B - G Confocal images of Prab - 3 : : GFP : : RAB - 3 in the nerve region corresponding to the dashed box in ( A ) of wild type ( B ) , cwn - 2 ( ok895 ) ( C ) , cfz - 2 ( ok1201 ) ( D ) , pop - 1 ( hu9 ) ( E ) , nlp - 40 ( tm4085 ) ( F ) and aex - 2 ( sa3 ) ( G ) mutants . H - I Quantification of the GFP : : RAB - 3 intensity in the nerve ring synaptic region for the indicated genotypes . Each dot represents one animal . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-G Confocal images of Prab-3::GFP::RAB-3 in the nerve region corresponding to the dashed box in (A) of wild type (B), cwn-2(ok895) (C), cfz-2(ok1201) (D), pop-1(hu9) (E), nlp-40(tm4085) (F) and aex-2(sa3) (G) mutants. H-I Quantification of the GFP::RAB-3 intensity in the nerve ring synaptic region for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines.


A APB Northern blot using a tRNAHis probe . m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points . Both queuosinylation and methylation levels could be restored by the addition of queuine . The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate , producing a single faster - migrating band ( ox ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A APB Northern blot using a tRNAHis probe. m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points. Both queuosinylation and methylation levels could be restored by the addition of queuine. The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate, producing a single faster-migrating band (ox). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa , whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Corresponding queuosinylation and methylation levels , as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C Representative metaplot of SF cells . 25 - 32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero . In - frame 27 - 30 mer ribosome footprints showing the correct periodicity .

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C Representative metaplot of SF cells. 25-32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero. In-frame 27-30 mer ribosome footprints showing the correct periodicity.


D Representative metagene plot of 27 - 30 mer ribosome footprints in S and SF culture conditions for two biological replicates ( rep1 and rep2 ) . Data information : SF ( serum free medium ) , q ( queuine )

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D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)


A Heatmap showing codon occupancy according to the color scale . Green bars indicate Q - decoded codons . The Asp - GAC codon translated by Q - and m5C38 - tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange . Lack of Q reduces ribosome translation speed at all Q dependent codons and at near - cognate decoded codons . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Heatmap showing codon occupancy according to the color scale. Green bars indicate Q-decoded codons. The Asp-GAC codon translated by Q- and m5C38-tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange. Lack of Q reduces ribosome translation speed at all Q dependent codons and at near-cognate decoded codons. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Translational speed of C ending codons relative to U ending codons . Error bars : ± SE of the permutated ratios of C - ending codon occupancy relative to U for the indicated conditions ; ( n = 4 ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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B Translational speed of C ending codons relative to U ending codons. Error bars: ±SE of the permutated ratios of C-ending codon occupancy relative to U for the indicated conditions; (n=4). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C Heatmap displaying increase or decrease of codon frequencies ( ‰ ) in SILAC down - regulated proteins relative to the average frequency of that codon in unchanged proteins .

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C Heatmap displaying increase or decrease of codon frequencies (‰) in SILAC down-regulated proteins relative to the average frequency of that codon in unchanged proteins.


D Codons translated by Q - tRNA are depleted in down - regulated proteins and increased in up - regulated proteins in both forward and reverse SILAC .

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D Codons translated by Q-tRNA are depleted in down-regulated proteins and increased in up-regulated proteins in both forward and reverse SILAC.


A The volcano plot shows differentially translated transcripts against adjusted p - values for SF compared to SF + q . Red dots indicate padj < 0 . 1 in multiple tests . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

A The volcano plot shows differentially translated transcripts against adjusted p-values for SF compared to SF+q. Red dots indicate padj< 0.1 in multiple tests. Data information: S (standard medium), SF (serum free medium), q (queuine)


C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q . As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified ; * p < 0 . 05 ( t - test ) ; n = 5 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , RO ( ribosome occupancy ) .

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C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q. As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified; *p <0.05 (t-test); n=5. Data information: S (standard medium), SF (serum free medium), q (queuine), RO (ribosome occupancy).


D Proliferation analysis of HeLa cells grown under the indicated culture conditions . Population doubling levels of three biological replicates were calculated for each time point . Error bars : ± SD ; n = 3 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

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D Proliferation analysis of HeLa cells grown under the indicated culture conditions. Population doubling levels of three biological replicates were calculated for each time point. Error bars: ±SD; n = 3. Data information: S (standard medium), SF (serum free medium), q (queuine)


A Representative fluorescence confocal images of live HeLa cells expressing Fluc - EGFP and FlucR188Q - EGFP maintained in SF compared to SF + q . Scale bars , 10 μm . Data information : SF ( serum free medium ) , q ( queuine )

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A Representative fluorescence confocal images of live HeLa cells expressing Fluc-EGFP and FlucR188Q-EGFP maintained in SF compared to SF+q. Scale bars, 10 μm. Data information: SF (serum free medium), q (queuine)


B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell . The number of cells showing at least one aggregate is indicated . 10 cells were imaged for Fluc - EGFP and 20 cells for FlucR188Q - EGFP from 3 independent transfections ; * p < 0 . 05 ( t - test ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , AU ( arbitrary units ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell. The number of cells showing at least one aggregate is indicated. 10 cells were imaged for Fluc-EGFP and 20 cells for FlucR188Q-EGFP from 3 independent transfections; *p < 0.05 (t-test). Data information: S (standard medium), SF (serum free medium), q (queuine), AU (arbitrary units).


