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[Background]: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. [Results]: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. [Conclusions]: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival., Consejería de Educación, Junta de Castilla y León, Grant/Award Number: CSI143P20; Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León, Grant/Award Numbers: CCVC8485, CLU-2019-02; MEC, Grant/Award Number: BFU2004-00860/BFI

Glial‐derived neurotrophic factor (GDNF) has been proposed as a potent neurotrophic factor with the potential to cure neurodegenerative diseases. In the cochlea, GDNF has been detected in auditory neurons and sensory receptor cells and its expression is upregulated upon trauma. Moreover, the application of GDNF in different animal models of deafness has shown its capacity to prevent hearing loss and promoted its future use in therapeutic trials in humans. In the present study we have examined the endogenous requirement of GDNF during auditory development in mice. Using a lacZ knockin allele we have confirmed the expression of GDNF in the cochlea including its sensory regions during development. Global inactivation of GDNF throughout the hearing system using a Foxg1‐Cre line causes perinatal lethality but reveals no apparent defects during formation of the cochlea. Using TrkC‐Cre and Atoh1‐Cre lines, we were able to generate viable mutants lacking GDNF in auditory neurons or both auditory neurons and sensory hair cells. These mutants show normal frequency‐dependent auditory thresholds. However, mechanoelectrical response properties of outer hair cells (OHCs) in TrkC‐Cre GDNF mutants are altered at low thresholds. Furthermore, auditory brainstem wave analysis shows an abnormal increase of wave I. On the other hand, Atoh1‐Cre GDNF mutants show normal OHC function but their auditory brainstem wave pattern is reduced at the levels of wave I, III and IV. These results show that GDNF expression during the development is required to maintain functional hearing at different levels of the auditory system., This work was supported by Ciberned, TerCel (RD06/0010/0000) and Red de Terapia Célular de la Junta de Castilla y León.

Numerous studies indicate that deficits in the proper integration or migration of specific GABAergic precursor cells from the subpallium to the cortex can lead to severe cognitive dysfunctions and neurodevelopmental pathogenesis linked to intellectual disabilities. A different set of GABAergic precursors cells that express Pax2 migrate to hindbrain regions, targeting, for example auditory or somatosensory brainstem regions. We demonstrate that the absence of BDNF in Pax2-lineage descendants of BdnfPax2KOs causes severe cognitive disabilities. In BdnfPax2KOs, a normal number of parvalbumin-positive interneurons (PV-INs) was found in the auditory cortex (AC) and hippocampal regions, which went hand in hand with reduced PV-labeling in neuropil domains and elevated activity-regulated cytoskeleton-associated protein (Arc/Arg3.1; here: Arc) levels in pyramidal neurons in these same regions. This immaturity in the inhibitory/excitatory balance of the AC and hippocampus was accompanied by elevated LTP, reduced (sound-induced) LTP/LTD adjustment, impaired learning, elevated anxiety, and deficits in social behavior, overall representing an autistic-like phenotype. Reduced tonic inhibitory strength and elevated spontaneous firing rates in dorsal cochlear nucleus (DCN) brainstem neurons in otherwise nearly normal hearing BdnfPax2KOs suggests that diminished fine-grained auditory-specific brainstem activity has hampered activity-driven integration of inhibitory networks of the AC in functional (hippocampal) circuits. This leads to an inability to scale hippocampal post-synapses during LTP/LTD plasticity. BDNF in Pax2-lineage descendants in lower brain regions should thus be considered as a novel candidate for contributing to the development of brain disorders, including autism.

© 2020 by the authors., Age-related decoupling of auditory nerve fibers from hair cells (cochlear synaptopathy) has been linked to temporal processing deficits and impaired speech recognition performance. The link between both is elusive. We have previously demonstrated that cochlear synaptopathy, if centrally compensated through enhanced input/output function (neural gain), can prevent age-dependent temporal discrimination loss. It was also found that central neural gain after acoustic trauma was linked to hippocampal long-term potentiation (LTP) and upregulation of brain-derived neurotrophic factor (BDNF). Using middle-aged and old BDNF-live-exon-visualization (BLEV) reporter mice we analyzed the specific recruitment of LTP and the activity-dependent usage of Bdnf exon-IV and -VI promoters relative to cochlear synaptopathy and central (temporal) processing. For both groups, specimens with higher or lower ability to centrally compensate diminished auditory nerve activity were found. Strikingly, low compensating mouse groups differed from high compensators by prolonged auditory nerve latency. Moreover, low compensators exhibited attenuated responses to amplitude-modulated tones, and a reduction of hippocampal LTP and Bdnf transcript levels in comparison to high compensators. These results suggest that latency of auditory nerve processing, recruitment of hippocampal LTP, and Bdnf transcription, are key factors for age-dependent auditory processing deficits, rather than cochlear synaptopathy or aging per se., We acknowledge grants from the Deutsche Forschungsgemeinschaft FOR 2060 project RU 713/3-2 (W.S., L.R., D.M.), Projektnummer 335549539/GRK 2381 (P.M.), SPP 1608 RU 316/12-1 (L.R.), and KN 316/12-1 (M.M, M.K.), Siegmund Kiener Stiftung (D.S.) and BFU2016-76580-P (T.S).

