Publications by authors named "Benjamin Kopecky"

19 Publications

  • Page 1 of 1

Interaction with ectopic cochlear crista sensory epithelium disrupts basal cochlear sensory epithelium development in Lmx1a mutant mice.

Cell Tissue Res 2020 Jun 13;380(3):435-448. Epub 2020 Jan 13.

Department of Biology, University of Iowa, Iowa City, IA, 52242-1324, USA.

The LIM homeodomain transcription factor Lmx1a shows a dynamic expression in the developing mouse ear that stabilizes in the non-sensory epithelium. Previous work showed that Lmx1a functional null mutants have an additional sensory hair cell patch in the posterior wall of a cochlear duct and have a mix of vestibular and cochlear hair cells in the basal cochlear sensory epithelium. In E13.5 mutants, Sox2-expressing posterior canal crista is continuous with an ectopic "crista sensory epithelium" located in the outer spiral sulcus of the basal cochlear duct. The medial margin of cochlear crista is in contact with the adjacent Sox2-expressing basal cochlear sensory epithelium. By E17.5, this contact has been interrupted by the formation of an intervening non-sensory epithelium, and Atoh1 is expressed in the hair cells of both the cochlear crista and the basal cochlear sensory epithelium. Where cochlear crista was formerly associated with the basal cochlear sensory epithelium, the basal cochlear sensory epithelium lacks an outer hair cell band, and gaps are present in its associated Bmp4 expression. Further apically, where cochlear crista was never present, the cochlear sensory epithelium forms a poorly ordered but complete organ of Corti. We propose that the core prosensory posterior crista is enlarged in the mutant when the absence of Lmx1a expression allows JAG1-NOTCH signaling to propagate into the adjacent epithelium and down the posterior wall of the cochlear duct. We suggest that the cochlear crista propagates in the mutant outer spiral sulcus because it expresses Lmo4 in the absence of Lmx1a.
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http://dx.doi.org/10.1007/s00441-019-03163-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393901PMC
June 2020

Role of donor macrophages after heart and lung transplantation.

Am J Transplant 2020 05 29;20(5):1225-1235. Epub 2020 Jan 29.

Department of Medicine, Washington University, Saint Louis, Missouri, USA.

Since the 1960s, heart and lung transplantation has remained the optimal therapy for patients with end-stage disease, extending and improving quality of life for thousands of individuals annually. Expanding donor organ availability and immunologic compatibility is a priority to help meet the clinical demand for organ transplant. While effective, current immunosuppression is imperfect as it lacks specificity and imposes unintended adverse effects such as opportunistic infections and malignancy that limit the health and longevity of transplant recipients. In this review, we focus on donor macrophages as a new target to achieve allograft tolerance. Donor organ-directed therapies have the potential to improve allograft survival while minimizing patient harm related to global suppression of recipient immune responses. Topics highlighted include the role of ontogenically distinct donor macrophage populations in ischemia-reperfusion injury and rejection, including their interaction with allograft-infiltrating recipient immune cells and potential therapeutic approaches. Ultimately, a better understanding of how donor intrinsic immunity influences allograft acceptance and survival will provide new opportunities to improve the outcomes of transplant recipients.
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http://dx.doi.org/10.1111/ajt.15751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202685PMC
May 2020

Molecular Imaging Visualizes Recruitment of Inflammatory Monocytes and Macrophages to the Injured Heart.

Circ Res 2019 03;124(6):881-890

Department of Medicine (B.K., G.F., G.B., S.L.B., K.J.L.), Washington University School of Medicine, St. Louis, MO.

Rationale: Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2- (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy.

Objective: To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart.

Methods And Results: We synthesized and tested the performance of a positron emission tomography radiotracer (Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2 mice, demonstrating target specificity. Autoradiography demonstrated that Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance.

Conclusions: These findings demonstrate the sensitivity and specificity of Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435034PMC
March 2019

The Macrophage in Cardiac Homeostasis and Disease: JACC Macrophage in CVD Series (Part 4).