C Specific luminescence activity of EGFP - tagged sensor proteins maintained in SF compared to SF + q . Luci ferase activity was normalized to the luciferase protein content in the sample . Error bars : ± SD ; * p < 0 . 05 ( t - test ) ; n = 8 . Data information : SF ( serum free medium ) , q ( queuine )

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C Specific luminescence activity of EGFP-tagged sensor proteins maintained in SF compared to SF+q. Luciferase activity was normalized to the luciferase protein content in the sample. Error bars: ±SD; *p < 0.05 (t-test); n = 8. Data information: SF (serum free medium), q (queuine)


A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion . Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks . Scale bar 250 nm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

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A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm. Data information: S (standard medium), SF (serum free medium), q (queuine).


B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q . Anti - KDEL ( ER stress marker ) ; anti - eIF2 - pS51 ( ER stress - induced inhibitor of translation ) ; anti - ATF4 ( transcription factor that activates ER stress - responsive genes ) ; anti - Hps70 ( chaperone ) . Nuclei were stained with Hoechst . Scale bar 10 µm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm. Data information: S (standard medium), SF (serum free medium), q (queuine).


A APB Northern blot using a tRNAHis probe . m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue . Both queuosinylation and methylation levels could be restored by the addition of q to the feed . The results are shown for three biological replicates . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A APB Northern blot using a tRNAHis probe. m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue. Both queuosinylation and methylation levels could be restored by the addition of q to the feed. The results are shown for three biological replicates. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q - free synthetic diet . Addition of q to the q - free diet significantly rescued the translation rate . n = 3 . Asterisks indicate statistically significant ( p < 0 . 05 , t - test ) differences . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q-free synthetic diet. Addition of q to the q-free diet significantly rescued the translation rate. n=3. Asterisks indicate statistically significant (p<0.05, t-test) differences. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


C Box plot showing the body weight of mice fed a q - free synthetic diet for 60 days , center lines show the medians ; box limits indicate the 25th and 75th percentiles , whiskers extend 1 . 5 times the interquartile range ; n = 3 . Addition of q to the q - free diet significantly rescued body weights . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

C Box plot showing the body weight of mice fed a q-free synthetic diet for 60 days, center lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range; n=3. Addition of q to the q-free diet significantly rescued body weights. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


D Dimethyl - labeling analysis of - q versus + q liver tissue . Top 10 % of deregulated proteins are indicated in red and blue . UPR effectors : HSPA5 / BiP , HSP90b1 and CALR are in orange . Data information : - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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D Dimethyl-labeling analysis of -q versus +q liver tissue. Top 10% of deregulated proteins are indicated in red and blue. UPR effectors: HSPA5/BiP, HSP90b1 and CALR are in orange. Data information: -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


F Liver sections from indicated mice were stained with anti - KDEL antibody . Arrowheads indicate ER stressed hepatocytes . Scale bar 25 µm . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


( A ) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ .

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(A) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ.


( B ) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells . Note that Meis2 mRNA positive LRCs have low ( no ) MEIS2 protein .

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(B) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells. Note that Meis2 mRNA positive LRCs have low (no) MEIS2 protein.


( C IHC labeling for MEIS2 ( C ) of LRCs positive for BrdU only and neuroblasts marked by DCX .

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(C IHC labeling for MEIS2 (C) of LRCs positive for BrdU only and neuroblasts marked by DCX.


D ) IHC labeling for MCM6 ( D ) of LRCs positive for BrdU only and neuroblasts marked by DCX .

D) IHC labeling for MCM6 (D) of LRCs positive for BrdU only and neuroblasts marked by DCX.


( E ) Dot plot showing the proportion of BrdU + LRCs negative for MEIS2 or MCM6 protein .

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(E) Dot plot showing the proportion of BrdU+ LRCs negative for MEIS2 or MCM6 protein.


( F ) Dot plot depicting the expression of miR - 204 in prospectively isolated cells of neural lineage .

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(F) Dot plot depicting the expression of miR-204 in prospectively isolated cells of neural lineage.


( B ) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi .

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(B) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi.


( C , D ) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF ( control , C ) or AntimiR204 ( D ) . White arrows indicate LRCs without detectable MEIS2 protein . The white arrowheads are pointing out the loss of neurogenic priming ( LRCs positive for MEIS2 protein ) upon the antagomir treatments .

