1993 — 2001 |
Mansour, Suzanne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genes Involved in Inner Ear Development
Congenital deafness with a genetic origin affects approximately 1 in 2000 children born every year. Studies of the origins of genetic deafness, and potential treatments, would be greatly facilitated by the development of mouse models of human deafness. Many mouse mutants with auditory impairment already exist, but the majority of the responsible genes are not readily accessible to molecular cloning. To accelerate the understanding of the genetic basis of hearing and deafness, a scheme to simultaneously identify and mutate genes involved in the development of the murine auditory system is proposed here. New lacZ gene trap vectors designed to increase the number of detectable genomic integration sites, and facilitate the identification of beta-galactosidase (beta-gal)- expressing cells will be constructed using molecular cloning techniques. These vectors will be introduced into pluripotent murine embryonic stem (ES) cells, and the resulting cell lines will be screened, both before and after in vitro differentiation,f or beta-gal activity that results from vector integration into an expressed gene. ES cell clones that show a regulated pattern of beta-gal activity in vitro will be used to generate chimeric embryos which will be analyzed for beta-gal activity at various developmental stages. Clones that give rise to beta-gal expression in components of the developing auditory system and/or the surrounding tissues thought to induce the auditory system, will be used to generate mouse strains that carry the gene trap insertions. A genetic analysis will be used to determine whether the disrupted genes give rise to overt mutant phenotypes in the embryonic and/or adult auditory system. Genes important for auditory system development, identified using this approach, can be cloned easily by virtue of their linkage to lacZ sequences, and will subsequently be identified by DNA sequence analysis. The DNA probes that result from this research could potentially be used in future linkage studies to identify genes involved in human disorders.
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1 |
2002 — 2004 |
Mansour, Suzanne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Conditional Gene Trapping
DESCRIPTION (provided by applicant): Gene trapping is a method of random insertional mutagenesis that permits visualization of trapped gene expression. Since gene trapping can be carried out in mouse embryonic stem (ES) cells, it is theoretically possible to create and permanently store a collection of mutant alleles representing every gene in the mouse genome. ES cells carrying a gene trap insertion can be used to generate the corresponding mouse strain. Thus, such a resource would be invaluable for exploring the expression and function of mammalian genes, individually and collectively, in a convenient model system. Since current evidence suggests that many genes have multiple roles, beyond the ones evident by assessing a null allele, it is highly desirable that the insertion mutations be conditional. In this way, the null genotype could be induced at specific times and places within the animal. The standard methods for identification of trapped genes are RNA-based, utilizing expensive and technically challenging RT-PCR techniques. With the immanent completion of the mouse genome it should be possible to use cheaper and simpler DNA-based techniques to specify the location of gene trap insertions. Another important consideration in the design of a library of gene trap insertions is the strain of mouse from which the ES cell line is derived. The mutations should be generated in a genetic background that is suitable for the widest array of applications. Currently, the most widely used ES cell lines are derived from various 129-derived strains, which are not appropriate for many studies. The specific aims of this proposal are designed to 1) generate and test multifunctional, conditional gene trap vectors, 2) develop robotic, DNA-based methods of identifying gene trap insertions and 3) generate and characterize new ES cell lines with genetic backgrounds compatible with a wide array of studies, including those of the auditory system. Successful completion of these aims will set the stage for large-scale gene trap cell line production, which will, in turn, accelerate the production of mouse models of human genetic disease.
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1 |
2004 — 2012 |
Mansour, Suzanne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genes Involved in Ear Development
DESCRIPTION (provided by applicant): Normal development of the peripheral auditory system requires coordinated deployment of signaling molecules to direct appropriate transcriptional responses in the myriad tissues that contribute to the ear. Fibroblast growth factors (FGFs) are among the important signals required for normal ear development and reductions in FGF signaling cause sensorineural and/or conductive hearing loss in several human syndromes. Over activation of FGF signaling is also detrimental. We found that loss of mouse DUSP6/MKP3, an ERK phosphatase that functions as a negative feedback regulator of FGF signaling, and is expressed in lineages giving rise to all three divisions of the ear, causes variably penetrant and expressive small size, craniosynostosis and conductive hearing loss. These phenotypes are similar to those caused by activating mutations in human FGFRs 1-3, but are unexplored from an auditory perspective in mouse models. To address the overall hypothesis that inappropriate activation of FGF signaling adversely affects development of the ear, we will use mouse genetics, gene and protein localization studies, audiometrics and skeletal analyses to determine the otic and other phenotypes of mice bearing combinations of Fgfr gain-of-function and Dusp6 null alleles and determine the unique and redundant roles of other ERK phosphatases in otic development. Our findings will help to focus attention on the likelihood that different activators and inhibitors of FGF signaling may currently be unrecognized as causal or modifying for various human hearing loss syndromes and that pharmacologic treatments aimed at targeted modulation of FGF signaling for amelioration of other FGFR-mediated conditions may have additional clinical applications for hearing loss prevention. Finally, a detailed understanding of the consequences of modulating FGF signaling is likely to be an important parameter in developing strategies for repair or regeneration of inner ear cell types. PUBLIC HEALTH RELEVANCE Hearing loss is the most common human sensory deficit and can be caused by mutations that change the levels of Fibroblast Growth Factor (FGF) signaling during development. We are using mice to model genetic cases of inappropriate activation of FGF signaling in order to understand the development of hearing loss in these conditions. These studies are expected to contribute knowledge that will be used in developing therapies for hearing loss.