J Am Coll Cardiol 2018 10;72(18):2213-2230

Zena and Michael A. Wiener Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address:

Macrophages are integral components of cardiac tissue and exert profound effects on the healthy and diseased heart. Paradigm shifting studies using advanced molecular techniques have revealed significant complexity within these macrophage populations that reside in the heart. In this final of a 4-part review series covering the macrophage in cardiovascular disease, the authors review the origins, dynamics, cell surface markers, and respective functions of each cardiac macrophage subset identified to date, including in the specific scenarios of myocarditis and after myocardial infarction. Looking ahead, a deeper understanding of the diverse and often dichotomous functions of cardiac macrophages will be essential for the development of targeted therapies to mitigate injury and orchestrate recovery of the diseased heart. Moreover, as macrophages are critical for cardiac healing, they are an emerging focus for therapeutic strategies aimed at minimizing cardiomyocyte death, ameliorating pathological cardiac remodeling, and for treating heart failure and after myocardial infarction.
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http://dx.doi.org/10.1016/j.jacc.2018.08.2149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209119PMC
October 2018

Canertinib induces ototoxicity in three preclinical models.

Hear Res 2015 Oct 7;328:59-66. Epub 2015 Jul 7.

Department of Otolaryngology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, USA. Electronic address:

Neuregulin-1 (NRG1) ligand and its epidermal growth factor receptor (EGFR)/ERBB family regulate normal cellular proliferation and differentiation in many tissues including the cochlea. Aberrant NRG1 and ERBB signaling cause significant hearing impairment in mice. Dysregulation of the same signaling pathway in humans is involved in certain types of cancers such as breast cancer or non-small cell lung cancer (NSCLC). A new irreversible pan-ERBB inhibitor, canertinib, has been tested in clinical trials for the treatment of refractory NSCLC. Its possible ototoxicity was unknown. In this study, a significant dose-dependent canertinib ototoxicity was observed in a zebrafish model. Canertinib ototoxicity was further confirmed in two mouse models with different genetic backgrounds. The data strongly suggested an evolutionally preserved ERBB molecular mechanism underlying canertinib ototoxicity. Thus, these results imply that clinical monitoring of hearing loss should be considered for clinical testing of canertinib or other pan-ERBB inhibitors.
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http://dx.doi.org/10.1016/j.heares.2015.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4581429PMC
October 2015

T-type calcium channel blockers as neuroprotective agents.

Pflugers Arch 2014 Apr 25;466(4):757-65. Epub 2014 Feb 25.

Department of Otolaryngology, Center for Aging, Washington University School of Medicine, 4560 Clayton Avenue, St. Louis, MO, 63110, USA.

T-type calcium channels are expressed in many diverse tissues, including neuronal, cardiovascular, and endocrine. T-type calcium channels are known to play roles in the development, maintenance, and repair of these tissues but have also been implicated in disease when not properly regulated. Calcium channel blockers have been developed to treat various diseases and their use clinically is widespread due to both their efficacy as well as their safety. Aside from their established clinical applications, recent studies have suggested neuroprotective effects of T-type calcium channel blockers. Many of the current T-type calcium channel blockers could act on other molecular targets besides T-type calcium channels making it uncertain whether their neuroprotective effects are solely due to blocking of T-type calcium channels. In this review, we discuss these drugs as well as newly developed chemical compounds that are designed to be more selective for T-type calcium channels. We review in vitro and in vivo evidence of neuroprotective effects by these T-type calcium channel blockers. We conclude by discussing possible molecular mechanisms underlying the neuroprotective effects by T-type calcium channel blockers.
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http://dx.doi.org/10.1007/s00424-014-1454-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005039PMC
April 2014

Evolution and development of the tetrapod auditory system: an organ of Corti-centric perspective.

Evol Dev 2013 Jan;15(1):63-79

Department of Biology, University of Iowa, CLAS, 143 BB, Iowa City, IA, 52242, USA.