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(C, D) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF (control, C) or AntimiR204 (D). White arrows indicate LRCs without detectable MEIS2 protein. The white arrowheads are pointing out the loss of neurogenic priming (LRCs positive for MEIS2 protein) upon the antagomir treatments.


( A , B ) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF ( control , A ) or AntimiR204 ( B ) . Boxed areas correspond to higher magnifications in adjacent panels ( A ' , B ' ) .

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(A, B) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF (control, A) or AntimiR204 (B). Boxed areas correspond to higher magnifications in adjacent panels (A', B').


Dot plots depicting the abundance of LRCs ( BrdU + , Ki67 - , DCX - ) in the SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of LRCs (BrdU+, Ki67-, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.


Dot plots depicting the abundance of aNSCs and TAPs ( BrdU - , Ki67 + , DCX - ) in the SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of aNSCs and TAPs (BrdU-, Ki67+, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.


Dot plots depicting the abundance of neuroblasts ( DCX + Ki67 + or DCX + Ki67 - ) in the SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of neuroblasts (DCX+ Ki67+ or DCX+ Ki67-) in the SEZ 7 days after aCSF or AntimiR204 injection.


( F , G ) Micrographs depicting the elbow - the most ventral region of the RMS ( F ) and OB neuronal layers ( G ) upon injection of artificial CSF ( control ) or AntimiR204 . ( H ) Dot plot showing the density of BrdU + cells ( progeny of NSCs ) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection .

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(F, G) Micrographs depicting the elbow - the most ventral region of the RMS (F) and OB neuronal layers (G) upon injection of artificial CSF (control) or AntimiR204. (H) Dot plot showing the density of BrdU+ cells (progeny of NSCs) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection.


( A ) Micrographs of in situ hybridization for miR - 204 in an adult mouse brain section . ( A ' and A ' ' ) are magnifications of boxed areas in ( A or A ' ) , respectively .

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(A) Micrographs of in situ hybridization for miR-204 in an adult mouse brain section. (A' and A'') are magnifications of boxed areas in (A or A'), respectively.


( B ) Dot plot showing miR - 204 levels in ChP and SEZ in the adult mouse brain measured by RT - qPCR . ( C ) Agarose gel of RT - qPCR product loaded after the saturation phase showing presence of miR - 204 and U6 in mouse ChP , SEZ and CSF .

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(B) Dot plot showing miR-204 levels in ChP and SEZ in the adult mouse brain measured by RT-qPCR. (C) Agarose gel of RT-qPCR product loaded after the saturation phase showing presence of miR-204 and U6 in mouse ChP, SEZ and CSF.


Plots depicting the number and size of EVs isolated from human CSF ( D )

Plots depicting the number and size of EVs isolated from human CSF (D)


Plots depicting the miR - 204 levels ( E ) in EVs and EV - free CSF ( n = 2 ) . ( F ) Agarose gel of RT - qPCR analysis depicting levels of miR - 204 in the EV - free supernatant and EV containing fraction of human CSF loaded at the saturation phase .

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Plots depicting the miR-204 levels (E) in EVs and EV-free CSF (n=2). (F) Agarose gel of RT-qPCR analysis depicting levels of miR-204 in the EV-free supernatant and EV containing fraction of human CSF loaded at the saturation phase.


( G ) Micrographs of transmission electron microscopy imaged extracellular vesicles ( EVs ) isolated from sham treated ( upper row ) and GW4869 treated animals ( lower row ) . ( G ' ' and G ' ' ' ) are magnifications of boxed areas in overview images to the left . ( H ) Dot plot depicting miR - 204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor .

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(G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left. (H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.


( A ) Gel pictures of the saturation phase of RT - qPCR analysis for miR - 204 in SEZ primary culture cells , ChP co - culture and ChP explants 7 day post preparation .

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(A) Gel pictures of the saturation phase of RT-qPCR analysis for miR-204 in SEZ primary culture cells, ChP co-culture and ChP explants 7 day post preparation.


( B ) Gel electrophoresis of RT - qPCR analysis loaded at the saturation phase for miR - 204 in SEZ culture medium and SEZ medium from the ChP co - culture .

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(B) Gel electrophoresis of RT-qPCR analysis loaded at the saturation phase for miR-204 in SEZ culture medium and SEZ medium from the ChP co-culture.


( C ) Dot plot showing the proportion of neural stem cells ( GFAP + ) out of DAPI + cells in primary SEZ / ChP co - culture with or without treatment with GW4869 inhibitor or AntimiR204 .

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(C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204.


( D - G ) Micrographs depicting the cellular compositions of SEZ cultures after co - culturing with control treated , GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating . Boxed areas correspond to the higher magnification images in adjacent panels ( D ' , E ' , F ' , G ' ) .

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(D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').


( B , C ) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle . ( B ' and C ' ) are magnifications of the SEZ and ( B ' ' and C ' ' ) of the ChP . ( D ) Dot plot depicting expression of miR - 204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR - 204 - specific TuD compared to scrambled control .