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2012 |
Mansour, Suzanne L |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
2012 Fibroblast Growth Factors in Development & Disease Gordon Research Conferenc @ Gordon Research Conferences
DESCRIPTION (provided by applicant): This application requests funds to support the 2012 (4th) Gordon Research Conference (GRC) on Fibroblast Growth Factors (FGFs) in Development & Disease at Les Diablerets Conference Center, Les Diablerets, Switzerland, May 13-18, 2012 and the associated (1st) Gordon Research Seminar (GRS) on Emerging Concepts in FGF Biology, May 12-13, 2012 at the same site. The FGFs comprise a family of growth and differentiation factors with essential functions in development, metabolism, and repair of tissues and organs. Dysregulation of FGF signaling is associated with human diseases, including several developmental/genetic diseases, metabolic disorders, and malignancies. The FGF- GRC will feature talks from invited and abstract-selected speakers as well as extended poster sessions encompassing the most recent and timely research in this highly diverse field. The conference will promote interdisciplinary interactions between academic scientists, clinicians and colleagues from industry studying all aspects of FGF biology. Topics covered in this unique conference include biochemistry of the FGF signaling complex and control of downstream signaling pathways and their impact on cell biology, physiology, endocrinology, development, pharmacology, pathology and regeneration/repair. For the first time, we will also offer a pre-conference GRS, organized by junior investigators at the postdoctoral level for investigators at the graduate student, postdoctoral and junior independent levels to present their research in a mentored environment, preparing them for full participation in the subsequent GRC. The goals of the FGF-GRC & GRS organizers are to: 1) Provide an international forum for talks and posters featuring mainly unpublished data representative of the diverse approaches taken by both junior and senior FGF biologists. 2) Encourage wide-ranging formal and informal discussions of the data, stimulating new and/or improved experimental directions for all attendees. 3) Encourage networking and new collaborations among attendees, particularly those in disparate fields of FGF biology and at different levels of advancement. 4) Provide junior investigators with an up-to-date background on all aspects of FGF biology and technology, and constructive feedback on their research projects from senior investigators. In support of these goals we propose to 1) support travel and/or registration costs for a diverse selection of 25 well-established invited speakers/discussion leaders plus the 4 GRC and GRS organizers and four junior independent investigators as speakers and 2) support travel and/or registration costs for 2 well-established invited speaker/mentors, 1 junior independent investigator and 10 trainees at the graduate student or postdoctoral levels to attend the GRS. These meetings will contribute significantly to the FGF field, through presentation of the most recent and significant advances and multidisciplinary networking of scientists at all levels, increasing opportunities for translaton of new insights into FGF biology into novel medical treatments for a broad range of diseases. PUBLIC HEALTH RELEVANCE: We request funds to partially support the 2012 Gordon Research Conference (GRC) and Gordon Research Seminar (GRS) on Fibroblast Growth Factors (FGFs) in Development & Disease. The FGFs are growth and differentiation factors with essential functions in development, metabolism and repair of tissues and organs and abnormal regulation of FGF signaling is associated with human diseases, including several developmental/genetic conditions, metabolic disorders and cancer. The FGF GRC and GRS will contribute significantly to advancing the FGF field, through presentation and discussion of the most recent and significant findings, and multidisciplinary networking of scientists at all levels, increasing opportunities for translation of new insights into FGF biology into novel medical treatments for a broad range of diseases.