Myc is considered an essential transcription factor for heart development, but cardiac defects have only been studied in global Myc loss-of-function models. Here, we eliminated Myc by recombining a Myc floxed allele with the Nkx2.5Cre driver. We observed no anatomical, cellular or functional alterations in either fetuses or adult cardiac Myc-deficient mice. We re-examined Myc expression during development and found no expression in developing cardiomyocytes. In contrast, we confirmed that Mycn is essential for cardiomyocyte proliferation and cardiogenesis. Mosaic Myc overexpression in a Mycn-deficient background shows that Myc can replace Mycn function, recovering heart development. We further show that this recovery involves the elimination of Mycn-deficient cells by cell competition. Our results indicate that Myc is dispensable in cardiomyocytes both during cardiogenesis and for adult heart homeostasis, and that Mycn is exclusively responsible for cardiomyocyte proliferation during heart development. Nonetheless, our results show that Myc can functionally replace Mycn We also show that cardiomyocytes compete according to their combined Myc and Mycn levels and that cell competition eliminates flawed cardiomyocytes, suggesting its relevance as a quality control mechanism in cardiac development., This work is supported by a grant from the Fondation Leducq [‘Redox Regulation of Cardiomyocyte Renewal’ 17CVD04] and by grants from the Ministerio de Ciencia, Innovación y Universidades [BFU2015-71519-P and RD16/0011/0019 (ISCIII)]. N.M.-M. was supported by a pre-doctoral contract from “la Caixa” Foundation [LACAIXA-SO14]. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

Myc is considered an essential transcription factor for heart development, but cardiac defects have only been studied in global Myc loss of function models. Here, we eliminated Myc by recombining aMycfloxed allele with theNkx2.5Credriver. We observed no anatomical, cellular or functional alterations in either fetuses or adult cardiac Myc-deficient mice. We re-examined Myc expression during development and found no expression in developing cardiomyocytes. In contrast, we confirmed thatMycnis essential for cardiomyocyte proliferation and cardiogenesis. Mosaic Myc overexpression in aMycn-deficient background, shows that Myc can replace Mycn function, recovering heart development. We further show that this recovery involves the elimination of Mycn-deficient cells by Cell Competition. Our results indicate thatMycis dispensable during cardiogenesis and adult heart homeostasis andMycnis exclusively responsible for cardiomyocyte proliferation during heart development. Nonetheless, our results show thatMyccan functionally replaceMycn. We also show that cardiomyocytes compete according to their overall Myc+Mycn levels and that Cell Competition eliminates flawed cardiomyocytes, suggesting its relevance as a quality control mechanism in cardiac development.

El pdf del artículo es la versión pre-print., Multiple Fgfs are expressed during formation and patterning of the telencephalon in vertebrates. Fgf8 has been shown to control the size of the telencephalon and the development of signaling centers in zebrafish and mouse. Next to Fgf8, Fgf3 also influences telencephalic gene expression in the zebrafish. Moreover, Fgf3 and Fgf8 have been shown to have combinatorial functions during forebrain development in this species. Here, we have examined telencephalic development in Fgf3 null mouse mutants and embryos that lack both Fgf3 and Fgf8 in their forebrain. In contrast to zebrafish, Fgf3 mutants show normal forebrain development and expression of telencephalic marker genes. Although double mutants for Fgf3 and Fgf8 show a further reduction of forebrain size no additional changes of telencephalic gene expression are observed compared with Fgf8 mutants. Therefore unlike in zebrafish, Fgf3 is not required for mouse forebrain development whereas Fgf8 has a central role during this process. © 2008 Wiley-Liss, Inc., We acknowledge the support of the Spanish Ministry of Education (BFU2007-60130), Ciberned, TerCel, and the Junta of Castilla y León to T.S and E.D-F.

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis. © 2008 Elsevier Inc. All rights reserved., This work was supported by the grant BFU2006-05604 from the Spanish Ministry of Education and Science to C.P. and BFU2007-61030, Tercel, Ciberned and Junta de Castilla y León to T.S. C.V.E. was supported by a FI fellowship from AGAUR (Generalitat de Catalunya), and by CONACYT (Mexico); E.D.F. was supported by a FPI fellowship from the MEC, Spain.

The identification of transcriptional differences has served as an important starting point in understanding the molecular mechanisms behind biological processes and systems. The developmental biology of the inner ear, the biology of hearing and of course the pathology of deafness are all processes that warrant a molecular description if we are to improve human health. To this end, technological innovation has meant that larger scale analysis of gene transcription has been possible for a number of years now, extending our molecular analysis of genes to beyond those that are currently in vogue for a given system. In this review, some of the contributions gene profiling has made to understanding developmental, pathological and physiological processes in the inner ear are highlighted., This work was supported by the Spanish MinEco (BFU2010-15477, BFU2013-40944), Fundació La Marató de TV3, TerCel (RD06/ 0010/0000) and Red de Terapia Cèular de la Junta de Castilla y León.