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(B, C) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle. (B' and C') are magnifications of the SEZ and (B'' and C'') of the ChP. (D) Dot plot depicting expression of miR-204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR-204-specific TuD compared to scrambled control.


Micrographs depicting immunoreactivity for MEIS2 ( E , F ) priming factors in the LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition and scrambled control White arrows point out the primed LRCs , the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR - 204 interference .

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Micrographs depicting immunoreactivity for MEIS2 (E, F) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR-204 interference.


Micrographs depicting immunoreactivity for MCM6 ( H , I ) priming factors in the LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition and scrambled control White arrows point out the primed LRCs , the arrowheads label the MCM6 + LRCs . Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 ( J ) after ChP specific miR - 204 interference .

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Micrographs depicting immunoreactivity for MCM6 (H, I) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MCM6+ LRCs. Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 (J) after ChP specific miR-204 interference.


( K , L ) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control ( K ) and miR - 204 specific TuD ( L ) . Note the reduction of primed LRCs ( BrdU + only , white arrows in K and L ) upon TuD - 204 injection . ( M ) Dot plot depicting the number of LRCs ( BrdU + only ) in the SEZ 7 days after miR - 204 inhibition .

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(K, L) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control (K) and miR-204 specific TuD (L). Note the reduction of primed LRCs (BrdU+ only, white arrows in K and L) upon TuD-204 injection. (M) Dot plot depicting the number of LRCs (BrdU+ only) in the SEZ 7 days after miR-204 inhibition.


D Heatmaps showing pairwise comparison of MPRA activity for all constructs ( mRNA / DNA ) between donors - left panel : resting CD4 T cells ; right panel : stimulated CD4 T cells .

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D Heatmaps showing pairwise comparison of MPRA activity for all constructs (mRNA/DNA) between donors - left panel: resting CD4 T cells; right panel: stimulated CD4 T cells.


B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells . GATA1 , NF - κB , and RUNX1 constructs were designed to include a binding site for the indicated transcription factors ( + ) or with that site disrupted ( - ) . Fixed effects meta - analysis P value is shown : * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 . Box and whisker plots represent median and IQR ( box ) and min - to - max ( whiskers ) .

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B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells. GATA1, NF-κB, and RUNX1 constructs were designed to include a binding site for the indicated transcription factors (+) or with that site disrupted (-). Fixed effects meta-analysis P value is shown: *<0.05; **<0.01, ***<0.001, ****<0.0001. Box and whisker plots represent median and IQR (box) and min-to-max (whiskers).


D Expression - modulating effect of each SNP [ log2 ( OR ) ] as measured in MPRA and validation experiments . OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele ( luciferase : n = 5 , MPRA : n = 12 ) .

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D Expression-modulating effect of each SNP [log2(OR)] as measured in MPRA and validation experiments. OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele (luciferase: n=5, MPRA: n=12).


A Pie chart depicting fine - mapping results ( Huang et al , 2017 ) ( posterior probabilities ) for an IBD - associated locus on 21q21 ( left panel ) . MPRA results in resting ( centre panel ) and stimulated CD4 T cells ( right panel ) showing that the putative causal variant has significant expression - modulating effect .

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A Pie chart depicting fine-mapping results (Huang et al, 2017) (posterior probabilities) for an IBD-associated locus on 21q21 (left panel). MPRA results in resting (centre panel) and stimulated CD4 T cells (right panel) showing that the putative causal variant has significant expression-modulating effect.


B Pie chart depicting Bayesian fine - mapping results for an AS - associated locus on 2p15 ( left panel ) . MPRA results in stimulated T cells ( right panel ) showing that rs6759298 has a significant expression - modulating effect ( the strongest of any variant at this locus ) while the other candidate SNPs have negligible effects .

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B Pie chart depicting Bayesian fine-mapping results for an AS-associated locus on 2p15 (left panel). MPRA results in stimulated T cells (right panel) showing that rs6759298 has a significant expression-modulating effect (the strongest of any variant at this locus) while the other candidate SNPs have negligible effects.


C GWAS results ( Liu et al , 2015 ) at a Crohn ' s disease and multiple sclerosis - associated locus on 6p23 ( left panel ) . MPRA for candidate SNPs in stimulated T cells ( right panel ) identifying a SNP ( rs34421390 ) with by far the greatest expression - modulating effect at this locus ( blue , risk allele reduces expression ; red , risk allele increases expression ) . Dotted horizontal line represents significance threshold ( corrected for multiple - testing ) .

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C GWAS results (Liu et al, 2015) at a Crohn's disease and multiple sclerosis-associated locus on 6p23 (left panel). MPRA for candidate SNPs in stimulated T cells (right panel) identifying a SNP (rs34421390) with by far the greatest expression-modulating effect at this locus (blue, risk allele reduces expression; red, risk allele increases expression). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


D GWAS results at a Type 1 Diabetes - associated locus on 14q32 ( Onengut - Gumuscu et al , 2015 ; left panel ) . MPRA for the candidate SNPs in stimulated T cells ( right panel ) . The construct with the largest and most significant effect contains the risk alleles for 2 SNPs ( rs1988588 and rs3902659 ) , each of which has a smaller effect when tested individually ( position indicated by vertical dotted line ) . Dotted horizontal line represents significance threshold ( corrected for multiple - testing ) .

