochlear, retinal and brain tissue. CHD7 is dosage dependent and the here described regulation by let-7 miRNAs may be critical to fine tune CHD7 protein levels during sensory and neuronal development.\n\nSIGNIFICANCEThe evolutionary highly conserved let-7 miRNAs are essential for proper timing of cell state transitions during embryogenesis. Even though abundantly expressed in the vertebrate auditory organ, surprisingly little is known about their function in auditory sensory differentiation. Here, we demonstrate that endogenous let-7 miRNAs are essential for limiting auditory sensory progenitor (pro-sensory) cell self-renewal. Furthermore, we find that precocious let-7 miRNAs expression interferes with auditory hair cell differentiation and identify chromatin remodeler CHD7 as a potential target gene of let-7 repressive function in HC differentiation.

developmental biology↗

bioRxiv2018

Activin signaling informs the graded pattern of terminal mitosis and hair cell differentiation in the mammalian cochlea

The mammalian auditory sensory epithelium has one of the most stereotyped cellular patterns known in vertebrates. Mechano-sensory hair cells are arranged in precise rows, with one row of inner and three rows of outer hair cells spanning the length of the spiral-shaped sensory epithelium. Aiding such precise cellular patterning, differentiation of the auditory sensory epithelium is precisely timed and follows a steep longitudinal gradient. The molecular signals that promote auditory sensory differentiation and instruct its graded pattern are largely unknown. Here, we identify Activin A as an activator of hair cell differentiation and show, using mouse genetic approaches, that a local gradient of Activin A signaling within the auditory sensory epithelium times the longitudinal gradient of hair cell differentiation. Furthermore, we provide evidence that Activin-type signaling regulates a radial gradient of terminal mitosis within the auditory sensory epithelium, which constitutes a novel mechanism for limiting the number of inner hair cells being produced.

developmental biology↗