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0.904 |
2012 — 2016 |
Mansour, Suzanne L Schoenwolf, Gary C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Signals Integrating Cellular Dynamics to Sculpt the Inner Ear (A1)
DESCRIPTION (provided by applicant): Misregulation of intercellular signaling disrupts inner ear morphogenesis in human subjects and animal models, leading to hearing and balance disorders. Normal morphogenesis of the inner ear's membranous labyrinth requires temporal integration of regional and cell fate specification with changes in cell behavior. Past work has begun to identify changes in otocyst gene expression downstream of signals, including BMP, FGF and SHH, but much less is known about the cell behaviors driving morphogenesis and how these behaviors are coordinated temporally with progressive restriction of cell fates to generate the mature vestibular and cochlear compartments. Using temporally and spatially controlled loss- and gain-of-function in chick and mouse embryos, we propose to test the general hypothesis that BMP/TGFss, FGF and HH signaling are integrated to regulate otocyst regional and cell fate specification and cell behavior to initiate normal morphogenesis of the vestibular and cochlear compartments of the developing membranous labyrinth. Our preliminary data provide proof-of-principle for our approach. In control chick embryos, we identified a columnar-to-squamous cell shape change in the dorsolateral otocyst epithelium that occurs concomitant with thinning and expansion to form the primordial canal pouch. Spatiotemporally controlled loss- and gain-of-function experiments showed that BMP/SMAD signaling is both necessary and sufficient for this cell shape change. In addition, similar chick misexpression experiments revealed a common intersection point regulating BMP/SHH signaling during early otocyst dorsoventral patterning and morphogenesis. In mouse, we found that otocyst-derived FGF3 and FGF10 signals, in addition to their well-known roles in vestibular morphogenesis, are required to initiate cochlear morphogenesis. However, these epithelial signals are not required for early otocyst regional patterning, which is normal in FGF-deficient otocysts. Therefore, we propose to test the specific hypotheses that 1) temporal integration of BMP/TGFss, FGF and HH signaling controls three key early steps of otocyst morphogenesis: primordial canal outgrowth, subdivision of the primordium into vertical and lateral canal pouches and initial cochlear outgrowth, and 2) that such signaling coordinates otocyst regional and/or cell fate specification with changes in relevant cell behaviors. Our proposal takes advantage of the unique expertise of a team of established investigators using state-of-the-art molecular genetic and embryologic techniques in two animal models with complementary strengths that will illuminate important differences and similarities in mechanisms driving key morphogenetic steps downstream of growth factor signaling. This will advance the field by providing a novel understanding of how signaling pathways are integrated to control initiation of vestibular and cochlear morphogenesis, and how these signals coordinate specification of cell fate and changes in cell behavior to initiate and sculpt a normally functioning membranous labyrinth. Such information provides an essential foundation for understanding and ultimately preventing human hearing loss.
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2015 — 2019 |
Mansour, Suzanne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Inducing Cochlear Sensory Cell Differentiation
? DESCRIPTION (provided by applicant): Irreparable neurodegeneration characterizes many neurologic diseases and contributes to the decline in nervous system function with age. Thus, uncovering mechanisms for neuronal replacement is a central challenge of therapeutics for many clinical presentations. This challenge is formidable in the CNS because of the variety and complexity of neurons, glia and their connections. Therefore, we are investigating a simpler model: the neuron-like sensory hair cells (HCs) of the auditory system, which are supported by glia-like cells and relay sound stimuli through auditory neurons to the brain. Genetic and environmental damage to HCs cause sensorineural hearing loss (SNHL), a common condition with significant healthcare costs. Regardless of initiating cause, in most cases of SNHL, the HCs die, whereas supporting cells (SCs) and auditory neurons can persist. However, as mammalian cochleae have no regenerative capacity, loss of HCs leads to permanent SNHL that is ameliorated, but not cured, by current therapies. Inhibition of Notch signaling has emerged as a means of inducing new HCs from residual SCs in deafened animals, but in mice it is inefficient after the embryonic stages when HCs normally arise, suggesting that regulation of additional developmental signals may be critical to HC regeneration and rewiring. Among the critical signals required for genesis of both HCs and SCs are members of the Fibroblast Growth Factor (FGF) family, which differ in their ability to activate different FGF receptors. FGF20/FGFR1 signaling promotes development of outer hair cells (OHCs), which amplify sound stimuli transduced by inner hair cells (IHCs); and FGF8/FGFR3 signaling promotes differentiation of pillar cells (PCs), SCs that separate IHCs from OHCs. Curiously, although genetic loss and gain of FGFR3 signaling have opposing effects on PC differentiation that cause SNHL, both changes induce ectopic OHCs supported by ectopic Deiters' cells (DCs). Fgfr3-/- mice show ectopic OHCs and DCs by late gestation. Our preliminary studies of a mouse FGFR3 gain-of-function mutation with altered ligand-binding specificity show that ectopic OHCs and DCs arise postnatally. Furthermore, their genesis depends on FGF10, the same unexpected ligand that alters RAS/MAPK target genes and induces the DC-to-PC fate transformation and plasticity we described previously. In this proposal we test the hypothesis that FGF/RAS/MAPK signaling is sufficient to induce OHC differentiation from DC progenitors both embryonically and postnatally, including in an in vivo model of OHC loss. We will 1) determine whether an ectopic gain-of-function mechanism drives ectopic OHCs and DCs in Fgfr3-/- mice; 2) determine the lineage of ectopic OHCs in FGFR3 loss- and gain-of-function mutants; and 3) determine whether forced activation of RAS/MAPK signaling in DCs of normal and OHC-depleted cochleae will induce OHCs. Our results will drive future studies addressing the long-term goal of harnessing the FGF pathway together with other signals to induce robust mammalian auditory HC regeneration and hearing restoration in damaged cochleae, and will inform efforts to manipulate CNS glia to differentiate as neurons.