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D GWAS results at a Type 1 Diabetes-associated locus on 14q32 (Onengut-Gumuscu et al, 2015; left panel). MPRA for the candidate SNPs in stimulated T cells (right panel). The construct with the largest and most significant effect contains the risk alleles for 2 SNPs (rs1988588 and rs3902659), each of which has a smaller effect when tested individually (position indicated by vertical dotted line). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


A IBD GWAS results ( Liu et al , 2015 ) at a multi - disease - associated locus on chromosome 6q23 .

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A IBD GWAS results (Liu et al, 2015) at a multi-disease-associated locus on chromosome 6q23.


C A single variant ( rs6927172 ) has the largest and most significant expression - modulating activity in resting ( left panel ) and stimulated CD4 T cells ( right panel ) with the risk allele reducing transcription . Plots represent median and IQR ( box ) and min - to - max ( whiskers ) . FDR - corrected meta - analysis P value shown .

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C A single variant (rs6927172) has the largest and most significant expression-modulating activity in resting (left panel) and stimulated CD4 T cells (right panel) with the risk allele reducing transcription. Plots represent median and IQR (box) and min-to-max (whiskers). FDR-corrected meta-analysis P value shown.


E Allele - specific NF - κB binding in CD4 T cells from rs6927172 heterozygotes , demonstrating reduced NF - κB binding to the risk allele following stimulation ( n = 8 ; one - sample t - test , two - tailed ) .

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E Allele-specific NF-κB binding in CD4 T cells from rs6927172 heterozygotes, demonstrating reduced NF-κB binding to the risk allele following stimulation (n=8; one-sample t-test, two-tailed).


F Allele - specific expression of enhancer RNA in heterozygous CD4 T cells . DNA used for technical control ( n = 6 ; paired t - test ; two - tailed ) .

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F Allele-specific expression of enhancer RNA in heterozygous CD4 T cells. DNA used for technical control (n=6; paired t-test; two-tailed).


G Genome - wide H3K27ac ChIP - seq in stimulated CD4 T cells from major - and minor - allele homozygotes at rs6927172 ( n = 6 ) . Upper panels show input - normalised H3K27ac signals plotted against enhancer rank . Super - enhancers are defined above the inflection point of the curve . Lower panels show H3K27ac reads from a major ( left ) and a minor ( risk ) allele homozygote ( right ) in a 9kb window around rs6927172 .

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G Genome-wide H3K27ac ChIP-seq in stimulated CD4 T cells from major- and minor-allele homozygotes at rs6927172 (n=6). Upper panels show input-normalised H3K27ac signals plotted against enhancer rank. Super-enhancers are defined above the inflection point of the curve. Lower panels show H3K27ac reads from a major (left) and a minor (risk) allele homozygote (right) in a 9kb window around rs6927172.


I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD , stratified by rs6927172 genotype ( qPCR ; one - way ANOVA ) . Error bars represent SD . Expression of IL20RA and IL22RAR2 not detected .

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I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD, stratified by rs6927172 genotype (qPCR; one-way ANOVA). Error bars represent SD. Expression of IL20RA and IL22RAR2 not detected.


B Primary CD4 T cell editing efficiency for indicated combinations of 5 ' and 3 ' gRNAs ( n = 6 for DB , DH , FH , and n = 2 for FB ) . Distribution of indels assessed using ICE .

B Primary CD4 T cell editing efficiency for indicated combinations of 5' and 3' gRNAs (n=6 for DB, DH, FH, and n=2 for FB). Distribution of indels assessed using ICE.


D Expression of genes on 6q23 in EU - containing mRNA ( EU added at time of stimulation ) showing that deletion of the NF - κB binding site specifically reduces transcription of TNFAIP3 , but not other genes at this locus ( n = 6 ; one sample t - test ) . Representative data from the DH gRNA combination .

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D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.


E Expression of CD69 , an activation marker , following CRISPR editing with indicated gRNA combinations or the non - targeting ( negative ) control ( NTC ) - data shown for ATTO - 550 positive ( RNP - containing ) and negative cells ( n = 6 ; paired t - test , one - tailed ) . Inset flow cytometry plot depicting representative gating of ATTO - 550 positive and negative cells .

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E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.


F Correlation between editing efficiency ( total indel rate ) and levels of phosphorylated IκBα in CD4 T cells ( normalised to the mean fluorescence intensity in NTC ; n = 9 , linear regression ) .

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F Correlation between editing efficiency (total indel rate) and levels of phosphorylated IκBα in CD4 T cells (normalised to the mean fluorescence intensity in NTC; n=9, linear regression).