The tetrapod auditory system transmits sound through the outer and middle ear to the organ of Corti or other sound pressure receivers of the inner ear where specialized hair cells translate vibrations of the basilar membrane into electrical potential changes that are conducted by the spiral ganglion neurons to the auditory nuclei. In other systems, notably the vertebrate limb, a detailed connection between the evolutionary variations in adaptive morphology and the underlying alterations in the genetic basis of development has been partially elucidated. In this review, we attempt to correlate evolutionary and partially characterized molecular data into a cohesive perspective of the evolution of the mammalian organ of Corti out of the tetrapod basilar papilla. We propose a stepwise, molecularly partially characterized transformation of the ancestral, vestibular developmental program of the vertebrate ear. This review provides a framework to decipher both discrete steps in development and the evolution of unique functional adaptations of the auditory system. The combined analysis of evolution and development establishes a powerful cross-correlation where conclusions derived from either approach become more meaningful in a larger context which is not possible through exclusively evolution or development centered perspectives. Selection may explain the survival of the fittest auditory system, but only developmental genetics can explain the arrival of the fittest auditory system. [Modified after (Wagner 2011)].
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http://dx.doi.org/10.1111/ede.12015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918746PMC
January 2013

Correct timing of proliferation and differentiation is necessary for normal inner ear development and auditory hair cell viability.

Dev Dyn 2013 Feb;242(2):132-47

University of Iowa, Department of Biology, Iowa City, IA 52242, USA.

Background: Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2-Cre N-Myc conditional knockout (CKO) mice highlighted the need of N-Myc for proper neurosensory development and possible redundancy with L-Myc. The late-onset hair cell death in the absence of early N-Myc expression could be due to mis-regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1.

Results: Pax2-Cre N-Myc L-Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation result in morphologically abnormal ears. In particular, the organ of Corti apex is re-patterned into a vestibular-like organization and the base is truncated and fused with the saccule.

Conclusions: These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix-Loop-Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development.
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http://dx.doi.org/10.1002/dvdy.23910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553268PMC
February 2013

N-Myc and L-Myc are essential for hair cell formation but not maintenance.

Brain Res 2012 Nov 25;1484:1-14. Epub 2012 Sep 25.

University of Iowa, Department of Biology, Iowa City, IA 52242, USA.

Sensorineural hearing loss results from damage to the hair cells of the organ of Corti and is irreversible in mammals. While hair cell regeneration may prove to be the ideal therapy after hearing loss, prevention of initial hair cell loss could provide even more benefit at a lower cost. Previous studies have shown that the deletion of Atoh1 results in embryonic loss of hair cells while the absence of Barhl1, Gfi1, and Pou4f3 leads to the progressive loss of hair cells in newborn mice. We recently reported that in the early embryonic absence of N-Myc (using Pax2-Cre), hair cells in the organ of Corti develop and remain until at least seven days after birth, with subsequent progressive loss. Thus, N-Myc plays a role in hair cell viability; however, it is unclear if this is due to its early expression in hair cell precursors and throughout the growing otocyst as it functions through proliferation or its late expression exclusively in differentiated hair cells. Furthermore, the related family member L-Myc is mostly co-expressed in the ear, including in differentiated hair cells, but its function has not been studied and could be partially redundant to N-Myc. To test for a long-term function of the Mycs in differentiated hair cells, we generated nine unique genotypes knocking out N-Myc and/or L-Myc after initial formation of hair cells using the well-characterized Atoh1-Cre. We tested functionality of the auditory and vestibular systems at both P21 and four months of age and under the administration of the ototoxic drug cisplatin. We conclude that neither N-Myc nor L-Myc is likely to play important roles in long-term hair cell maintenance. Therefore, it is likely that the late-onset loss of hair cells resulting from early deletion of the Mycs leads to an unsustainable developmental defect.
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http://dx.doi.org/10.1016/j.brainres.2012.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482343PMC
November 2012

Understanding the evolution and development of neurosensory transcription factors of the ear to enhance therapeutic translation.

