Dysfunction of programmed embryo senescence is linked to genetic developmental defects.

Developmental senescence is a form of programmed senescence that contributes to morphogenesis during embryonic development. We showed recently that the SIX1 homeoprotein, an essential regulator of organogenesis, is also a repressor of adult cellular senescence. Alterations in the SIX/EYA pathway are linked to the human branchio-oto-renal (BOR) syndrome, a rare congenital disorder associated with defects in the ears, kidneys and branchial arches. Here, we have used Six1-deficient mice, an animal model of the BOR syndrome, to investigate whether dysfunction of senescence underpins the developmental defects associated with SIX1 deficiency. We have focused on the developing inner ear, an organ with physiological developmental senescence that is severely affected in Six1-deficient mice and BOR patients. We show aberrant levels and distribution of senescence markers in Six1-deficient inner ears concomitant with defective morphogenesis of senescent structures. Transcriptomic analysis and ex vivo assays support a link between aberrant senescence and altered morphogenesis in this model, associated with deregulation of the TGFß/BMP pathway. Our results show that misregulation of embryo senescence may lead to genetic developmental disorders, significantly expanding the connection between senescence and disease.

Ceramide Kinase Inhibition Blocks IGF-1-Mediated Survival of Otic Neurosensory Progenitors by Impairing AKT Phosphorylation.

Sphingolipids are bioactive lipid components of cell membranes with important signal transduction functions in health and disease. Ceramide is the central building block for sphingolipid biosynthesis and is processed to form structurally and functionally distinct sphingolipids. Ceramide can be phosphorylated by ceramide kinase (CERK) to generate ceramide-1-phosphate, a cytoprotective signaling molecule that has been widely studied in multiple tissues and organs, including the developing otocyst. However, little is known about ceramide kinase regulation during inner ear development. Using chicken otocysts, we show that genes for CERK and other enzymes of ceramide metabolism are expressed during the early stages of inner ear development and that CERK is developmentally regulated at the otic vesicle stage. To explore its role in inner ear morphogenesis, we blocked CERK activity in organotypic cultures of otic vesicles with a specific inhibitor. Inhibition of CERK activity impaired proliferation and promoted apoptosis of epithelial otic progenitors. CERK inhibition also compromised neurogenesis of the acoustic-vestibular ganglion. Insulin-like growth factor-1 (IGF-1) is a key factor for proliferation, survival and differentiation in the chicken otocyst. CERK inhibition decreased IGF-1-induced AKT phosphorylation and blocked IGF-1-induced cell survival. Overall, our data suggest that CERK is activated as a central element in the network of anti-apoptotic pro-survival pathways elicited by IGF-1 during early inner ear development.

Otic Neurogenesis Is Regulated by TGFß in a Senescence-Independent Manner.

Cellular senescence has classically been associated with aging. Intriguingly, recent studies have also unraveled key roles for senescence in embryonic development, regeneration, and reprogramming. Developmental senescence has been reported during embryonic development in different organisms and structures, such as the endolymphatic duct during inner ear development of mammals and birds. However, there is no study addressing the possible role of senescence on otic neurogenesis. TGFß/SMAD is the best-known pathway linked to the induction of developmentally programmed cell senescence. Here, we studied if TGFß2 induces cellular senescence during acoustic-vestibular-ganglion (AVG) formation. Using organotypic cultures of AVG, and characterizing different stages of otic neurogenesis in the presence of TGFß2 and a selective TGF-ß receptor type-I inhibitor, we show that TGFß2 exerts a powerful action in inner ear neurogenesis but, contrary to what we recently observed during endolymphatic duct development, these actions are independent of cellular senescence. We show that TGFß2 reduces proliferation, and induces differentiation and neuritogenesis of neuroblasts, without altering cell death. Our studies highlight the roles of TGFß2 and cellular senescence in the precise regulation of cell fate within the developing inner ear and its different cell types, being their mechanisms of action highly cell-type dependent.

TGFß2-induced senescence during early inner ear development.

