Additional file 3: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNA raw counts. Raw counts from sequencing of each of the small miRNA libraries. The number of sequences that correspond to each miRNA is listed. (XLSM 60 kb

Additional file 4: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNAs normalized in reads-per-million (RPM). The number of reads of each miRNA was normalized by dividing the raw counts by the total number of million aligned reads per sample, i.e. reads per million (RPM). (XLSM 98 kb

Additional file 1: of MiR-210 promotes sensory hair cell formation in the organ of corti

Number of miRSeq reads and alignment statistics. (XLSM 8 kb

R-Spondin Potentiates Wnt/β-Catenin Signaling through Orphan Receptors LGR4 and LGR5

<div><p>The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4−/− crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.</p> </div

RSPO1 interacts with the extracellular domain of LGR4 and LGR5.

<p>(a) RSPO1 binds to cells overexpressing LGR4 or LGR5. HEK293T cells transiently overexpressing HA-tagged LGR4 or LGR5 were incubated with RSPO1-GFP-conditioned medium for 1 h at 37°C and subjected to immunofluorescence analysis. RSPO1-GFP only binds to cells overexpressing LGR4 or LGR5. Also note the co-localization of RSPO1-GFP and LGR4 or LGR5 in some intracellular vesicular structures. (b) Co-immunoprecipitation of LGR4-ECD and RSPO1. HEK293T cells were transiently transfected with plasmids expressing either C-terminally V5-6xHis-tagged LGR4-ECD or Fc-tagged RSPO1. Supernatants of these cultures and of control non-transfected HEK293T cells were mixed in combinations as indicated and subjected to Fc-pulldown experiments. Eluates (E), flow-through fractions (F) and input lysates (I) were analyzed by Western Blot analyses. Immunoreagents against IgG or V5 were applied to detect RSPO1 (a-Fc) and LGR4-ECD (α-V5), respectively. The position of the 62 kDa marker band is indicated on the right.</p

Lgr4-RSPO1-potentiated Wnt/β-catenin activation is critical for organoid survival.

<p>(a) Embryonic organoid cultures from Lgr5 (−/−), Lgr5 (−/+) and Lgr5 (+/+) are viable. Organoid cultures from E18.5 embryos of heterozygous Lgr5 KO timed matings were established. Depicted are representative organoids at day 1 (D1), day 2 (D2) and day 4 (D4) following passaging. (b) Lack of embryo Lgr4 (−/−) organoid culture growth. Organoid cultures from E16.5 embryos of heterozygous Lgr4 KO timed matings were established and allowed to grow in culture under standard growth conditions. Viability was scored by microscopic analysis over at least 2 weeks. Left column, representative E16.5 embryo of each genotype; columns 2–5, depicted are representative organoid cultures at day 1 (D1), day 4 (D4), day 6 (D6) and day 14 (D14) following passaging. Whereas wild-type (+/+) and heterozygous Lgr4 (−/+) cultures grew out, homozygous mutant Lgr4 (−/−) cultures could not be obtained. (c) The numbers shown in the table indicate the numbers of organoid cultures that grew out offset against the number of organoids prepared of each genotype; (*), one (+/+) culture grew for 1 week but subsequently lost viability for unknown reasons. (d) Lack of organoid growth upon RSPO1 withdrawal could be overcome by addition of a GSK3 inhibitor. Organoid cultures derived from adult C57Bl/6 mice were grown under standard conditions. At passage 2, multiple aliquots of sub-cultures were assessed for growth under the following conditions: No RSPO1, RSPO1 withdrawal; RSPO1, presence of 10 ng/ml RSPO1; No RSPO1+ CHIR99021, 5 uM CHIR99021 in the absence of RSPO1. Organoid growth was monitored over 8 days by microscopy. CHIR99021 was able to partially rescue organoid growth. Shown are 2 representative cultures for each condition (top and bottom, each). Note that the organoids treated with the GSK3 inhibitor reveal a cyst phenotype without crypts.</p

LGR4 is required for RSPO1-induced Wnt/β-catenin signaling.

<p>(a) Depletion of LGR4 inhibits RSPO1-, but not Wnt3a-, induced STF reporter activation. HEK293T-STF cells were transfected with pGL2, LGR4, LGR5, or LRP6 siRNA’s. 40 h after transfection, cells were treated with RSPO1 or Wnt3a-conditioned medium overnight, and luciferase was measured. pGL2 siRNA was used as negative control. (b) Expression of LGR4, LGR5 and LGR6 rescue LGR4 siRNA-elicited decrease in RSPO1-induced STF reporter activation. HEK293T cells stably expressing siRNA-resistant LGR4 (LGR4-R), LGR5, LGR6, LGR2 or vector control were transfected with pGL2 siRNA or LGR4 siRNA, and corresponding STF values were determined. Expression of LGR4-R, LGR5 and LGR6, but not LGR2, could rescue LGR4 loss. Luciferase values were normalized relative to pGL2 siRNA. (c) LGR4 siRNA specifically reduces RSPO1-induced LRP6 phosphorylation and β-catenin stabilization. HEK293T cells transfected with indicated siRNA’s were treated with RSPO1 or Wnt3a-conditioned medium overnight. Phospho-LRP6, cytosolic β-catenin and tubulin were analyzed by immunoblotting using indicated antibodies.</p

RSPO1-LGR4-induced β-catenin activation is not mediated by GPCR signaling.