Cell Tissue Res 2012 Aug 13;349(2):415-32. Epub 2012 Jun 13.

Department of Biology, University of Iowa, College of Liberal Arts and Sciences, Iowa City, IA 52242, USA.

Reconstructing a functional organ of Corti is the ultimate target towards curing hearing loss. Despite the impressive technical gains made over the last few years, many complications remain ahead for the two main restoration avenues: in vitro transformation of pluripotent cells into hair cell-like cells and adenovirus-mediated gene therapy. Most notably, both approaches require a more complete understanding of the molecular networks that ensure specific cell types form in the correct places to allow proper function of the restored organ of Corti. Important to this understanding are the basic helix-loop-helix (bHLH) transcription factors (TFs) that are highly diverse and serve to increase functional complexity but their evolutionary implementation in the inner ear neurosensory development is less conspicuous. To this end, we review the evolutionary and developmentally dynamic interactions of the three bHLH TFs that have been identified as the main players in neurosensory evolution and development, Neurog1, Neurod1 and Atoh1. These three TFs belong to the neurogenin/atonal family and evolved from a molecular precursor that likely regulated single sensory cell development in the ectoderm of metazoan ancestors but are now also expressed in other parts of the body, including the brain. They interact extensively via intracellular and intercellular cross-regulation to establish the two main neurosensory cell types of the ear, the hair cells and sensory neurons. Furthermore, the level and duration of their expression affect the specification of hair cell subtypes (inner hair cells vs. outer hair cells). We propose that appropriate manipulation of these TFs through their characterized binding sites may offer a solution by itself, or in conjunction with the two other approaches currently pursued by others, to restore the organ of Corti.
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http://dx.doi.org/10.1007/s00441-012-1454-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3508675PMC
August 2012

Mutational ataxia resulting from abnormal vestibular acquisition and processing is partially compensated for.

Behav Neurosci 2012 Apr 6;126(2):301-13. Epub 2012 Feb 6.

Department of Biology and Carver College of Medicine, Medical Scientist Training Program, University of Iowa, USA.

Due to the multisensory input into the balance system, the loss of one input, such as an ear, can generally be compensated for. However, when a mismatch or incomplete loss of inputs occurs, the ability to compensate for the stimulus misrepresentation may be compromised. The inner ear and cerebellum are important input and processing centers for balance but no genetic models have been generated to assess balance or compensation in the abnormal development of both these organs/brain areas. Important to their formation is regulation of proliferation mediated by the proto-oncogene N-Myc. Conditional knockouts (CKOs) of N-Myc using Tg(Pax2-Cre) have a misshapen and smaller ear with a fused utricle, saccule, and cochlea and absent horizontal canal, aberrant cochlear and vestibular innervations, and a size reduction in the cerebellum. CKOs are viable with obvious behavioral deficits, including circling behavior and unstable gait. To test the degree of ataxia and possible compensation of vestibular defects in these mutant mice, we use the Noldus Catwalk System to assess the gait of Tg(Pax2-Cre) N-Myc CKOs over five months. N-Myc CKOs perform worse than control littermates, in particular, in step regularity. We show that disrupting one member of the Myc family during embryonic development coincides with a differential loss of function in the cochlea compared to the vestibular apparatus. In addition, we show that the distortion in the ear morphology combined with a reduction of the cerebellum, rather than a complete loss of the vestibular-cerebellar pathway, leads to partial behavioral compensation that remains unchanged over time.
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http://dx.doi.org/10.1037/a0026896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314111PMC
April 2012

Expression of Neurog1 instead of Atoh1 can partially rescue organ of Corti cell survival.

PLoS One 2012 24;7(1):e30853. Epub 2012 Jan 24.

University of Iowa, Department of Biology, Iowa City, Iowa, United States of America.

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1(KINeurog1)) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1(+/KINeurog1)) and homozygous (Atoh1(KINeurog1/KINeurog1)) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete 'flat epithelium' in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1(f/+)), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1(KINeurog1) can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030853PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265522PMC
June 2012

A novel Atoh1 "self-terminating" mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability.

