Doctor of Philosophy, J W Goethe Universitat Frankfurt (2010)
Stefan Heller, Postdoctoral Faculty Sponsor
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
We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the dopamine-?-hydroxylase promoter. By crossing to the ROSA26 reporter mice we show that tamoxifen-induced Cre recombinase in adult mice specifically activates ?-galactosidase expression in differentiated noradrenergic neurons of the central and peripheral nervous system. Tamoxifen application in adult mice did not induce ?-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
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-2? and AP-2?, expressed in migrating neural crest cells (NCC) and maintained in sympathetic progenitors and differentiated neurons. The elimination of both AP-2? and AP-2? results in the virtually complete absence of sympathetic and sensory ganglia due to apoptotic cell death of migrating NCC. In the AP-2? 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-2? and AP-2? 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-2? also leads to the complete absence of noradrenergic neurons of the Locus coeruleus (LC). Whereas AP-2?/? 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