<p>(a) RSPO1-LGR4 does not signal via G_αs. (Left) HEK293 cells overexpressing LGR4 where incubated with IBMX (phosphodiesterases inhibitor) either alone or in presence of RSPO1. Isoproterenol (Iso), an agonist of the endogenously expressed β₂-adrenergic receptor, was used as a positive control of G_s signaling. cAMP accumulation was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040976#s4" target="_blank">Materials and Methods</a>. (Right) Similar as in (a), but without IBMX. (b) RSPO1-LGR4 does not signal via Gi. (Left) Forskolin (FSK, adenylyl cyclase stimulator)-stimulated cAMP levels are not reduced by RSPO1 in LGR4-overexpressing cells. Sphingosine-1-phosphate (S1P, agonist of the endogenous S1PRs) served as a positive G_i signaling control. cAMP accumulation was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040976#s4" target="_blank">Materials and Methods</a> section. (Right) LGR4 expression did not cause decreased cAMP levels when compared to LGR2 in FSK-treated cells. (c) RSPO1-LGR4 does not signal via G_αq. HEK293 cells overexpressing LGR4 where incubated with LiCl₂ (inositol phosphate phosphatases inhibitor) either alone or in presence of RSPO1. A compound mix (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040976#s4" target="_blank">Material and Methods</a>) served as a G_q signalling positive control. Inositol phosphate accumulation was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040976#s4" target="_blank">Materials and Methods</a> section. Similarly, no increase in calcium release was measured upon RSPO1 treatment (right) while a positive control (ATP, agonist of the endogenous P2RYs) is activating the cells. (d) HEK293T cells were transiently transfected with pSTF, pRenilla and cDNA constructs expressing LGR4 or the LacZ control. Cells were stimulated for 24 h as follows before luciferase values were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040976#pone-0040976-g002" target="_blank">Figure 2a:</a> Mock, medium; Iso, 1 uM Isoproterenol; RSPO1, 50 ng/ml RSPO1; RSPO1+Iso, 50 ng/ml RSPO1+1 µM Isoproterenol. (e) The same reporter and cDNA constructs were transfected into HEK293T cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040976#pone-0040976-g006" target="_blank">Figure 6d</a>, and luciferase values were determined after the following pre-treatments and subsequent stimulations, separated by a PBS wash, each: Mock+Mock, 4 h medium pre-treatment followed by 24 h medium treatment; PTX+Mock, 4 h pre-treatement with 100 ng/ml pertussis toxin (PTX) followed by 24 h medium treatment; Mock+RSPO1, 4 h medium pre-treatment followed by 24 h 50 ng/ml RSPO1 treatment; PTX+RSPO1, 4 h pre-treatment with 100 ng/ml PTX followed by 24 h 50 ng/ml RSPO1 treatment.</p

Secretomics screen on LGR4-STF HEK293 cells.

<p>A HEK293 cell line harboring stably integrated LGR4 and the STF reporter construct were incubated with conditioned media of a library of 2859 proteins known or predicted to be secreted. Firefly luciferase values were determined for each sample and normalized to Alamar blue values of the respective samples. Media + PBS and pcDNA DEST40: Negative controls, not expressing any secreted protein; recombinant RSPO1 samples, positive controls using recombinant RSPO1 at concentrations ranging from 0.1−240 ng/ml; RSPO1 pcDNA DEST40, positive control expressing RSPO1 cDNA; Sample: Combined dataset of all secreted (known or predicted) proteins, each protein assessed from conditioned media of duplicate cDNA transfections. RSPO1-4 proteins (black squares) scored highest. Then WNT proteins (red triangles) WNT1, WNT2, WNT3A, WNT7B and WNT8A reproducibly scored above a threshold of 10⁴.⁵ that marks the upper range of the bulk of datapoints, whereas WNT2B, WNT5A, WNT5B, WNT6, WNT8B, WNT9B, WNT10A, WNT10B, WNT11 and WNT16 scored below; WNT3, WNT4, WNT7A and WNT9A centered around the threshold. Each dot, triangle or square represents the luciferase value of a unicate transfected cDNA construct. Shown is an aggregate display of the primary screen and hit confirmation analysis.</p

Overexpressed LGR4 homologs sensitize cells to RSPO1-induced β-catenin signaling.

<p>(a) Exogenously expressed LGR4 homologs sensitize cells to RSPO1-induced STF reporter activation. HEK293T cells were transiently co-transfected with pSTF, pRenilla and a construct encoding either LGR4, LGR5, LGR6, LGR7 or as a control LacZ. One day later, cells were treated with increasing concentrations of either RSPO1 (left; ng/ml), Relaxin 2 (RLX030, right; nM) or just medium (Mock), and luminescence values were determined 18 h later. Firefly luciferase values were normalized according to their corresponding Renilla luciferase values. Results are shown as mean +/− standard deviation of triplicate transfections. (b) DKK1 blocks LGR4-RSPO-induced Wnt pathway activation. DKK1 efficiently blocked LGR4-RSPO1-induced Wnt reporter activation. In the LacZ control transfection, the mild stimulatory effect by RSPO1 via presumably endogenous LGR4 could similarly be reversed by DKK1. (c) Synergistic Wnt pathway activation by inducible LGR4 expression and low levels of RSPO1. Stable cell lines expressing LGR4, LGR5 or LGR2 upon doxycycline treatment were stimulated with 50 ng/ml RSPO1 or 10% Wnt3a-conditioned medium (CM). LGR4 and, to a much lesser extend, LGR5 expression sensitized the cells to RSPO1-, but not Wnt3a-induced STF activation, whereas LGR2 failed to do so.</p