PLoS One 2012 18;7(1):e30358. Epub 2012 Jan 18.

Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.

Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030358PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261193PMC
July 2012

Scanning thin-sheet laser imaging microscopy elucidates details on mouse ear development.

Dev Dyn 2012 Mar 23;241(3):465-80. Epub 2012 Jan 23.

Department of Biology, University of Iowa, Iowa City, Iowa 52242, USA.

Background: The mammalian inner ear is transformed from a flat placode into a three-dimensional (3D) structure with six sensory epithelia that allow for the perception of sound and both linear and angular acceleration. While hearing and balance problems are typically considered to be adult onset diseases, they may arise as a developmental perturbation to the developing ear. Future prevention of hearing or balance loss requires an understanding of how closely genetic mutations in model organisms reflect the human case, necessitating an objective multidimensional comparison of mouse ears with human ears that have comparable mutations in the same gene.

Results: Here, we present improved 3D analyses of normal murine ears during embryonic development using optical sections obtained through Thin-Sheet Laser Imaging Microscopy. We chronicle the transformation of an undifferentiated otic vesicle between mouse embryonic day 11.5 to a fully differentiated inner ear at postnatal day 15.

Conclusions: Our analysis of ear development provides new insights into ear development, enables unique perspectives into the complex development of the ear, and allows for the first full quantification of volumetric and linear aspects of ear growth. Our data provide the framework for future analysis of mutant phenotypes that are currently under-appreciated using only two dimensional renderings.
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http://dx.doi.org/10.1002/dvdy.23736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010664PMC
March 2012

The myc road to hearing restoration.

Cells 2012 Sep 25;1(4):667-98. Epub 2012 Sep 25.

Department of Biology, 143 Biology Building, University of Iowa, Iowa City, IA 52242, USA.

Current treatments for hearing loss, the most common neurosensory disorder, do not restore perfect hearing. Regeneration of lost organ of Corti hair cells through forced cell cycle re-entry of supporting cells or through manipulation of stem cells, both avenues towards a permanent cure, require a more complete understanding of normal inner ear development, specifically the balance of proliferation and differentiation required to form and to maintain hair cells. Direct successful alterations to the cell cycle result in cell death whereas regulation of upstream genes is insufficient to permanently alter cell cycle dynamics. The Myc gene family is uniquely situated to synergize upstream pathways into downstream cell cycle control. There are three Mycs that are embedded within the Myc/Max/Mad network to regulate proliferation. The function of the two ear expressed Mycs, N-Myc and L-Myc were unknown less than two years ago and their therapeutic potentials remain speculative. In this review, we discuss the roles the Mycs play in the body and what led us to choose them to be our candidate gene for inner ear therapies. We will summarize the recently published work describing the early and late effects of N-Myc and L-Myc on hair cell formation and maintenance. Lastly, we detail the translational significance of our findings and what future work must be performed to make the ultimate hearing aid: the regeneration of the organ of Corti.
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http://dx.doi.org/10.3390/cells1040667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901154PMC
September 2012

Regeneration of Hair Cells: Making Sense of All the Noise.

Pharmaceuticals (Basel) 2011 Jun;4(6):848-879

Department of Biology, University of Iowa, Iowa City, IA, 52242, USA.

Hearing loss affects hundreds of millions of people worldwide by dampening or cutting off their auditory connection to the world. Current treatments for sensorineural hearing loss (SNHL) with cochlear implants are not perfect, leaving regenerative medicine as the logical avenue to a perfect cure. Multiple routes to regeneration of damaged hair cells have been proposed and are actively pursued. Each route not only requires a keen understanding of the molecular basis of ear development but also faces the practical limitations of stem cell regulation in the delicate inner ear where topology of cell distribution is essential. Improvements in our molecular understanding of the minimal essential genes necessary for hair cell formation and recent advances in stem cell manipulation, such as seen with inducible pluripotent stem cells (iPSCs) and epidermal neural crest stem cells (EPI-NCSCs), have opened new possibilities to advance research in translational stem cell therapies for individuals with hearing loss. Despite this, more detailed network maps of gene expression are needed, including an appreciation for the roles of microRNAs (miRs), key regulators of transcriptional gene networks. To harness the true potential of stem cells for hair cell regeneration, basic science and clinical medicine must work together to expedite the transition from bench to bedside by elucidating the full mechanisms of inner ear hair cell development, including a focus on the role of miRs, and adapting this knowledge safely and efficiently to stem cell technologies.
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http://dx.doi.org/10.3390/ph4060848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180915PMC
June 2011

Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear.

Dev Dyn 2011 Jun 29;240(6):1373-90. Epub 2011 Mar 29.

Department of Biology, University of Iowa, Iowa City, Iowa, USA.

Ear development requires interactions of transcription factors for proliferation and differentiation. The proto-oncogene N-Myc is a member of the Myc family that regulates proliferation. To investigate the function of N-Myc, we conditionally knocked out N-Myc in the ear using Tg(Pax2-Cre) and Foxg1(KiCre). N-Myc CKOs had reduced growth of the ear, abnormal morphology including fused sensory epithelia, disrupted histology, and disorganized neuronal innervation. Using Thin-Sheet Laser Imaging Microscopy (TSLIM), 3D reconstruction and quantification of the cochlea revealed a greater than 50% size reduction. Immunochemistry and in situ hybridization showed a gravistatic organ-cochlear fusion and a "circularized" apex with no clear inner and outer hair cells. Furthermore, the abnormally developed cochlea had cross innervation from the vestibular ganglion near the basal tip. These findings are put in the context of the possible functional relationship of N-Myc with a number of other cell proliferative and fate determining genes during ear development.
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http://dx.doi.org/10.1002/dvdy.22620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092837PMC
June 2011

Dissecting the molecular basis of organ of Corti development: Where are we now?

Hear Res 2011 Jun 21;276(1-2):16-26. Epub 2011 Jan 21.

Department of Biology, College of Liberal Arts and Sciences, 143 BB, University of Iowa, Iowa City, IA 52242, USA.

This review summarizes recent progress in our understanding of the molecular basis of cochlear duct growth, specification of the organ of Corti, and differentiation of the different types of hair cells. Studies of multiple mutations suggest that developing hair cells are involved in stretching the organ of Corti through convergent extension movements. However, Atoh1 null mutants have only undifferentiated and dying organ of Corti precursors but show a near normal extension of the cochlear duct, implying that organ of Corti precursor cells can equally drive this process. Some factors influence cochlear duct growth by regulating the cell cycle and proliferation. Shortened cell cycle and premature cell cycle exit can lead to a shorter organ of Corti with multiple rows of hair cells (e.g., Foxg1 null mice). Other genes affect the initial formation of a cochlear duct with or without affecting the organ of Corti. Such observations are consistent with evolutionary data that suggest some developmental uncoupling of cochlear duct from organ of Corti formation. Positioning the organ of Corti requires multiple genes expressed in the organ of Corti and the flanking region. Several candidate factors have emerged but how they cooperate to specify the organ of Corti and the topology of hair cells remains unclear. Atoh1 is required for differentiation of all hair cells, but regulation of inner versus outer hair cell differentiation is still unidentified. In summary, the emerging molecular complexity of organ of Corti development demands further study before a rational approach towards regeneration of unique types of hair cells in specific positions is possible.
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http://dx.doi.org/10.1016/j.heares.2011.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097286PMC
June 2011

Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti.

Hear Res 2011 May 10;275(1-2):66-80. Epub 2010 Dec 10.

University of Iowa, Department of Biology, 143 BB, Iowa City, IA 52242, USA.

Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell death. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), the remaining cells of the organ of Corti are transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, except for the apex and few fibers in the base. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.
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http://dx.doi.org/10.1016/j.heares.2010.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065550PMC
May 2011