A decline in neuronal plasticity during the adult life span has been proposed to be associated with a reduced level of the effectors of plasticity responses (e.g., BDNF). Alteration of plasticity is also correlated with age-related hearing loss (presbycusis), but to date no detailed studies of BDNF expression have been performed in the young or aging mature cochlea. We have used rat and gerbil animal models for presbycusis, which displayed hearing loss in the final third of the animals' natural life span. We demonstrate for the first time a co-localization of BDNF protein, transcripts III and IV in cochlear neurons with a declining distribution towards low-frequency processing cochlear turns. BDNF protein was also found within the neuronal projections of the cochlea. A significant reduction of BDNF transcripts in high-frequency processing cochlear neurons was observed during aging, though this did not coincide with a major reduction of BDNF protein. In contrast, BDNF protein in peripheral and central projections was drastically reduced. Our results suggest that reduced BDNF protein levels in auditory nerves over age may be a crucial factor in the altered brainstem plasticity observed during presbycusis. © 2006 Elsevier Inc. All rights reserved., This work was supported by the Deutsche Forschungsgemeinschaft DFG-SFB444, DFG Kni 316/3-2 and Fortüne Kni-F1251236, Fortüne Kni-F12512 79, Fortüne 816-0-0 and the Spanish MEC.

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re- specification of non-sensory areas into sensory epithelia, and the loss of Reissner’s membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror- image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea., This work was supported by the Spanish MinEco [BFU2010-15477, BFU2011-24057, BFU2013-40944]; Fundació La Maratód e TV3; TerCel [RD06/0010/0000]; and Red de Terapia Célular de la Junta de Castilla y León.

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease., This work was supported by the Center for Cancer Research of the Intramural Research Program of the National Institutes of Health through the National Cancer Institute.

Fibroblast growth factor-2 (FGF2) has been postulated to be a key regulator involved in the proliferation, differentiation, and regeneration of sensory hair cells. Here we have addressed the potential functions of FGF2 during the formation and regeneration of the auditory epithelium in chicken and mice. By using viral gene transfer, based on herpes simplex type 1 virus (HSV-1), we show that ectopically applied FGF2 drastically increases the number of cells expressing early hair cell markers during embryonic development in avians. Intriguingly, FGF2 does not stimulate cell division during this process. These data suggest that FGF2 plays a role during differentiation of sensory hair cells in avians. To address the potential functions of FGF2 during murine inner ear development, we analyzed FGF2 mouse mutants. Mice lacking FGF2 showed normal formation of the inner ear, and no abnormalities were observed at the adult stage. Moreover, FGF2 mouse mutants showed similar hearing thresholds compared with those observed in control mice before and after noise damage. Therefore, endogenous FGF2 appears not to be essential for the development or functional maintenance of the auditory organ in mammals. In light of these results, the differential roles of FGF2 in the vertebrate inner ear are discussed with respect to its previously postulated functions.

The otic vesicle (otocyst) occupies a pivotal position in inner ear development, bridging the gap between otic placode determination, and morphogenesis of vestibular and auditory compartments. The molecular mechanisms underlying the progressive subdivision of the developing inner ear into different compartments, and the molecular control and execution of the different developmental processes involved, are largely unknown. Since relatively few genes have been implicated in these processes, we have undertaken this study to identify genes involved in these early embryonic stages. We have used cDNA subtractions of mouse otic vesicle against adult liver cDNA, and describe a set of 280 candidate genes. We have also performed otic vesicle RNA hybridizations against DNA chips to not only confirm the efficacy of the library approach, but also to investigate the utility of DNA array alternatives. To begin to dissect potential developmental roles, we investigated the spatial pattern of gene expression for a selected set of 80 genes in developing mouse embryos at mid-gestation by whole-mount in situ hybridization. These data illustrate the compartmentalisation of gene expression in the otic vesicle for the majority of genes tested, and furthermore, implicate many of the genes tested with distinct developmental subprocesses.

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea,whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea,which may also be of relevance for treatment of hearing loss in humans.

El pdf del artículo es la versión pre-print.-- et al., Neurotrophin-3 (NT-3) is one of the most potent stimulators for survival of auditory sensory neurons. Viral transfer of neurotrophins into auditory neurons may offer a route to provide a permanent supply of the growth factor and guarantee their long-term survival. Herpes simplex virus type 1 (HSV-1)-based vectors have demonstrated their effectiveness to transfer genes into peripheral sensory neurons. In the present report, we have produced a HSV-1-based amplicon vector expressing NT-3. This vector efficiently infects isolated auditory neurons and stimulates their survival during distinct developmental stages of the inner ear. Therefore, this vector may present a unique entry point to develop therapies preventing or treating hearing impairment caused by the degeneration of auditory neurons. © 2002 Elsevier Science Ireland Ltd. All rights reserved., This research was supported by DGCYT and the Junta of Castilla y León.

