PhD Biological StructureUniversity of Washington, School of Medicine, Seattle, WA 2009
BA Biology Indiana University, Bloomington, IN 2004
BA Communications StudiesUniversity of Michigan, Ann Arbor, MI 2000
2017-present Instructor at Stanford University School of Medicine, Department of Otolaryngology
2016-2017 Research Associate at Stanford University School of Medicine, Department of Otolaryngology
Mentor: Stefan Heller
2010-2016 Postdoctoral Fellow at Stanford University School of Medicine, Department of Otolaryngology
Mentor: Stefan Heller, External Co-Mentor Andy Groves
2009-2010 Postdoctoral Fellow at University of Washington Institute for Stem Cells and Regenerative Medicine
Mentor: Olivia Bermingham-McDonogh
2004-2009 Doctoral Student at University of Washington, School of Medicine, Department of Biological Structure Advisor: Tom Reh.
2017-2020 NIDCD Early Career Research (ECR) Award (R21): 1 R21 DC016125-01
Title: Otic Sensory Lineage Specification and Gene Regulation
PI: Hartman, BH
Direct Costs: $100,000/year.
Project Period: April 1, 2017 - March 31, 2020
Total Cumulative Federal Direct and Indirect for Project Period: $484,037
2013-2016 NIDCD Postdoctoral Individual F32 National Research Service Award (NRSA) Title: Genetic Control of Otic Induction Stanford
2011-2013 Extramural Pediatric Loan Repayment Program Award, NIH/NIDCD
2006-2009 Predoctoral Fellowship, NIH/NICHD Institutional T32 NRSA in Developmental Biology, UW
2004-2005 Predoctoral Fellowship, NIH/NIDCD Institutional T32 NRSA in Speech and Hearing Sciences, UW
*Hartman BH, Heller S. Proximal and Distal Cis Elements Regulate Expression of the Inner Ear-Specific Ubiquitin Ligase Subunit Fbx2. In Preparation
16 Hartman BH, Bӧscke R, Ellwanger D, Keymeulen S, Scheibinger M, Heller S. Fbxo2VHC mouse and embryonic stem cell reporter lines delineate in vitro-generated inner ear sensory epithelia cells and enable otic lineage selection and Cre-recombination. Developmental Biology, Epub Ahead of Print, Sept 1, 2018
15 Lee J, Bӧscke R, Tang PC, Hartman BH, Heller S, Koehler KR. Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids. Cell Reports 2018 Jan 2;22(1):242-254
14 Hartman BH, Durruthy-Durruthy R, Laske RD, Losorelli S, Heller S. Identification and characterization of mouse otic sensory lineage genes. Frontiers in Cellular Neuroscience 2015 Mar 19;9:79.
13 Durruthy-Durruthy R, Gottlieb A, Hartman BH, Waldhaus J, Laske RD, Altman R, Heller S. Reconstruction of the Mouse Otocyst and Early Neuroblast Lineage at Single-Cell Resolution. Cell 2014 May 8;157(4):964-78.
12 Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Intrinsic regenerative potential of murine cochlear supporting cells. Scientific Reports 2011;1:26.
11 Nelson BR, Ueki Y, Reardon S, Karl MO, Georgi S, Hartman BH, Lamba DA, Reh TA. Genome-wide analysis of Müller glial differentiation reveals a requirement for Notch signaling in postmitotic cells to maintain the glial fate. PLoS One 2011;6(8):e22817.
10 Hartman BH, Reh TA, Bermingham-McDonogh O. Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear. Proceedings of the National Academy of Sciences U S A. 2010 Sep 7;107(36):15792-7. * 100+ citations
9 Hartman BH, Nelson BR, Reh TA, and Bermingham-McDonogh O. Delta/Notch-Like EGF-related Receptor (DNER) is expressed in hair cells and neurons in the developing and adult mouse inner ear. Journal for the Association of Research in Otolaryngology 2010 Jan 8.
Instructor, Otolaryngology - Head & Neck Surgery Divisions
Current Research and Scholarly Interests
My long-term research goal is to develop translatable regenerative medicine for the inner ear. In essence, the mammalian organ of Corti lacks the capacity to regenerate because it’s cells become terminally differentiated and lose the ability to behave as prosensory progenitors. In contrast, supporting cells of birds and other non-mammals retain some properties of their prosensory origins and, in response to hair cell damage, are capable of producing new hair cells through division and/or transdifferentiation.
My long-term research strategy is aimed at the following big picture questions, related to understanding the prosensory state:
What are the key transcriptional and epigenetic features of prosensory cells and how can we restore these features to the damaged adult organ of Corti to enable regeneration?