Embryonic development requires the coordinated regulation of apoptosis, survival, autophagy, proliferation and differentiation programs. Senescence has recently joined the cellular processes required to master development, in addition to its well-described roles in cancer and ageing. Here, we show that senescent cells are present in a highly regulated temporal pattern in the developing vertebrate inner ear, first, surrounding the otic pore and, later, in the otocyst at the endolymphatic duct. Cellular senescence is associated with areas of increased apoptosis and reduced proliferation consistent with the induction of the process when the endolymphatic duct is being formed. Modulation of senescence disrupts otic vesicle morphology. Transforming growth factor beta (TGFß) signaling interacts with signaling pathways elicited by insulin-like growth factor type 1 (IGF-1) to jointly coordinate cellular dynamics required for morphogenesis and differentiation. Taken together, these results show that senescence is a natural occurring process essential for early inner ear development.

Complementary and distinct roles of autophagy, apoptosis and senescence during early inner ear development.

The development of the inner ear complex cytoarchitecture and functional geometry requires the exquisite coordination of a variety of cellular processes in a temporal manner. At early stages of inner ear development several rounds of cell proliferation in the otocyst promote the growth of the structure. The apoptotic program is initiated in exceeding cells to adjust cell type numbers. Apoptotic cells are cleared by phagocytic cells that recognize the phosphatidylserine residues exposed in the cell membrane thanks to the energy supplied by autophagy. Specific molecular programs determine hair and supporting cell fate, these populations are responsible for the functions of the adult sensory organ: detection of sound, position and acceleration. The neurons that transmit auditory and balance information to the brain are also born at the otocyst by neurogenesis facilitated by autophagy. Cellular senescence participates in tissue repair, cancer and aging, situations in which cells enter a permanent cell cycle arrest and acquire a highly secretory phenotype that modulates their microenvironment. More recently, senescence has also been proposed to take place during vertebrate development in a limited number of transitory structures and organs; among the later, the endolymphatic duct in the inner ear. Here, we review these cellular processes during the early development of the inner ear, focusing on how the most recently described cellular senescence participates and cooperates with proliferation, apoptosis and autophagy to achieve otic morphogenesis and differentiation.

Autophagy in the Vertebrate Inner Ear.

Autophagy is a conserved catabolic process that results in the lysosomal degradation of cell components. During development, autophagy is associated with tissue and organ remodeling, and under physiological conditions it is tightly regulated as it plays a housekeeping role in removing misfolded proteins and damaged organelles. The vertebrate inner ear is a complex sensory organ responsible for the perception of sound and for balance. Cell survival, death and proliferation, as well as cell fate specification and differentiation, are processes that are strictly coordinated during the development of the inner ear in order to generate the more than a dozen specialized cell types that constitute this structure. Here, we review the existing evidence that implicates autophagy in the generation of the vertebrate inner ear. At early stages of chicken otic development, inhibiting autophagy impairs neurogenesis and causes aberrant otocyst morphogenesis. Autophagy provides energy for the clearing of dying cells and it favors neuronal differentiation. Moreover, autophagy is required for proper vestibular development in the mouse inner ear. The autophagy-related genes Becn1, Atg4g, Atg5, and Atg9, are expressed in the inner ear from late developmental stages to adulthood, and Atg4b mutants show impaired vestibular behavior associated to defects in otoconial biogenesis that are also common to Atg5 mutants. Autophagic flux appears to be age-regulated, augmenting from perinatal stages to young adulthood in mice. This up-regulation is concomitant with the functional maturation of the hearing receptor. Hence, autophagy can be considered an intracellular pathway fundamental for in vertebrate inner ear development and maturation.

Age-regulated function of autophagy in the mouse inner ear.

Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles during early development in the chicken embryo and in the response to otic injury in the adult mouse. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation. We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the autophagy machinery expression in the inner ear is regulated with age but is not compromised by the chronic absence of IGF-1. Our data also strongly support that the up-regulation of autophagy machinery genes is concomitant with the functional maturation of the inner ear.

C-Raf deficiency leads to hearing loss and increased noise susceptibility.

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K(+) channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf (+/-) mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.

Early development of the vertebrate inner ear.

This is a review of the biological processes and the main signaling pathways required to generate the different otic cell types, with particular emphasis on the actions of insulin-like growth factor I. The sensory organs responsible of hearing and balance have a common embryonic origin in the otic placode. Lineages of neural, sensory, and support cells are generated from common otic neuroepithelial progenitors. The sequential generation of the cell types that will form the adult inner ear requires the coordination of cell proliferation with cell differentiation programs, the strict regulation of cell survival, and the metabolic homeostasis of otic precursors. A network of intracellular signals operates to coordinate the transcriptional response to the extracellular input. Understanding the molecular clues that direct otic development is fundamental for the design of novel treatments for the protection and repair of hearing loss and balance disorders.