G Secretion of effector cytokines following deletion of the NF - κB binding site - Th1 ( left panel , IFNγ ) , Th17 ( centre panel , IL - 17A ) and Th2 subsets ( right panel , IL - 4 ) ( n = 6 , paired t - test , one - tailed ) .

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G Secretion of effector cytokines following deletion of the NF-κB binding site - Th1 (left panel, IFNγ), Th17 (centre panel, IL-17A) and Th2 subsets (right panel, IL-4) (n=6, paired t-test, one-tailed).


d , ratio between the CPD ( copy per droplets , normalized to γ − actin and Hprt reference genes relative expression ) of structural [ N , nucleocapsid ] and non - structural [ IP4 : RdRp , RNA - dependent RNA polymerase ] viral gene expression determined by digital droplet PCR ( ddPCR ) in the nasal turbinates and in the lungs at 4 dpi . Data information : Horizontal lines indicate medians . The p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney test M : male hamsters ; F : female hamsters . Data were obtained from two independent experiments for each sex .

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d, ratio between the CPD (copy per droplets, normalized to γ−actin and Hprt reference genes relative expression) of structural [N, nucleocapsid] and non-structural [IP4: RdRp, RNA-dependent RNA polymerase] viral gene expression determined by digital droplet PCR (ddPCR) in the nasal turbinates and in the lungs at 4 dpi. Data information: Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


f , cytokines and chemokines transcripts in the nasal turbinates at 4dpi in male and female SARS - CoV - 2 infected hamsters , treated with saline or with 400 µg / kg ivermectin . Data information : Horizontal lines indicate medians . The p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney test M : male hamsters ; F : female hamsters . Data were obtained from two independent experiments for each sex .

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f, cytokines and chemokines transcripts in the nasal turbinates at 4dpi in male and female SARS-CoV-2 infected hamsters, treated with saline or with 400 µg/kg ivermectin. Data information: Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


b , Validation targets in the lung at 4 dpi . Horizontal lines indicate medians . The p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney test . Data information : M : male hamsters ; F : female hamsters . Data were obtained from two independent experiments for each sex .

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b, Validation targets in the lung at 4 dpi. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test. Data information: M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


a , representative histopathology photomicrographies of lungs according to the different groups : mock _ saline , CoV _ saline and CoV _ ivermectin . Top panels : whole lung sections . Bottom panels : high magnification . CoV _ saline section exhibits important congestion ( * ) , edema associated with few mononuclear cells ( white arrowheads ) . Note the thickening of the alveolar walls . CoV _ ivermectin section exhibits important amounts of mononuclear cells ( black arrowheads ) and less marked signs of congestion or edema . Hematoxylin and Eosin . Scale bars = 1 mm ( top panels ) and 20 μm ( bottom panels ) .

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a, representative histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline and CoV_ivermectin. Top panels: whole lung sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion or edema. Hematoxylin and Eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels).


b , representative immunofluorescence photomicrographies of neutrophils ( Ly - 6G ) , monocytes / macrophages ( Iba1 ) , M2 macrophages ( Arg1 ) and SARS - CoV - 2 ( NP ) in the lung . Scale bars = 50 μm .

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b, representative immunofluorescence photomicrographies of neutrophils (Ly-6G), monocytes/macrophages (Iba1), M2 macrophages (Arg1) and SARS-CoV-2 (NP) in the lung. Scale bars = 50 μm.


c , quantification of Iba1 + cells , Arg1 + cells and Iba1 + Arg1 + cells in the lungs . mock _ saline n = 3 ( males ) , mock _ ivermectin n = 4 ( males ) , CoV _ saline n = 9 ( 6 males and 3 females ) , CoV _ ivermectin n = 6 ( 4 males and 2 females ) . Data information : M : male hamsters ; F : female hamsters . Horizontal lines indicate medians . The p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, quantification of Iba1+ cells, Arg1+ cells and Iba1+Arg1+ cells in the lungs. mock_saline n=3 (males), mock_ivermectin n=4 (males), CoV_saline n=9 (6 males and 3 females), CoV_ivermectin n=6 (4 males and 2 females). Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test


B . qRT - PCR expression analysis of lncMN2 isoforms ( left panel ) and embedded miRNAs ( right panel ) in mESC and EBs differentiated for 6 days ( EB6 ) . Data , that are expressed as mean ( error bars represent SD ) , were normalized over Atp50 mRNA ( left ) and snoRNA U25 ( right ) levels and reported as RNA fold over the control ( mESC ) , set as 1 . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. qRT-PCR expression analysis of lncMN2 isoforms (left panel) and embedded miRNAs (right panel) in mESC and EBs differentiated for 6 days (EB6). Data, that are expressed as mean (error bars represent SD), were normalized over Atp50 mRNA (left) and snoRNA U25 (right) levels and reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