The aim of gene transfer to the cochlea and vestibular organ is to protect the inner ear from different disorders. Although various vectors for gene delivery have been used with some success, there remains a need for a reliable transfer of genes into the inner ear without damaging cochlear function. Here, we have tested a novel application method for gene transfer into the rat inner ear in vivo using herpes simplex virus type-1(HSV-1)-based amplicon vectors. Our goal was to find an entry route into the inner ear that leaves its function intact. Besides other non-invasive and invasive application techniques, we applied the viral vector via injection through a small opening of the utriculus. Using this method, efficient β-galactosidase reporter gene expression was achieved in nearly all neurons in the vestibulum and cochlea, without functional hearing deficits. At the time point of maximal expression 5 days after injection, β-galactosidase activity was also observed in axonal fibres and synaptic endings close to inner and outer hair cells. Our results thus describe an efficient and reliable protocol for short-term expression of potential therapeutic genes in the neuronal compartment of the inner ear.

Brain-derived neurotrophic factor, BDNF, is one of the most important neurotrophic factors acting in the peripheral and central nervous system. In the auditory system its function was initially defined by using constitutive knockout mouse mutants and shown to be essential for survival of neurons and afferent innervation of hair cells in the peripheral auditory system. Further examination of BDNF null mutants also revealed a more complex requirement during re-innervation processes involving the efferent system of the cochlea. Using adult mouse mutants defective in BDNF signaling, it could be shown that a tonotopical gradient of BDNF expression within cochlear neurons is required for maintenance of a specific spatial innervation pattern of outer hair cells and inner hair cells. Additionally, BDNF is required for maintenance of voltage-gated potassium channels (KV) in cochlear neurons, which may form part of a maturation step within the ascending auditory pathway with onset of hearing and might be essential for cortical acuity of sound-processing and experience-dependent plasticity. A presumptive harmful role of BDNF during acoustic trauma and consequences of a loss of cochlear BDNF during aging are discussed in the context of a partial reversion of this maturation step. We compare the potentially beneficial and harmful roles of BDNF for the mature auditory system with those BDNF functions known in other sensory circuits, such as the vestibular, visual, olfactory, or somatosensory system.

The importance of individual members of the fibroblast growth factor gene family during innervation of the vertebrate inner ear is not clearly defined. Here we address the role of fibroblast growth factor 2 (FGF-2 or basic FGF) during development of the chicken inner ear. We found that FGF-2 stimulated survival of isolated cochlear and vestibular neurons during distinct phases of inner ear innervation. The potential neurotrophic role of FGF-2 was confirmed by its expression in the corresponding sensory epithelia and the detection of one of its high-affinity receptors in inner ear neurons. Finally, we have analysed the potential of the amplicon system based on defective herpes simplex virus type 1 (HSV-1) vectors to express FGF-2 in cochlear neurons. Overexpression of FGF-2 in cochlear neurons resulted in neuronal differentiation demonstrating the presence of biologically active growth factor. This study underlines the potential of FGF-2 to control innervation and development of sensory epithelia in the avian inner ear. Furthermore, amplicon vectors may provide a useful tool to analyse gene function in isolated neurons of the vertebrate inner ear.

Various mutations of the hairless (hr) gene of mice result in hair loss and other integument defects. To examine the role of the hr gene in mouse development, the expression profile of hr has been determined by in situ hybridisation and correlated to the nature of genetic changes and morphological abnormalities in different mutant animals. Four variant alleles have been characterised at the molecular level. hr/hr mice produce reduced, but significant, levels of hr mRNA whereas other alleles contain mutations which would be expected to preclude the synthesis of functional product, demonstrating a correlation between allelic variation at the hr locus and phenotypic severity. hr expression was shown to be widespread and temporally regulated. It was identified in novel tissues such as cartilage, developing tooth, inner ear, retina, and colon as well as in skin and brain. Analysis of mice homozygous for the rhino allele of hairless revealed that, although no morphological defects were detectable in many tissues normally expressing hr, previously undescribed abnormalities were present in several tissues including inner ear, retina, and colon. These findings indicate that the hairless gene product plays a wider role in development than previously suspected. Dev Dyn 1999;216:113-126.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License., N-myc belongs to the myc proto-oncogene family, which is involved in numerous cellular processes such as proliferation, growth, apoptosis, and differentiation. Conditional deletion of N-myc in the mouse nervous system disrupted brain development, indicating that N-myc plays an essential role during neural development. How the development of the olfactory epithelium and neurogenesis within are affected by the loss of N-myc has, however, not been determined. To address these issues, we examined an N-mycFoxg1Cre conditional mouse line, in which N-myc is depleted in the olfactory epithelium. First changes in N-myc mutants were detected at E11.5, with reduced proliferation and neurogenesis in a slightly smaller olfactory epithelium. The phenotype was more pronounced at E13.5, with a complete lack of Hes5-positive progenitor cells, decreased proliferation, and neurogenesis. In addition, stereological analyses revealed reduced cell size of post-mitotic neurons in the olfactory epithelium, which contributed to a smaller olfactory pit. Furthermore, we observed diminished proliferation and neurogenesis also in the vomeronasal organ, which likewise was reduced in size. In addition, the generation of gonadotropin-releasing hormone neurons was severely reduced in N-myc mutants. Thus, diminished neurogenesis and proliferation in combination with smaller neurons might explain the morphological defects in the N-myc depleted olfactory structures. Moreover, our results suggest an important role for N-myc in regulating ongoing neurogenesis, in part by maintaining the Hes5-positive progenitor pool. In summary, our results provide evidence that N-myc deficiency in the olfactory epithelium progressively diminishes proliferation and neurogenesis with negative consequences at structural and cellular levels. © The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc., Contract grant sponsor: The Swedish Research Council, Umeå University , Märta Lundqvist foundation and the Kempe foundation. Contract grant sponsor: Spanish MinEco; contract grant number: BFU2010–15477.