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2015 — 2016 |
Mansour, Suzanne L |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
New Mouse Models For Inducible Cell-Specific Ablation
? DESCRIPTION (provided by applicant): Sensorineural hearing loss (SNHL) is a significant healthcare problem, with large social and financial costs. SNHL occurs when the auditory sensory hair cells of the inner ear, or the primary neural pathways connecting these cells to the brain, cease to function appropriately. Such damage occurs consequent to genetic and/or environmental insults. SNHL is permanent because of subsequent sensory and/or neural cell death, and the inability of such cells to regenerate. There are many ideas about how to provoke regeneration of lost auditory hair cells and neurons in mammals. These include stimulating regeneration of hair cells from residual endogenous supporting cells, as occurs normally in fish and birds. Similarly, spiral ganglion neurons might be regenerated from glia. In both cases, manipulating the signals and transcription factors normally involved in the development of these cells is under intensive investigation. Stem cell-based replacement strategies are also being pursued. Regardless of the preferred approach, any proposed therapy for SNHL will require testing in an in vivo mammalian model. However, existing mammalian models of sensory and neural cell loss in the peripheral auditory system are not ideal. For example, both ototoxic drugs and noise exposure can be controlled temporally to cause hearing loss, but these treatments affect the entire inner ear, not only the auditory hair cells and/or spiral ganglion neurons, and this can confound treatment studies. Inducible single recombinase-based methods of ablating inner ear sensory or neural cells are promising, but also frequently impact other cells/tissues that are essential for viability of the animal, thus limiting their utility. In this application, w propose to develop and validate an improved system, called intersectional and inducible cell-specific ablation (IICSA), for inducing precise and reproducible ablation of mouse auditory hair cells or spiral ganglion neurons at any stage of interest. This will enable modeling of inner hair cell, outer hair cell or spiral ganglion neuron loss at different stages, and provide a platform fo testing hearing restoration therapies in a mammal. IICSA is a significant advance over current technology because it will enable doxycycline-inducible cell ablation with the potential for reduced or absent off-target effects. We will use state- of-the-art targeted transgenesis and genome editing techniques to generate the necessary IICSA driver and effector mouse strains. This technology will be developed and tested for inducible ablation of specific cochlear cell types, but is adaptable to any target cells of interest, enabling modeling of any number of degenerative conditions for both basic and translational studies.