AKT signaling mediates IGF-I survival actions on otic neural progenitors.

BACKGROUND: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts. METHODOLOGY/PRINCIPAL FINDINGS: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27(Kip)-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC. CONCLUSIONS/SIGNIFICANCE: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development.

Autophagy during vertebrate development.

Autophagy is an evolutionarily conserved catabolic process by which cells degrade their own components through the lysosomal machinery. In physiological conditions, the mechanism is tightly regulated and contributes to maintain a balance between synthesis and degradation in cells undergoing intense metabolic activities. Autophagy is associated with major tissue remodeling processes occurring through the embryonic, fetal and early postnatal periods of vertebrates. Here we survey current information implicating autophagy in cellular death, proliferation or differentiation in developing vertebrates. In developing systems, activation of the autophagic machinery could promote different outcomes depending on the cellular context. Autophagy is thus an extraordinary tool for the developing organs and tissues.

RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development.

BACKGROUND: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. CONCLUSIONS/SIGNIFICANCE: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.

Squeeze involvement in the specification of Drosophila leucokinergic neurons: Different regulatory mechanisms endow the same neuropeptide selection.

One of the most widely studied phenomena in the establishment of neuronal identity is the determination of neurosecretory phenotype, in which cell-type-specific combinatorial codes direct distinct neurotransmitter or neuropeptide selection. However, neuronal types from divergent lineages may adopt the same neurosecretory phenotype, and it is unclear whether different classes of neurons use different or similar components to regulate shared features of neuronal identity. We have addressed this question by analyzing how differentiation of the Drosophila larval leucokinergic system, which is comprised of only four types of neurons, is regulated by factors known to affect expression of the FMRFamide neuropeptide. We show that all leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on LK expression of loss- and gain-of-function mutations, we can describe three types of Lk regulation. In the brain LHLK cells, both Sqz and Apterous (Ap) are required for LK expression, but surprisingly, high levels of either Sqz or Ap alone are sufficient to restore LK expression in these neurons. In the suboesophageal SELK cells, Sqz, but not Ap, is required for LK expression. In the abdominal ABLK neurons, inhibition of retrograde axonal transport reduces LK expression, and although sqz is dispensable for LK expression in these cells, it can induce ectopic leucokinergic ABLK-like cells when over-expressed. Thus, Sqz appears to be a regulatory factor for neuropeptidergic identity common to all leucokinergic cells, whose function in different cell types is regulated by cell-specific factors.

Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development.

Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. Here we analyze the role of nab in Drosophila development. By employing genetic approaches we found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. We identified two partners of Nab: the zinc-finger transcription factors Rotund and Squeeze. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. Our results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. We also showed by pull-down assays that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. We propose two mechanisms by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing.

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway.

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.

Neurosecretory identity conferred by the apterous gene: lateral horn leucokinin neurons in Drosophila.

The LIM-HD protein Apterous has been shown to regulate expression of the FMRFamide neuropeptide in Drosophila neurons (Benveniste et al. [1998] Development 125:4757-4765). To test whether Apterous has a broader role in controlling neurosecretory identity, we analyzed the expression of several neuropeptides in apterous (ap) mutants. We show that Apterous is necessary for expression of the Leucokinin neuropeptide in a pair of brain neurons located in the lateral horn region of the protocerebrum (LHLK neurons). ap null mutants are depleted of Leucokinin in these cells, whereas hypomorphic mutants show reduced Leucokinin expression. Other Leucokinin-containing neurons are not affected by mutations in ap gene. Co-expression of apterous and Leucokinin is observed exclusively in the LHLK neurons, from larval stages to adulthood. Rescue assays performed in null ap mutants, by expressing Apterous protein under apGAL4 and elavGAL4 drivers, demonstrate the recovery of Leucokinin in the LHLK neurons. These results reinforce the emerging role of the LIM-HD proteins in determining neuronal identity. They also clarify the neuroendocrine phenotype of apterous mutants.