Analysis of trkB−/−; trkC−/− double mutant mice revealed that peripheral and central inner ear sensory neurons are affected in these mice. However, a substantial amount of cochlear and vestibular neurons survive, possibly due to maintenance or upregulation of TrkA expression. To clarify the function of the TrkA receptor during development of the cochlear and vestibular ganglion we analysed trkA−/− mice and the expression of this receptor in inner ear sensory neurons of trkB−/−; trkC−/− animals. TrkA homozygous mutant mice showed normal numbers of neurons and no TrkA expression was detected in neurons of trkB−/−; trkC−/− double mutant mice. We conclude that TrkA is not essential for inner ear development and that in the absence of any of the known catalytic Trk receptors peripheral inner ear sensory neurons are prone to undergo cell death or must use a different signaling mechanism to survive.

12 páginas, 6 figuras.-- et al., Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27Kip1 and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear., This work was supported by Ministerio de Ciencia e Innovación Grants BFU2010-15477 and PLE 2009-0098, Ciberned, TerCel, and Red de Terapia Célular de Castilla y León to T.S., and Grant R37CA57138 to R.N.E.

Cell cycle exit and acquirement of a postmitotic state is essential for the proper development of organs. In the present review, we examine the role of the cell cycle control in the sensory epithelia of the mammalian inner ear. We describe the roles of the core cell cycle regulators in the proliferation of prosensory cells and in the initiation and maintenance of terminal mitosis of the sensory epithelia. We also discuss how other intracellular signalling may influence the cell cycle. Finally, we address the question of whether manipulations of the cell cycle may have the potential to create replacement cells for the damaged inner sensory epithelia. © 2013 Elsevier Ltd., The work of the authors was supported by grants from the Academy of Finland and Jane and Aatos Erkko Foundation (U.P.), and the Spanish MinEco (BFU2010-15477, PLE-2009-0098), TerCel (RD06/0010/0000) and Red de Terapia Célular de la Junta de Castilla y León (T.S.).

Transcription factors (TFs) participate during various processes throughout inner ear development such as induction, morphogenesis and determination of cell fate and differentiation. The analysis of mouse mutants has been essential to define the requirement of different members of TF families during these processes. Next to their roles during normal development TFs have also been tested for their capacity to induce differentiation or reprogram cells upon misexpression. Recently the capacity of TFs to transdifferentiate easily accessible cells such as fibroblasts to highly specialized cell types has opened a new pathway for regenerative therapies. In this review the influence of TFs acting during different phases and processes of inner ear development will be summarized. A special focus will be given to TFs with a potential to reprogram or transdifferentiate cells to sensory cell types of the inner ear such as hair cells or neurons and thus may form part of future protocols directed to generate replacement cells in a clinical context. © 2012 Elsevier B.V., Supported by the Spanish MinEco (BFU2010-15477, PLE-2009-0098), TerCel (RD06/0010/0000), Ciberned, Fundació La Marató de TV3 and Red de Terapia Célular de la Junta de Castilla y León.

Fgf and Wnt signalling have been shown to be required for formation of the otic placode in vertebrates. Whereas several Fgfs including Fgf3, Fgf8 and Fgf10 have been shown to participate during early placode induction, Wnt signalling is required for specification and maintenance of the otic placode, and dorsal patterning of the otic vesicle. However, the requirement for specific members of the Wnt gene family for otic placode and vesicle formation and their potential interaction with Fgf signalling has been poorly defined. Due to its spatiotemporal expression during placode formation in the hindbrain Wnt8a has been postulated as a potential candidate for its specification. Here we have examined the role of Wnt8a during formation of the otic placode and vesicle in mouse embryos. Wnt8a expression depends on the presence of Fgf3 indicating a serial regulation between Fgf and Wnt signalling during otic placode induction and specification. Wnt8a by itself however is neither essential for placode specification nor redundantly required together with Fgfs for otic placode and vesicle formation. Interestingly however, Wnt8a and Fgf3 are redundantly required for expression of Fgf15 in the hindbrain indicating additional reciprocal interactions between Fgf and Wnt signalling. Further reduction of Wnt signalling by the inactivation of Wnt1 in a Wnt8a mutant background revealed a redundant requirement for both genes during morphogenesis of the dorsal portion of the otic vesicle. © 2012 Elsevier Ireland Ltd., Supported by the Spanish MinEco (BFU2007-61030 and BFU2010-15477), TerCel (RD06/0010/0000), Ciberned and Red de Terapia Célular de la Junta de Castilla y León.

et al., Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury. © 2012 Springer Science+Business Media New York., This work was supported by the Marie Curie Research Training Network CavNET MRTN-CT-2006-035367, Deutsche Forschungsgemeinschaft DFG-Kni-316-4-1, and Hahn Stiftung (Index AG).

