Doctor of Philosophy, Goethe University Frankfurt (2010)
Diplom, Goethe University Frankfurt (2006)
Honors & Awards
Research Fellowship, German Research Foundation (DFG) (2011 - 2014)
Single-cell proteomics reveals changes in expression during hair-cell development.
Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.
View details for DOI 10.7554/eLife.50777
View details for PubMedID 31682227
- In vitro Methods to Cultivate Spiral Ganglion Cells, and Purification of Cellular Subtypes for Induced Neuronal Reprogramming FRONTIERS IN NEUROSCIENCE 2018; 12
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 PubMedID 30179592
Transcriptional Dynamics of Hair-Bundle Morphogenesis Revealed with CellTrails.
2018; 23 (10): 2901–14.e14
Protruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics. Utilizing quantitative gene-expression data for key bundle proteins from single cells of the developing chick utricle, we reconstructed de novo a bifurcating trajectory that spanned from progenitor cells to mature striolar and extrastriolar hair cells. Extraction and alignment of developmental trails and association of pseudotime with bundle length measurements linked expression dynamics of individual genes with bundle growth stages. Differential trail analysis revealed high-resolution dynamics of transcripts that control striolar and extrastriolar bundle development, including those that encode proteins that regulate [Ca2+]i or mediate crosslinking and lengthening of actin filaments.
View details for PubMedID 29874578
Aminoglycoside Damage and Hair Cell Regeneration in the Chicken Utricle.
Journal of the Association for Research in Otolaryngology : JARO
In this study, we present a systematic characterization of hair cell loss and regeneration in the chicken utricle in vivo. A single unilateral surgical delivery of streptomycin caused robust decline of hair cell numbers in striolar as well as extrastriolar regions, which in the striola was detected very early, 6 h post-insult. During the initial 12 h of damage response, we observed global repression of DNA replication, in contrast to the natural, mitotic hair cell production in undamaged control utricles. Regeneration of hair cells in striolar and extrastriolar regions occurred via high rates of asymmetric supporting cell divisions, accompanied by delayed replenishment by symmetric division. While asymmetric division of supporting cells is the main regenerative response to aminoglycoside damage, the detection of symmetric divisions supports the concept of direct transdifferentiation where supporting cells need to be replenished after their phenotypic conversion into new hair cells. Supporting cell divisions appear to be well coordinated because total supporting cell numbers throughout the regenerative process were invariant, despite the initial large-scale loss of hair cells. We conclude that a single ototoxic drug application provides an experimental framework to study the precise onset and timing of utricle hair cell regeneration in vivo. Our findings indicate that initial triggers and signaling events occur already within a few hours after aminoglycoside exposure. Direct transdifferentiation and asymmetric division of supporting cells to generate new hair cells subsequently happen largely in parallel and persist for several days.
View details for PubMedID 29134476
A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.
2013; 8 (6)
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.
View details for DOI 10.1371/journal.pone.0066026
View details for PubMedID 23750277
View details for PubMedCentralID PMC3672136
Generation of the tamoxifen-inducible DBH-Cre transgenic mouse line DBH-CT
2011; 49 (12): 935-941
We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the dopamine-β-hydroxylase promoter. By crossing to the ROSA26 reporter mice we show that tamoxifen-induced Cre recombinase in adult mice specifically activates β-galactosidase expression in differentiated noradrenergic neurons of the central and peripheral nervous system. Tamoxifen application in adult mice did not induce β-galactosidase activity in parasympathetic neurons that transiently express DBH during development. Thus, this transgenic mouse line represents a valuable tool to study gene function in mature noradrenergic neurons by conditional inactivation.