What mechanisms regulate gene expression in prosensory cells and the adult cochlea and how can we exploit these to develop targeted and effective gene therapy for inner ear regeneration?
How can we best utilize and manipulate otic development in embryonic stem cell models to better understand and control the prosensory state?
What biological and engineering principles can we utilize to advance embryonic stem cell models toward controlled in vitro production of functional sensory organs?
Fbxo2VHC mouse and embryonic stem cell reporter lines delineate in vitro-generated inner ear sensory epithelia cells and enable otic lineage selection and Cre-recombination.
While the mouse has been a productive model for inner ear studies, a lack of highly specific genes and tools has presented challenges. The absence of definitive otic lineage markers and tools is limiting in vitro studies of otic development, where innate cellular heterogeneity and disorganization increase the reliance on lineage-specific markers. To address this challenge in mice and embryonic stem (ES) cells, we targeted the lineage-specific otic gene Fbxo2 with a multicistronic reporter cassette (Venus/Hygro/CreER = VHC). In otic organoids derived from ES cells, Fbxo2VHC specifically delineates otic progenitors and inner ear sensory epithelia. In mice, Venus expression and CreER activity reveal a cochlear developmental gradient, label the prosensory lineage, show enrichment in a subset of type I vestibular hair cells, and expose strong expression in adult cerebellar granule cells. We provide a toolbox of multiple spectrally distinct reporter combinations for studies that require use of fluorescent reporters, hygromycin selection, and conditional Cre-mediated recombination.
View details for DOI 10.1016/j.ydbio.2018.08.013
View details for PubMedID 30179592
Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids.
2018; 22 (1): 242–54
The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.
View details for DOI 10.1016/j.celrep.2017.12.007
View details for PubMedID 29298425
Identification and characterization of mouse otic sensory lineage genes
FRONTIERS IN CELLULAR NEUROSCIENCE
Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells.
View details for DOI 10.3389/fncel.7015.00079
View details for Web of Science ID 000352432300001
View details for PubMedID 25852475
Reconstruction of the Mouse Otocyst and Early Neuroblast Lineage at Single-Cell Resolution
2014; 157 (4): 964-978
The otocyst harbors progenitors for most cell types of the mature inner ear. Developmental lineage analyses and gene expression studies suggest that distinct progenitor populations are compartmentalized to discrete axial domains in the early otocyst. Here, we conducted highly parallel quantitative RT-PCR measurements on 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling-pathway-associated transcripts, and novel otic-specific genes. By applying multivariate cluster, principal component, and network analyses to the data matrix, we were able to readily distinguish the delaminating neuroblasts and to describe progressive states of gene expression in this population at single-cell resolution. It further established a three-dimensional model of the otocyst in which each individual cell can be precisely mapped into spatial expression domains. Our bioinformatic modeling revealed spatial dynamics of different signaling pathways active during early neuroblast development and prosensory domain specification. PAPERFLICK:
View details for DOI 10.1016/j.cell.2014.03.036
View details for Web of Science ID 000335765500022
View details for PubMedID 24768691
Genome-Wide Analysis of Muller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate
2011; 6 (8): e22817
Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10-14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.
View details for DOI 10.1371/journal.pone.0022817
View details for Web of Science ID 000293511900015
View details for PubMedID 21829655
View details for PubMedCentralID PMC3149061
Intrinsic regenerative potential of murine cochlear supporting cells
The lack of cochlear regenerative potential is the main cause for the permanence of hearing loss. Albeit quiescent in vivo, dissociated non-sensory cells from the neonatal cochlea proliferate and show ability to generate hair cell-like cells in vitro. Only a few non-sensory cell-derived colonies, however, give rise to hair cell-like cells, suggesting that sensory progenitor cells are a subpopulation of proliferating non-sensory cells. Here we purify from the neonatal mouse cochlea four different non-sensory cell populations by fluorescence-activated cell sorting (FACS). All four populations displayed proliferative potential, but only lesser epithelial ridge and supporting cells robustly gave rise to hair cell marker-positive cells. These results suggest that cochlear supporting cells and cells of the lesser epithelial ridge show robust potential to de-differentiate into prosensory cells that proliferate and undergo differentiation in similar fashion to native prosensory cells of the developing inner ear.