Cell therapy is one of the most promising future techniques in the medical arsenal for the repair of damaged or destroyed tissue. The diseases which cell therapy can target are very varied: Hormonal dysfunction, such as diabetes and growth hormone deficiency; neurodegenerative diseases, such as Parkinson's, Alzheimer's and Huntington's; and cardiovascular lesions, such as myocardial infarction, peripheral vascular ischaemia; as well as lesions in the cornea, skeletal muscle, skin, joints and bones etc. The objective of cell therapy is to restore the lost function rather than produce a new organ, which could cause duplicity and undesirable effects. Several resources of cells can be used to restore the damaged tissue, such as resident stem cells, multipotent adult progenitor cells or embryonic stem cells. Some cell therapies have been established and approved for clinical use, such as artificial skin derived from keratinocytes, derived from chondrocyte, cells of the corneal limbus or pancreatic islet transplantation. These therapies have had good results, although the scarcity of the starting material may represent a serious limitation. Other therapies under research, using pluripotent stem cells, have been modest so it is useful to review the protocols and try to improve the outcomes. In this chapter we will review the new advances made in this way.

Cell therapy is one of the most promising future techniques in the medical arsenal for the repair of damaged or destroyed tissue. The diseases which cell therapy can target are very varied: Hormonal dysfunction, such as diabetes and growth hormone deficiency; neurodegenerative diseases, such as Parkinson's, Alzheimer's and Huntington's; and cardiovascular lesions, such as myocardial infarction, peripheral vascular ischaemia; as well as lesions in the cornea, skeletal muscle, skin, joints and bones etc. The objective of cell therapy is to restore the lost function rather than produce a new organ, which could cause duplicity and undesirable effects. Several resources of cells can be used to restore the damaged tissue, such as resident stem cells, multipotent adult progenitor cells or embryonic stem cells. Some cell therapies have been established and approved for clinical use, such as artificial skin derived from keratinocytes, derived from chondrocyte, cells of the corneal limbus or pancreatic islet transplantation. These therapies have had good results, although the scarcity of the starting material may represent a serious limitation. Other therapies under research, using pluripotent stem cells, have been modest so it is useful to review the protocols and try to improve the outcomes. In this chapter we will review the new advances made in this way. © 2012 Landes Bioscience and Springer Science+Business Media., We are garteful for the financial backing from the Cell Therapy Network of the Instituto de Salud Carlos III and the Centre for Regenerative Medicine and Cell Therapy of Castilla y León.

et al., The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNF Pax2 KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNF Pax2 KO IHCs showed a normal maturation. Likewise, in BDNF Pax2 KO membrane capacitance (ΔC m) as a function of inward calcium current (I Ca) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔC m, but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNF Pax2 KOIHCs. Maximal ΔC m correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNF Pax2 KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure. © 2012 the authors., This work was supported by the Marie Curie Research Training Network CavNET MRTN-CT-2006-035367, the Deutsche Forschungsgemeinschaft DFG-Kni-316-4-1 and Hahn Stiftung (Index AG), and by the Wellcome Trust, 088719 and 091895 (to W.M.). W.M. and S.L.J. are Royal Society University Research Fellows.

[Aim]: Hearing loss is the most common sensory disorder in humans, its main cause being the loss of cochlear hair cells. We studied the potential of human mesenchymal stem cells (hMSCs) to differentiate towards hair cells and auditory neurons. [Materials & methods]: hMSCs were first differentiated to neural progenitors and subsequently to hair cell- or auditory neuron-like cells using in vitro culture methods. [Results]: Differentiation of hMSCs to an intermediate neural progenitor stage was critical for obtaining inner ear sensory lineages. hMSCs generated hair cell-like cells only when neural progenitors derived from nonadherent hMSC cultures grown in serum-free medium were exposed to EGF and retinoic acid. Auditory neuron-like cells were obtained when treated with retinoic acid, and in the presence of defined growth factor combinations containing Sonic Hedgehog. [Conclusion]: The results show the potential of hMSCs to give rise to inner ear sensory cells. © 2012 Future Medicine Ltd.

