gene-transfer-into-chicken-auditory-organ-ovo-micro

Gene Transfer into the Chicken Auditory Organ by <em>In Ovo</em> Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

The Johns Hopkins University, School of Medicine

Thymidine kinase (TK) is a growth factor-inducible enzyme that is highly expressed in proliferating mammalian cells. Expression of mouse TK mRNA is controlled by transcriptional and posttranscriptional mechanisms including antisense transcription. Here we report the identification of a novel gene that is divergently transcribed from the bidirectional TK promoter. This gene encodes kynurenine formamidase (KF), an enzyme of the tryptophan metabolism. Whereas the TK gene is induced upon interleukin-2-mediated activation of resting T cells, the KF gene becomes simultaneously repressed. The TK promoter is regulated by E2F, SP1, histone acetyltransferases, and deacetylases. The binding site for the growth-regulated transcription factor E2F is beneficial for TK promoter activity but not required for KF expression. In contrast, the SP1 binding site is crucial for transcription in both directions. Inhibition of histone deacetylases by trichostatin A leads to increased histone acetylation at the TK/KF promoter and thereby to selective activation of the TK promoter and simultaneous shut-off of KF expression. Similarly, TK gene activation by interleukin-2 is linked to histone hyperacetylation, whereas KF expression correlates with reduced histone acetylation. The KF gene is the rare example of a mammalian gene whose expression is linked to histone hypoacetylation at its promoter.

Alternate activation of two divergently transcribed mouse genes from a bidirectional promoter is linked to changes in histone modification.

The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.

The tumor suppressor p53 and histone deacetylase 1 are antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 gene.

Math1 is a bHLH transcription factor expressed in neural progenitor cells in multiple regions of the nervous system. Previously we identified a Math1 enhancer that directs expression of reporter genes in a Math1 specific pattern [Development 127 (2000) 1185]. We have used a portion of this enhancer to drive expression of a nuclear GFP reporter in the Math1 lineage in transgenic mice. In this transgenic mouse strain, GFP is expressed in Math1 domains in the (1). developing spinal cord in progenitors to dI1 dorsal interneurons, (2). granule-cell progenitors in the developing cerebellum, (3). Merkel cells in the skin, and (4). hair cells in the developing vestibular and auditory systems. Furthermore, non-Math1 related expression is detected that is likely due to the absence of inhibitory regulatory sequences from the transgene. These expression domains include (1). the apical ectodermal ridge in developing limbs, (2). post-mitotic cells in the developing cortex and spinal cord, (3). the dentate gyrus, (4). retina, and (5). olfactory epithelium. Because GFP marks specific neuronal cell types in living tissue, this transgenic strain is a powerful tool for future studies on the development and electrophysiological properties of distinct cell types in the central nervous system and in sensory systems.

Math1-driven GFP expression in the developing nervous system of transgenic mice.

The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.

In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme.

Expression of the cyclin-dependent kinase inhibitor p27(Kip1) defines a post-mitotic population of cells in the embryonic mammalian cochlea that constitutes the nascent organ of Corti. Here, we describe techniques to purify these precursors using a transgenic p27/GFP reporter and fluorescence activated cell sorting (FACS). We demonstrate that these cells express other markers of the sensory lineage, such as Sox2, and when placed in dissociated cell culture differentiate as hair cells and supporting cells. The purified sensory progenitors thus obtained provide a means of studying the process of hair cell and supporting cell differentiation in vitro, as well as providing a means of analyzing the molecular and physiological properties of this unique population of cells.

Prospective identification and purification of hair cell and supporting cell progenitors from the embryonic cochlea.

Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.

Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells.

Sensory hair cells of the auditory organ are generated during embryogenesis and remain postmitotic throughout life. Previous work has shown that inactivation of the cyclin-dependent kinase inhibitor (CKI) p19(Ink4d) leads to progressive hearing loss attributable to inappropriate DNA replication and subsequent apoptosis of hair cells. Here we show the synergistic action of another CKI, p21(Cip1), on cell cycle reactivation. The codeletion of p19(Ink4d) and p21(Cip1) triggered profuse S-phase entry of auditory hair cells during a restricted period in early postnatal life, leading to the transient appearance of supernumerary hair cells. In addition, we show that aberrant cell cycle reentry leads to activation of a DNA damage response pathway in these cells, followed by p53-mediated apoptosis. The majority of hair cells were absent in adult cochleas. These data, together with the demonstration of changing expression patterns of multiple CKIs in auditory hair cells during the stages of early postnatal maturation, show that the maintenance of the postmitotic state is an active, tissue-specific process, cooperatively regulated by several CKIs, and is critical for the lifelong survival of these sensory cells.

p19(Ink4d) and p21(Cip1) collaborate to maintain the postmitotic state of auditory hair cells, their codeletion leading to DNA damage and p53-mediated apoptosis.

The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that-unlike other Hes/Hey factors-Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.

Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti.

In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.

Notch signaling limits supporting cell plasticity in the hair cell-damaged early postnatal murine cochlea.