C . qRT - PCR expression analysis of lncMN2 - 202 / 203 / 204 isoforms ( left panel ) , miR - 325 - 3p ( middle panel ) and miR - 384 - 5p ( right panel ) in WT , KO1 and KO2 EB6 . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. qRT-PCR expression analysis of lncMN2-202/203/204 isoforms (left panel), miR-325-3p (middle panel) and miR-384-5p (right panel) in WT, KO1 and KO2 EB6. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


D . Cytofluorimetric analysis of cells from dissociated EB6 . The left diagram reports the distribution of cells ( percentage of the peak ) according to GFP levels , from WT ( gray ) , KO1 ( orange ) and KO2 ( blue ) EB . In the histogram aside , the percentage of GFP + cells in WT , KO1 and KO2 is reported . Data are expressed as mean and error bars represent SD . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Cytofluorimetric analysis of cells from dissociated EB6. The left diagram reports the distribution of cells (percentage of the peak) according to GFP levels, from WT (gray), KO1 (orange) and KO2 (blue) EB. In the histogram aside, the percentage of GFP+ cells in WT, KO1 and KO2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


E . Left : representative immunofluorescence of the MN marker Isl1 ( red signals ) in cells maintained in vitro for 14 days after EB6 dissociation ( DIV14 ) . DAPI counterstaining in blue . Scale bar = 100 mm . . Right : box plot shows the distribution of Isl1 + nuclei with respect to the total cell number marked with DAPI staining in WT and in lncMN2 KO mutants ( clone 1 and clone 2 ) . Data are expressed as mean and error bars represent SD . The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value . N = 3 biological replicates and 10 fields were analyzed for each replicate . Scale bar = 100 mm . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm.. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in lncMN2 KO mutants (clone 1 and clone 2). Data are expressed as mean and error bars represent SD. The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Scale bar=100 mm. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


B . qRT - PCR expression analysis of lncMN2 - 203 isoform , lncMN2 - 202 / 204 , miR - 325 - 3p and miR - 384 - 5p embedded miRNAs in WT , ∆ SP1 and ∆ SP2 EB6 . Data , that are expressed as mean ( error bars represent SD ) , are normalized over Atp5o mRNA levels ( for lncRNA isoform quantification ) or over snoRNA U25 levels ( for miRNA quantification ) and are reported as RNA fold over the control ( mESC ) , set as 1 . N = 3 biological replicates . Data are expressed as mean and error bars represent SD . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. qRT-PCR expression analysis of lncMN2-203 isoform, lncMN2-202/204, miR-325-3p and miR-384-5p embedded miRNAs in WT, ∆SP1 and ∆SP2 EB6. Data, that are expressed as mean (error bars represent SD), are normalized over Atp5o mRNA levels (for lncRNA isoform quantification) or over snoRNA U25 levels (for miRNA quantification) and are reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


C . Cytofluorimetric analysis of cells from dissociated EB6 . The right diagram reports the cell distribution ( percentage of the peak ) according to GFP levels , from WT ( gray line ) , ∆ SP1 ( purple line ) and ∆ SP2 ( blue line ) EB6 . In the histogram aside , the percentage of GFP + cells in WT , ∆ SP1 and ∆ SP2 is reported . Data are expressed as mean and error bars represent SD . N = 3 biological replicates . Data are expressed as mean and error bars represent SD . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Cytofluorimetric analysis of cells from dissociated EB6. The right diagram reports the cell distribution (percentage of the peak) according to GFP levels, from WT (gray line), ∆SP1 (purple line) and ∆SP2 (blue line) EB6. In the histogram aside, the percentage of GFP+ cells in WT, ∆SP1 and ∆SP2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


D . Left : representative immunofluorescence of the MN marker Isl1 ( red signals ) in cells maintained in vitro for 14 days after EB6 dissociation ( DIV14 ) . DAPI counterstaining in blue . Scale bar = 100 mm . Right : box plot shows the distribution of Isl1 + nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆ SP mutants ( clone 1 and clone 2 ) . The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value . N = 3 biological replicates and 10 fields were analyzed for each replicate . Data are expressed as mean and error bars represent SD . Scale bar = 100 mm . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆SP mutants (clone 1 and clone 2). The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Data are expressed as mean and error bars represent SD. Scale bar=100 mm. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


B - E ) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation ( pH3 , red ) in the adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-E) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.


withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation ( pH3 , red ) in the adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.


wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , and significantly increased by dietary histidine depletion ( 25 % his ) , quantified in ( F ) ( n = 28 , 22 , 7 ) , scale bar = 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n=28,22,7), scale bar = 50 μm


F - H ) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , and significantly increased by dietary histidine depletion ( 25 % his ) ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his),


I - K ) Representaitve pictures showing that adult nerfin - 1 clonal growth was significantly reduced on - his diet compared to CDD ( measured at day 0 , and day 9 ) quantified in ( K ) ( n = 27 , 110 , 136 ) . Scale bar = 75μm . * * * p < 0 . 001 L - N ) Representaitve pictures showing that larval nerfin - 1 clonal growth was significantly reduced on 25 % his diet compared to CDD ( after 6 days ) , quantified in ( N ) ( n = 53 , 36 ) . Scale bar = 50 μm .