11 páginas, 5 figuras, 1 tabla.-- et al.-- El pdf del artículo es la versión post-print., [Introduction]: Tff3 peptide exerts important functions in cytoprotection and restitution of the gastrointestinal (GI) tract epithelia. Moreover, its presence in the rodent inner ear and involvement in the hearing process was demonstrated recently. However, its role in the auditory system still remains elusive. Our previous results showed a deterioration of hearing with age in Tff3-deficient animals. [Results]: Present detailed analysis of auditory brain stem response (ABR) measurements and immunohistochemical study of selected functional proteins indicated a normal function and phenotype of the cochlea in Tff3 mutants. However, a microarray-based screening of tissue derived from the auditory central nervous system revealed an alteration of securin (Pttg1) and serpina3n expression between wild-type and Tff3 knock-out animals. This was confirmed by qRT-PCR, immunostaining and western blots., This work was supported by Ciberned, MiCINN and Red de Terapia Célular de Castilla y León.

El pdf del artículo es el manuscrito de autor., Several members of the FGF gene family have been shown to intervene from various tissue sources to direct otic placode induction and otic vesicle formation. In this study we define the roles of FGF8, found in different expression domains during this process, in mice and chickens. By conditional inactivation of Fgf8 in distinct tissue compartments we demonstrate that Fgf8 is required in the mesoderm and endoderm during early inner ear development. In the chicken embryo, overexpression of Fgf8 from various tissue sources during otic specification leads to a loss of otic tissue. In contrast ectopic overexpression of Fgf10, a major player during murine otic induction, does not influence otic vesicle formation in chicken embryos but results in the formation of ectopic structures with a non-otic character. This study underlines the crucial role of a defined Fgf8 expression pattern controlling inner ear formation in vertebrates. © 2009 Elsevier Ireland Ltd. All rights reserved., Supported by the Spanish MiCINN (BFU2007-61030), TerCel, Ciberned, Junta de Castilla y León and the DFG (SFB 444).

El pdf del artículo es la versión post-print.-- et al., A central challenge in evolutionary biology is understanding how genetic mutations underlie morphological changes. Because highly calcified enamel enables preservation of detailed dental features, studying tooth morphology enables this question to be addressed in both extinct and extant species. Previous studies have found that mutant mice can have severe abnormalities in tooth morphology, and several authors have explored the evolutionary implications of tooth number modifications in mutants. However, although they can potentially shed much light on evolutionary mechanisms, anomalies in tooth shape remain poorly studied. Here, we report that alterations in dosage of the Fgf3 gene cause morphological changes in both genetically engineered mutant mice and in human patients. By comparing the dental morphologies in mice and humans carrying Fgf3 mutations with primitive rodent and primate fossils, we determined that decreases in dosage of Fgf3 lead to phenotypes that resemble the progressive reappearance of ancestral morphologies. We propose that modifications in the FGF signaling pathway have played an important role in evolution of mammalian dentition by giving rise to new cusps and interconnecting cusps by new crests. We anticipate that our multidisciplinary study will advance the detailed correlation of subtle dental modifications with genetic mutations in a variety of mammalian lineages., V.L. is a research fellow of Alexander von Humboldt Foundation. Scientific missions of C.C. and L.V. have been supported by the French National Research Agency Quenottes program and National Science Foundation Revealing Hominid Origins Initiative Small Mammals (Award BCS-0321893). Support for this research was provided by a Sandler Foundation grant and National Institutes of Health Grant K08-DE017654 (to O.K.) and by the Spanish Ministerio de Ciencia e Innovación Grant BFU2007-61030 (to T.S.).

El pdf del artículo es la versión pre-print.-- et al., [Conclusion]: Cell transplantation into the utriculus provides an efficient and non-invasive route to introduce embryonic stem (ES) cells into the vestibular and cochlear portions of the inner ear. [Objective]: The transfer of stem cells into the inner ear for therapeutic purposes is an important approach to cure damage to the cochlea and vestibulum. A key issue is to provide an entry point for cell transplants into the inner ear that does not affect its physiologic functions. The aim of this study was to examine the feasibility of transferring ES cells into the inner ear via the utriculus. [Materials and methods]: ES cells were injected via utriculostomy into the mouse inner ear. The distribution of the injected cells was determined using a beta-galactosidase marker gene expressed by the ES cells. [Results]: Injected ES cells were found within the perilymph of the scala tympani and vestibuli. Moreover, ES cells were detected close to the cochlear sensory epithelium and spiral limbus., Supported by the Instituto de Salud Carlos III (Red de Terapia Celular).

Classical studies have postulated the action of an endomesodermal signal initiating inner ear induction, subsequently followed by a neural tube-derived signal to complete the process of otic placode formation in the surface ectoderm. Members of the Fibroblast growth factor (FGF) gene family have been implicated in these processes. In this review, expression analysis and recent experimental evidence for candidate inner ear FGF ligands during inner ear induction is discussed. Careful examination of the spatiotemporal expression patterns of different FGFs during inner ear induction reveals that the sequential appearance of FGF members in the endoderm and/or mesoderm is followed by expression in the posterior hindbrain in all vertebrate species analysed to date. Experimental manipulations have demonstrated the sufficiency and/or necessity of some FGFs during different steps of inner ear induction in vitro and in vivo. Combining the advantages of the molecular tools and approaches available in different experimental systems such as zebrafish, chicken or mouse will eventually lead to a complete understanding of how FGFs control inner ear induction in vertebrates. © UBC Press., Supported by the Spanish MEC (BFU2004-860) and the DFG (SFB 444).