Mechano-sensory hair cells (HCs), housed in the inner ear cochlea, are critical for the perception of sound. In the mammalian cochlea, differentiation of HCs occurs in a striking basal-to-apical and medial-to-lateral gradient, which is thought to ensure correct patterning and proper function of the auditory sensory epithelium. Recent studies have revealed that Hedgehog signaling opposes HC differentiation and is critical for the establishment of the graded pattern of auditory HC differentiation. However, how Hedgehog signaling interferes with HC differentiation is unknown. Here, we provide evidence that in the murine cochlea, Hey1 and Hey2 control the spatiotemporal pattern of HC differentiation downstream of Hedgehog signaling. It has been recently shown that HEY1 and HEY2, two highly redundant HES-related transcriptional repressors, are highly expressed in supporting cell (SC) and HC progenitors (prosensory cells), but their prosensory function remained untested. Using a conditional double knock-out strategy, we demonstrate that prosensory cells form and proliferate properly in the absence of Hey1 and Hey2 but differentiate prematurely because of precocious upregulation of the pro-HC factor Atoh1. Moreover, we demonstrate that prosensory-specific expression of Hey1 and Hey2 and its subsequent graded downregulation is controlled by Hedgehog signaling in a largely FGFR-dependent manner. In summary, our study reveals a critical role for Hey1 and Hey2 in prosensory cell maintenance and identifies Hedgehog signaling as a novel upstream regulator of their prosensory function in the mammalian cochlea. The regulatory mechanism described here might be a broadly applied mechanism for controlling progenitor behavior in the central and peripheral nervous system.

Hey1 and Hey2 control the spatial and temporal pattern of mammalian auditory hair cell differentiation downstream of Hedgehog signaling.

Proper tissue development requires strict coordination of proliferation, growth, and differentiation. Strict coordination is particularly important for the auditory sensory epithelium, where deviations from the normal spatial and temporal pattern of auditory progenitor cell (prosensory cell) proliferation and differentiation result in abnormal cellular organization and, thus, auditory dysfunction. The molecular mechanisms involved in the timing and coordination of auditory prosensory proliferation and differentiation are poorly understood. Here we identify the RNA-binding protein LIN28B as a critical regulator of developmental timing in the murine cochlea. We show that Lin28b and its opposing let-7 miRNAs are differentially expressed in the auditory sensory lineage, with Lin28b being highly expressed in undifferentiated prosensory cells and let-7 miRNAs being highly expressed in their progeny-hair cells (HCs) and supporting cells (SCs). Using recently developed transgenic mouse models for LIN28B and let-7g, we demonstrate that prolonged LIN28B expression delays prosensory cell cycle withdrawal and differentiation, resulting in HC and SC patterning and maturation defects. Surprisingly, let-7g overexpression, although capable of inducing premature prosensory cell cycle exit, failed to induce premature HC differentiation, suggesting that LIN28B's functional role in the timing of differentiation uses let-7 independent mechanisms. Finally, we demonstrate that overexpression of LIN28B or let-7g can significantly alter the postnatal production of HCs in response to Notch inhibition; LIN28B has a positive effect on HC production, whereas let-7 antagonizes this process. Together, these results implicate a key role for the LIN28B/let-7 axis in regulating postnatal SC plasticity.

The RNA-binding protein LIN28B regulates developmental timing in the mammalian cochlea.

The auditory sensory epithelium, composed of mechano-sensory hair cells (HCs) and highly specialized glial-like supporting cells (SCs), is critical for our ability to detect sound. SCs provide structural and functional support to HCs and play an essential role in cochlear development, homeostasis and repair. Despite their importance, however, surprisingly little is known about the molecular mechanisms guiding SC differentiation. Here, we provide evidence that in addition to its well-characterized inhibitory function, canonical Notch signaling plays a positive, instructive role in the differentiation of SCs. Using γ-secretase inhibitor DAPT to acutely block canonical Notch signaling, we identified a cohort of Notch-regulated SC-specific genes, with diverse functions in cell signaling, cell differentiation, neuronal innervation and synaptogenesis. We validated the newly identified Notch-regulated genes in vivo using genetic gain (Emx2(Cre/+); Rosa26(N1ICD/+)) and loss-of-function approaches (Emx2(Cre/+); Rosa26(DnMAML1/+)). Furthermore, we demonstrate that Notch over-activation in the differentiating murine cochlea (Emx2(Cre/+); Rosa26(N1ICD/+)) actively promotes a SC-specific gene expression program. Finally, we show that outer SCs -so called Deiters' cells are selectively lost by prolonged reduction (Emx2(Cre/+); Rosa26(DnMAML1/+/+)) or abolishment of canonical Notch signaling (Fgfr3-iCreER; Rbpj(-/Δ)), indicating a critical role for Notch signaling in Deiters' cell development.

Article	Year
Alternate activation of two divergently transcribed mouse genes from a bidirectional promoter is linked to changes in histone modification. The Journal of biological chemistry\| PubMed ID: 12411446	2003
The tumor suppressor p53 and histone deacetylase 1 are antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 gene. Molecular and cellular biology\| PubMed ID: 12665570	2003
Math1-driven GFP expression in the developing nervous system of transgenic mice. Gene expression patterns : GEP\| PubMed ID: 12915300	2003
In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme. Developmental biology\| PubMed ID: 15282159	2004
Prospective identification and purification of hair cell and supporting cell progenitors from the embryonic cochlea. Brain research\| PubMed ID: 16616734	2006
Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. Nature\| PubMed ID: 16791196	2006
p19(Ink4d) and p21(Cip1) collaborate to maintain the postmitotic state of auditory hair cells, their codeletion leading to DNA damage and p53-mediated apoptosis. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 17287518	2007
Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti. Developmental cell\| PubMed ID: 19154718	2009
Notch signaling limits supporting cell plasticity in the hair cell-damaged early postnatal murine cochlea. PloS one\| PubMed ID: 24023676	2013
Hey1 and Hey2 control the spatial and temporal pattern of mammalian auditory hair cell differentiation downstream of Hedgehog signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 25232121	2014
The RNA-binding protein LIN28B regulates developmental timing in the mammalian cochlea. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 26139524	2015
Canonical Notch signaling plays an instructive role in auditory supporting cell development. Scientific reports\| PubMed ID: 26786414	2016

Angelika Doetzlhofer

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