Emx2 defines bidirectional polarity of neuromasts by changing hair-bundle orientation and not hair-cell positions
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA640655 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12055985.fastq.gz | Fastqsanger.gz | |||
| SRR12055986.fastq.gz | Fastqsanger.gz | |||
| SRR12055987.fastq.gz | Fastqsanger.gz | |||
| SRR12055988.fastq.gz | Fastqsanger.gz |
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Project description:The bidirectional sensitivity of neuromasts to water flow in the zebrafish lateral line system is attributed to the opposite orientation of the hair bundles on top of sensory hair cells (HC) within a neuromast. After each HC precursor divides to form two nascent HCs, HCs of the same bundle orientation are positioned on one side of the neuromast, across from HCs with opposite bundle orientation. The transcription factor emx2 is expressed in only one of the sibling HCs. Loss or gain of function of emx2 in HCs causes unidirectional hair bundle orientation in neuromasts. It is not clear whether Emx2 is required specifically in establishing hair bundle orientation after HC formation or Emx2 has an earlier role in HC fate and/or positioning, which indirectly changes hair bundle orientation. A phenomenon, in which nascent sibling HCs exchange positions with each other, has been postulated to be the mechanism for HCs to acquire their designated positions within the neuromast. We asked whether Emx2 is involved in positional acquisition of HCs within the neuromast. Using live-imaging and our emx2 reporter we find that the HC rearrangement, redefined as two processes named Rock & Roll are required for HCs to acquire their positions. Although Emx2 regulates the duration of the Rock and the frequency of Roll of nascent HCs, it is not required for their positional acquisition. Instead, Emx2 regulates the morphology of nascent HCs, which facilitates the rearrangement process.
2021-01-19 | GSE152859 | GEO
Project description:The asymmetric location of stereociliary bundle (hair bundle) on the apical surface of mechanosensory hair cells (HCs) dictates the direction in which a given HC can respond to cues such as sound, head movements, and water pressure. Notably, vestibular sensory organs of the inner ear, the maculae, exhibit a line of polarity reversal (LPR) across which, hair bundles are polarized in a mirror-image pattern. Similarly, HCs in neuromasts of the zebrafish lateral line system are generated as pairs, and two sibling HCs develop opposite hair bundle orientations. Within these sensory organs, expression of the transcription factor Emx2 is restricted to only one side of the LPR in the maculae or one of the two sibling HCs in neuromasts. Emx2 mediates hair bundle polarity reversal in these restricted subsets of HCs and generates the mirror-image pattern of the sensory organs. Downstream effectors of Emx2 control bundle polarity cell-autonomously via heterotrimeric G proteins.
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Project description:This dataset consists of single-cell RNA-seq (mcSCRBseq) data of neuromast hair cells from wild type and Emx2 mutant zebrafish larvae. Posterior lateral-line neuromast hair cells were isolated by fluorescence activated cell sorting (FACS) from the dissociated trunks of wild type and Emx2-mutant Tg[myo6b:actb1-EGFP] transgenic zebrafish larvae expressing the green fluorescent protein EGFP in hair cells and unipotent hair-cell progenitors (UHCPs).
2020-03-05 | GSE143663 | GEO
Project description:Emx2 is a homeobox transcription factor that plays a critical role in development. Olfactory sensory neuron axons from Emx2 knockout mice fail to innervate their target tissue, the olfactory bulb. Homeobox transcription factors may also play an important role in olfactory receptor expression. We used microarrays to analyze differences in mRNA abundance in the olfactory epithelium of Emx2 wildtype and knockout embryonic day 18.5 mice. Keywords: Knockout-Wildtype comparision Total RNA from 9 Emx2 wildtype and 9 Emx2 knockout mice was analyzed. Equal amounts of RNA from 3 animals was pooled to create one biological sample, 3 pools per genotype, 6 chips total. RNA was hybridized to the Affymetrix Mouse Exon 1.0 ST array.
2008-10-15 | E-GEOD-12135 | biostudies-arrayexpress
Project description:Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.
| S-EPMC8129141 | biostudies-literature
Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.
2013-01-08 | PXD000104 | Pride
Project description:Purified hair bundles and utricular epithelium from E19-E20 chick inner ears were analyzed by LC-MS/MS to determine the abundant and enriched proteins of the hair bundle. This dataset used a Thermo LTQ Velos mass spectrometer for protein detection.
2015-11-17 | PXD002414 | Pride
Project description:Purified utricle hair bundles and utricular epithelium from P3-P7 rat inner ears were analyzed by LC-MS/MS to determine the abundant and enriched proteins of the hair bundle. This dataset used a Thermo LTQ mass spectrometer for protein detection.
2015-11-17 | PXD002416 | Pride
Project description:Purified hair bundles and utricular epithelium from E19-E20 chick inner ears were analyzed by LC-MS/MS to determine the abundant and enriched proteins of the hair bundle. This dataset used a Thermo LTQ mass spectrometer for protein detection.
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