View details for DOI 10.1038/srep00026
View details for Web of Science ID 000296046900002
View details for PubMedID 22355545
View details for PubMedCentralID PMC3216513
- Three-Dimensional Imaging of the Mouse Organ of Corti Cytoarchitecture for Mechanical Modeling 11th International Workshop on the Mechanics of Hearing AMER INST PHYSICS. 2011
Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (36): 15792-15797
During inner ear morphogenesis, the process of prosensory specification defines the specific regions of the otic epithelium that will give rise to the six separate inner ear organs essential for hearing and balance. The mechanism of prosensory specification is not fully understood, but there is evidence that the Notch intercellular signaling pathway plays a critical role. The Notch ligand Jagged1 (Jag1) is expressed in the prosensory domains, and mutation of Jag1 impairs sensory formation. Furthermore, pharmacological inhibition of Notch in vitro during prosensory specification disrupts the prosensory process. Additionally, activation of Notch by cDNA electroporation in chick otocysts results in formation of ectopic sensory patches. Here we test whether Notch activity is sufficient for prosensory specification in the mouse, using a Cre-/loxP approach to conditionally activate the Notch pathway in nonsensory regions of the inner ear epithelia during different stages of otic vesicle morphogenesis. We find that broad ectopic activation of Notch at very early developmental stages causes induction of prosensory markers throughout the entire otic epithelium. At later stages of development, activation of Notch in nonsensory regions leads to induction of sensory patches that later differentiate to form complete ectopic sensory structures. Activation of Notch in isolated nonsensory cells results in lateral induction of Jag1 expression in neighboring cells and spreading of prosensory specification to the adjacent cells through an intercellular mechanism. These results support a model where activation of Notch and propagation through lateral induction promote prosensory character in specific regions of the developing otocyst.
View details for DOI 10.1073/pnas.1002827107
View details for Web of Science ID 000281637800032
View details for PubMedID 20798046
View details for PubMedCentralID PMC2936601
Delta/Notch-Like EGF-Related Receptor (DNER) is Expressed in Hair Cells and Neurons in the Developing and Adult Mouse Inner Ear
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2010; 11 (2): 187-201
The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER-/- mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.
View details for DOI 10.1007/s10162-009-0203-x
View details for Web of Science ID 000279397500004
View details for PubMedID 20058045
View details for PubMedCentralID PMC2862923
Hes5 Expression in the Postnatal and Adult Mouse Inner Ear and the Drug-Damaged Cochlea
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2009; 10 (3): 321-340
The Notch signaling pathway is known to have multiple roles during development of the inner ear. Notch signaling activates transcription of Hes5, a homologue of Drosophila hairy and enhancer of split, which encodes a basic helix-loop-helix transcriptional repressor. Previous studies have shown that Hes5 is expressed in the cochlea during embryonic development, and loss of Hes5 leads to overproduction of auditory and vestibular hair cells. However, due to technical limitations and inconsistency between previous reports, the precise spatial and temporal pattern of Hes5 expression in the postnatal and adult inner ear has remained unclear. In this study, we use Hes5-GFP transgenic mice and in situ hybridization to report the expression pattern of Hes5 in the inner ear. We find that Hes5 is expressed in the developing auditory epithelium of the cochlea beginning at embryonic day 14.5 (E14.5), becomes restricted to a particular subset of cochlear supporting cells, is downregulated in the postnatal cochlea, and is not present in adults. In the vestibular system, we detect Hes5 in developing supporting cells as early as E12.5 and find that Hes5 expression is maintained in some adult vestibular supporting cells. In order to determine the effect of hair cell damage on Notch signaling in the cochlea, we damaged cochlear hair cells of adult Hes5-GFP mice in vivo using injection of kanamycin and furosemide. Although outer hair cells were killed in treated animals and supporting cells were still present after damage, supporting cells did not upregulate Hes5-GFP in the damaged cochlea. Therefore, absence of Notch-Hes5 signaling in the normal and damaged adult cochlea is correlated with lack of regeneration potential, while its presence in the neonatal cochlea and adult vestibular epithelia is associated with greater capacity for plasticity or regeneration in these tissues; which suggests that this pathway may be involved in regulating regenerative potential.
View details for DOI 10.1007/s10162-009-0162-2
View details for Web of Science ID 000268495600002
View details for PubMedID 19373512
View details for PubMedCentralID PMC2757554
Acheate-scute like 1 (Ascl1) Is Required for Normal Delta-like (Dll) Gene Expression and Notch Signaling During Retinal Development
2009; 238 (9): 2163–78
Delta gene expression in Drosophila is regulated by proneural basic helix-loop-helix (bHLH) transcription factors, such as acheate-scute. In vertebrates, multiple Delta-like and proneural bHLH genes are expressed during neurogenesis, especially in the retina. We recently uncovered a relationship between Acheate-scute like 1 (Ascl1), Delta-like genes, and Notch in chick retinal progenitors. Here, we report that mammalian retinal progenitors are also the primary source of Delta-like genes, likely signaling through Notch among themselves, while differentiating neurons expressed Jagged2. Ascl1 is coexpressed in Delta-like and Notch active progenitors, and required for normal Delta-like gene expression and Notch signaling. We also reveal a role for Ascl1 in the regulation of Hes6, a proneurogenic factor that inhibits Notch signaling to promote neural rather than glial differentiation. Thus, these results suggest a molecular mechanism whereby attenuated Notch levels coupled with reduced proneurogenic activity in progenitors leads to increased gliogenesis and decreased neurogenesis in the Ascl1-deficient retina.