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2016 — 2020 |
Mansour, Suzanne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Auditory Supporting Cell Differentiation and Plasticity
? DESCRIPTION (provided by applicant): Sensorineural hearing loss (SNHL) affects a large proportion of the population, generating significant social and health care costs. Many forms of SNHL feature damage to or loss of cochlear sensory hair cells (HCs), which do not regenerate in mammals. Strategies for hearing restoration are informed by studies of birds and fish, which, unlike mammals, spontaneously regenerate HCs from residual supporting cells (SCs). Notch signaling inhibition has emerged as a promising means of regenerating HCs. In mouse models, however, this approach is inefficient after embryonic stages, suggesting that manipulating additional developmental signals may be required. The FGF signaling system is a promising candidate because tight regulation of FGF signaling is critical to all stages of inner ear development, including HC and SC differentiation. We showed previously that mice with an FGFR3-activating mutation modeling Muenke syndrome, have dominant hearing loss associated with a SC fate switch of two Deiters' cells (DCs) to two pillar cells (PCs). The cell fate switch occurs perinatally and is associated with an expansion of FGF/RAS/MAPK signaling into the prospective DC region. Unexpectedly, hearing and SC fate are restored in these Fgfr3 mutants following genetic reduction of FGF10, a ligand that does not normally activate FGFR3. Remarkably, the SC fate switch still occurs in these rescued animals, but is resolved over time. This is associated with restoration of normal patterns of FGF signaling and shows that seemingly fully differentiated cochlear SCs can reversibly switch fates in an FGF-regulated manner. Although genetic data clearly implicate FGF8 as a ligand for FGFR3 in normal PC differentiation, the SC phenotype of Fgf8 otic conditional knockout mice is weaker than that of the Fgfr3 null mice, suggesting that additional Fgfs are involved. Furthermore, the rescue of Muenke syndrome model phenotypes by Fgf10 heterozygosity begs the question of the normal role of cochlear Fgf10 in the perinatal period. Fgf3 is also expressed near developing SCs, but its role in their development is unknown. These data collectively lead to the hypothesis that FGF10 signals are required for development of Fgfr3P244R/+ phenotypes and that Fgf10 and/or Fgf3 are required together with Fgf8 for normal PC differentiation. This will be tested by temporal and spatial regulation FGF signaling in Muenke syndrome model and wild type mice (Aim 1). Our finding of FGF-regulated supporting cell plasticity and the observations by others that DCs express Fgfr3 into adulthood and that Notch inhibition can promote HC regeneration from SC progenitors, suggest the hypothesis that normal perinatal DCs can be transformed into PCs by forced activation of the RAS/MAPK pathway and that Notch inhibition induced in the context of FGF/RAS/MAPK activation will promote HC differentiation. This will be tested using spatial and temporal modulation of the two signaling pathways in vivo (Aim 2). Completion of the Aims will impact the development of strategies that employ developmental signals for hearing restoration.
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2021 |
Mansour, Suzanne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Inner Ear Development by Fgf Signals and Effectors
Morphogenesis of the inner ear epithelium requires coordinated deployment of several signaling pathways and disruptions cause abnormalities of hearing and/or balance. With the advent of cochlear implantation to treat hearing loss even in cases of inner ear malformation, it is critical to understand exactly how such malformations affect the auditory ganglia and innervation. Also, in light of the intense focus on in vitro generation of inner ear cell types for transplantation and in vivo manipulation of developmental signaling molecules to promote differentiation of various inner ear cells for hearing restoration, elucidating the roles and regulation of such signals and their effectors governing otic differentiation and morphogenesis are necessary to advance treatment. The genes encoding FGF3 and FGF10, ligands that signal through FGFR2b and FGFR1b, are expressed dynamically throughout otic development in both epithelial and ganglion domains. Studies conducted by the Mansour Lab of both conventional Fgf3 and Fgf10 conditional knockout mice and those expressing a doxycycline-inducible ligand trap (dnFGFR2b) that rapidly inhibits signaling through both FGFR1b and FGFR2b, showed that Fgf3 and Fgf10 are not required in the placode lineage for otocyst formation, but are required subsequently for otocyst patterning, neuroblast maintenance, epithelial proliferation and both vestibular and cochlear morphogenesis. Furthermore, the first genome wide analyses of otocyst mRNA revealed FGFR2b/1b signaling targets that define novel candidates for genes involved in otic morphogenesis and function. This proposal has two Aims addressing the hypotheses that 1) FGFR2b/1b signaling is required continuously for both otic neuroblast specification and maintenance, and that at later stages, mesenchymal signaling, as well as that in the epithelial and ganglion domains, is required for cochlear epithelial differentiation and ganglion maintenance and 2) FGFR2b/1b downstream target genes mediate some or all of the effects of FGFR2b/1b signaling on otic morphogenesis and gangliogenesis. To determine the early role of FGFR2b/1b signaling in otic ganglion formation and its later role in epithelial differentiation and ganglion maintenance, DOX-induced ubiquitous and CRE-limited expression of dnFGFR2b will be employed and morphology and molecular markers of otic patterning, proliferation and survival in both tissues will be assessed. To determine the roles of downstream targets of FGFR2b/1b signaling, two genes encoding transcription factors that are activated by FGFR2b/1b signaling and one gene encoding a BMP signaling regulator that is repressed by FGFR2b/1b signaling will be studied. Otic conditional mutants will be generated for each gene, and their morphologic and functional development will be assessed. In addition, the extent to which the BMP regulator contributes to the dnFGFR2b phenotypes and the effects of overexpressing the BMP regulator will be assessed. The results will contribute new knowledge that will facilitate future efforts to manipulate the FGF signaling system for hearing restoration.
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