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species., Author Summary Stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the so-called cervical loops. We have used a combination of mouse mutant analyses, organ culture experiments, and gene expression studies to identify the key signaling molecules that regulate epithelial stem cell proliferation in the cervical loop stem cell niche. We show that signals from the adjacent mesenchymal tissue regulate epithelial stem cells and form a complex regulatory network with epithelial signals. Stem cell proliferation is stimulated by fibroblast growth factor 3 (FGF3), and bone morphogenetic protein 4 (BMP4) represses Fgf3 expression. In turn, Activin inhibits the repressive effect of BMP4 and Follistatin antagonizes the activity of Activin. We also show that spatial differences in the levels of Activin and Follistatin expression contribute to the characteristic asymmetry of rodent incisors, which are covered by enamel only on their labial (front) side. We suggest that subtle variations in this or related regulatory networks may explain the different regenerative capacities and asymmetric development of various organs and animal species., A network comprising Activin, BMP, FGF, and Follistatin regulate incisor stem cell proliferation in the niche and account for asymmetric organogenesis.

El pdf del artículo es la versión pre-print., FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development. © 2007 Elsevier Inc. All rights reserved., Supported by the Spanish MEC (BFU2004-860) and the DFG (SFB 444).

The 22q11 deletion syndrome (22q11DS) is characterized by abnormal development of the pharyngeal apparatus. Mouse genetic studies have identified Tbx1 as a key gene in the etiology of the syndrome, in part, via interaction with the fibroblast growth factor (Fgf) genes. Three murine Fgfs, Fgf3, Fgf8 and Fgf10 are coexpressed in different combinations with Tbx1. They are all strongly downregulated in Tbx1-/- embryos, implicating epistatic interactions. Supporting this, Tbx1 and Fgf8 have been shown to genetically interact in the development of the fourth pharyngeal arch artery (PAA) and Fgf10 was identified to be a direct downstream target of Tbx1. To dissect the epistatic relationships of these genes during embryonic development and the molecular pathogenesis of the Tbx1 mutant phenotype, we generated Fgf10+/-;Tbx1+/- and Fgf3-/-;Tbx1+/- mice. Despite strong hypotheses that Fgf10 is the key gene downstream of Tbx1 in the development of the anterior heart field, we do not find evidence for genetic interaction between Tbx1 and Fgf10. Also, the Fgf3-/-;Tbx1+/- mutant mice do not show an additive phenotype. Furthermore, more severe defects do not occur in Fgf8+/-;Tbx1+/- mutants by crossing in the Fgf3 null allele. There is a possible additive effect only in PAA remodeling in the Fgf10+/-;Tbx1+/-;Fgf8+/- embryos. Our findings underscore the importance of potential functional redundancy with additional Fgfs in the development of the pharyngeal apparatus and cardiovascular system via Tbx1. This redundancy should be considered when looking at individual FGF genes as modifiers of 22q11DS.

The derivation of specialized differentiated cells from embryonic stem (ES) cells is now a major focus for future therapies involving cell and organ replacement in humans. To obtain populations of differentiated cells for transplantation into the human body, highly optimized protocols are needed that allow direction of the development of ES cells and their derivatives. These protocols mostly include the use of a combination of growth factors that control the differentiation of ES cells into highly specialized cells found in adult organs. The introduction of different growth factors into ES cells and their derivatives via viral gene transfer may greatly facilitate the optimization of these protocols. In this chapter, we describe a method based on herpes simplex virus type 1, which allows efficient gene transfer during several steps of a protocol, directed to obtain neural progenitors from ES cells. This protocol therefore may allow the study of potential factors influencing the cell fate or differentiation of ES cells and their derivatives.

Fibroblast growth factor-2 (FGF2) has been postulated to be a key regulator involved in the proliferation, differentiation, and regeneration of sensory hair cells. Here we have addressed the potential functions of FGF2 during the formation and regeneration of the auditory epithelium in chicken and mice. By using viral gene transfer, based on herpes simplex type 1 virus (HSV-1), we show that ectopically applied FGF2 drastically increases the number of cells expressing early hair cell markers during embryonic development in avians. Intriguingly, FGF2 does not stimulate cell division during this process. These data suggest that FGF2 plays a role during differentiation of sensory hair cells in avians. To address the potential functions of FGF2 during murine inner ear development, we analyzed FGF2 mouse mutants. Mice lacking FGF2 showed normal formation of the inner ear, and no abnormalities were observed at the adult stage. Moreover, FGF2 mouse mutants showed similar hearing thresholds compared with those observed in control mice before and after noise damage. Therefore, endogenous FGF2 appears not to be essential for the development or functional maintenance of the auditory organ in mammals. In light of these results, the differential roles of FGF2 in the vertebrate inner ear are discussed with respect to its previously postulated functions.