View details for DOI 10.1002/dvdy.21848
View details for Web of Science ID 000269686400005
View details for PubMedID 19191219
View details for PubMedCentralID PMC2905851
Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea
2008; 316 (1): 87-99
In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.
View details for DOI 10.1016/j.ydbio.2008.01.006
View details for Web of Science ID 000254845200008
View details for PubMedID 18291358
View details for PubMedCentralID PMC2362132
NeuroD1 regulates expression of thyroid hormone receptor beta 2 and cone opsins in the developing mouse retina
JOURNAL OF NEUROSCIENCE
2008; 28 (3): 749–56
The correct patterning of opsin expression in cone photoreceptors is critical for normal color vision. Thyroid hormone, and one of its receptors [thyroid hormone receptor beta2 (TRbeta2)], is an important regulator of opsin expression during cone photoreceptor development. Mice have two genes, encoding medium-wavelength (M) and short-wavelength (S) cone opsins. Targeted deletion of TRbeta2 leads to a uniform expression of S-opsin in all cone photoreceptors and a loss of M-opsin. The control of expression of TRbeta2 is therefore central to cone differentiation, yet there is little known about its regulation in the retina. We now report that the proneural bHLH (basic helix-loop-helix) transcription factor, NeuroD1, is necessary for sustained expression of TRbeta2 in immature cone photoreceptors. Mice deficient in NeuroD1 develop an opsin phenotype virtually identical with that of TRbeta2-deficient mice: all cones express S-opsin, and none expresses M-opsin. The introduction of NeuroD1 into embryonic retinal explants from NeuroD1-/- mice restores TRbeta2 expression. NeuroD1 binds an E-box in the intron control region of the TRbeta2 gene that mediates cone-specific expression, suggesting that NeuroD1 is a critical contributory factor to the expression of TRbeta2 in cones. These results thus connect the proneural pathway with opsin selection to ensure correct cone patterning during retinal development.
View details for DOI 10.1523/JNEUROSCI.4832-07.2008
View details for Web of Science ID 000252432700018
View details for PubMedID 18199774
Dll3 is expressed in developing hair cells in the mammalian cochlea
2007; 236 (10): 2875-2883
Notch mediates the process of lateral inhibition that controls the production of hair cells in the inner ear. Hair cells are known to express Notch ligands Dll1 and Jag2, which signal through Notch1 in adjacent supporting cells. However, recent genetic and pharmacological studies indicate that the level of Notch-mediated lateral inhibition is greater than can be accounted for by Dll1 and Jag2. Here, we report that another Notch ligand, Dll3, is expressed in developing hair cells, in a pattern that overlaps that of Dll1 and Jag2. We analyzed the cochleae of Dll3(pu) mutant mice, but did not detect any abnormalities. However, earlier studies have demonstrated that there is functional redundancy among Notch ligands in cochlear development and loss of one ligand can be at least partially compensated for by another. Thus Dll3 may play a role in lateral inhibition similar to that of Dll1 and Jag2.
View details for DOI 10.1002/dvdy.21307
View details for Web of Science ID 000250192100016
View details for PubMedID 17823936
Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells
2007; 304 (2): 479-498
In the developing nervous system, the balance between proliferation and differentiation is critical to generate the appropriate numbers and types of neurons and glia. Notch signaling maintains the progenitor pool throughout this process. While many components of the Notch pathway have been identified, the downstream molecular events leading to neural differentiation are not well understood. We have taken advantage of a small molecule inhibitor, DAPT, to block Notch activity in retinal progenitor cells, and analyzed the resulting molecular and cellular changes over time. DAPT treatment causes a massive, coordinated differentiation of progenitors that produces cell types appropriate for their developmental stage. Transient exposure of retina to DAPT for specific time periods allowed us to define the period of Notch inactivation that is required for a permanent commitment to differentiate. Inactivation of Notch signaling revealed a cascade of proneural bHLH transcription factor gene expression that correlates with stages in progenitor cell differentiation. Microarray/QPCR analysis confirms the changes in Notch signaling components, and reveals new molecular targets for investigating neuronal differentiation. Thus, transient inactivation of Notch signaling synchronizes progenitor cell differentiation, and allows for a systematic analysis of key steps in this process.
View details for DOI 10.1016/j.ydbio.2007.01.001
View details for Web of Science ID 000245819600004
View details for PubMedID 17280659