View details for DOI 10.1002/dvg.20773
View details for Web of Science ID 000297634000006
View details for PubMedID 21634003
The transcription factors AP-2 beta and AP-2 alpha are required for survival of sympathetic progenitors and differentiated sympathetic neurons
2011; 355 (1): 89-100
Differentiation of sympathetic neurons is controlled by a group of transcription factors, including Phox2b, Ascl1, Hand2 and Gata3, induced by bone morphogenetic proteins (BMPs) in progenitors located in ganglion primordia at the dorsal aorta. Here, we address the function of the transcription factors AP-2β and AP-2α, expressed in migrating neural crest cells (NCC) and maintained in sympathetic progenitors and differentiated neurons. The elimination of both AP-2α and AP-2β results in the virtually complete absence of sympathetic and sensory ganglia due to apoptotic cell death of migrating NCC. In the AP-2β knockout only sympathetic ganglia (SG) are targeted, leading to a reduction in ganglion size by about 40%, which is also caused by apoptotic death of neural crest progenitors. The conditional double knockout of AP-2α and AP-2β in sympathetic progenitors and differentiated noradrenergic neurons results in a further decrease in neuron number, leading eventually to small sympathetic ganglion rudiments postnatally. The elimination of AP-2β also leads to the complete absence of noradrenergic neurons of the Locus coeruleus (LC). Whereas AP-2α/β transcription factors are in vivo not required for the onset or maintenance of noradrenergic differentiation, their essential survival functions are demonstrated for sympathetic progenitors and noradrenergic neurons.
View details for DOI 10.1016/j.ydbio.2011.04.011
View details for Web of Science ID 000291454000009
View details for PubMedID 21539825
Neuroblastoma Phox2b Variants Stimulate Proliferation and Dedifferentiation of Immature Sympathetic Neurons
JOURNAL OF NEUROSCIENCE
2010; 30 (3): 905-915
Neuroblastoma is a pediatric tumor that is thought to arise from autonomic precursors in the neural crest. Mutations in the PHOX2B gene have been observed in familial and sporadic forms of neuroblastoma and represent the first defined genetic predisposition for neuroblastoma. Here, we address the mechanisms that may underlie this predisposition, comparing the function of wild-type and mutant Phox2b proteins ectopically expressed in proliferating, embryonic sympathetic neurons. Phox2b displays a strong antiproliferative effect, which is lost in all Phox2b neuroblastoma variants analyzed. In contrast, an increase in sympathetic neuron proliferation is elicited by Phox2b variants with mutations in the homeodomain when endogenous Phox2b levels are lowered by siRNA-mediated knockdown to mimic the situation of heterozygous PHOX2B mutations in neuroblastoma. The increased proliferation is blocked by Hand2 knockdown and the antiproliferative Phox2b effects are rescued by Hand2 overexpression, implying Hand2 in Phox2b-mediated proliferation control. A Phox2b variant with a nonsense mutation in the homeodomain elicits, in addition, a decreased expression of characteristic marker genes. Together, these results suggest that PHOX2B mutations predispose to neuroblastoma by increasing proliferation and promoting dedifferentiation of cells in the sympathoadrenergic lineage.
View details for DOI 10.1523/JNEUROSCI.5368-09.2010
View details for Web of Science ID 000273779200012
View details for PubMedID 20089899
In vivo role for CREB signaling in the noradrenergic differentiation of sympathetic neurons
MOLECULAR AND CELLULAR NEUROSCIENCE
2009; 42 (2): 142-151
Signaling pathways involving cAMP and CREB have been implicated in several aspects of sympathetic neuron differentiation. Here, we used in vivo loss-of-function approaches in both mouse and chick embryos to characterize the physiological role of cAMP/CREB. Whereas sympathetic neuron development proceeds normally in CREB-deficient mouse embryos, a decrease in noradrenergic differentiation (TH, DBH) was observed in chick sympathetic ganglia in response to ACREB, a dominant-negative CREB variant which interferes with the function of all CREB family members. In contrast, expression of the generic neuronal marker SCG10 was not affected by ACREB. As the decrease in noradrenergic gene expression is compensated at later stages of development and TH expression in differentiated neurons is not CREB-dependent, a transient role for CREB is proposed, accelerating noradrenergic but not generic neuronal differentiation of sympathetic neurons.
View details for DOI 10.1016/j.mcn.2009.06.007
View details for Web of Science ID 000269171700007
View details for PubMedID 19545628
The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons
2009; 329 (2): 191-200
The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.
View details for DOI 10.1016/j.ydbio.2009.02.020
View details for Web of Science ID 000266048300003
View details for PubMedID 19254708
View details for PubMedCentralID PMC2746555