I-K) Representaitve pictures showing that adult nerfin-1 clonal growth was significantly reduced on -his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n= 27, 110, 136). Scale bar = 75μm. ***p<0.001 L-N) Representaitve pictures showing that larval nerfin-1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n=53,36). Scale bar = 50 μm.


O - Q ) Representaitve pictures showing that adult pros clonal growth was not significantly altered on - his diet compared to CDD ( after 7 days ) , quantified in ( Q ) ( n = 12 , 23 , 16 ) . Scale bar = 75μm . R - T ) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction ( 25 % his ) compared to CDD ( after 6 days ) , quantified in ( T ) ( n = 19 , 13 ) . Scale bar = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O-Q) Representaitve pictures showing that adult pros clonal growth was not significantly altered on -his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23,16). Scale bar = 75μm. R-T) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n=19,13). Scale bar = 50 μm.


U - W ) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction ( 25 % his ) compared to CDD , quantified in ( W ) ( n = 27 , 19 ) . Scale bar = 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U-W) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n=27,19). Scale bar = 100 μm.


B ) Concentration of histidine and 3 - methyl - L - histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or - his diet ( n = 3 , 3 ) . Both compounds are reduced to below detection levels ( < 0 . 3mM ) upon dietary his depletion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion.


C ) Dietary supplementation of 3 - methyl - L - his for 4 days significantly increased adult nerfin - 1 clonal growth , and rescued nerfin - 1 growth inhibition due to his dietary withdrawal ( n = 18 , 14 , 7 , 9 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9).


D ) Dietary supplementation of histamine does not significantly increase adult nerfin - 1 clone size , but can rescue nerfin - 1 clonal growth inhibition due to his dietary withdrawal ( n = 42 , 14 , 23 , 25 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25).


E ) Larval nerfin - 1 clonal growth is significantly decreased upon overexpression of HDC RNAi ( n = 23 , 36 ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

E) Larval nerfin-1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n=23,36).


G ) Hdc inhibition significantly reduced the percentage of Dpn + NBs ( n = 16 , 17 ) , and significantly increased the percentage of Elav + neurons per clone ( n = 16 , 17 ) .

O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

G) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17).


H ) HdcRi overexpression reduced cellular growth of nerfin - 1 NBs ( n = 17 , 19 ) and neurons ( n = 36 , 19 ) measured as the ratio of nucleolus / nuclear volume .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H) HdcRi overexpression reduced cellular growth of nerfin-1 NBs (n=17,19) and neurons (n=36,19) measured as the ratio of nucleolus/nuclear volume.


A - E ) Representative images of nerfin - 1 clones in the adult VNC after 6 days of feeding on CDD or - his diet , scale bar = 100μm . Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in ( C ) ( n = 6 , 7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).


Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav + neurons ( red ) quantified in ( D ) ( n = 16 , 17 ) and significantly fewer Mira + NBs ( blue ) , quantified in ( E ) ( n = 12 , 22 ) ,

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22),


F - G ) Larval nerfin - 1 clones consisted of significantly greater proportion of differentiated neurons ( n = 10 , 7 ) and significantly reduced proportion of NBs per clone upon his dietary depletion ( 25 % his , n = 9 , 11 ) .

F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11).


Representative images of adult nerfin - 1 ( I - J ' , green ) and pros clones ( K - L ' , green ) , scale bar = 5μm . In nerfin - 1 clones , the size of the NBs ( Ase + , blue ) was significantly reduced upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 33 , 20 ) . In pros clones , the size of the NBs ( Ase + blue ) , and cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 9 , 8 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8)


Cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was also significantly reduced , quantified in ( M ) ( n = 33 , 20 ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n=33,20).


Cellular growth measured as the ratio of nucleolus / nuclear volume was significantly reduced in larval nerfin - 1 NBs after 6 days his dietary reduction ( 25 % his compared to CDD , n = 14 , 6 ) quantified in ( N ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N).


In pros clones , the size of the NBs ( Ase + blue ) , and cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( O ) ( n = 9 , 7 ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (O) (n=9,7).


Panel ( P ) shows that the larval nerfin - 1 neuron to NB reversion requires 15 - fold increase in cellular volume ( n = 10 ) and pros GMC to NB reversion requires approximately 3 . 5 - fold increase in cellular volume ( n = 31 ) .

Panel (P) shows that the larval nerfin-1 neuron to NB reversion requires 15-fold increase in cellular volume (n=10) and pros GMC to NB reversion requires approximately 3.5-fold increase in cellular volume (n=31).


A - D ) Representative pictures showing that pros NBs and GMCs are smaller than wildtype ( NBs and GMCs are distinguished at telophase , as a doublet of unequal size ) , quantified in C ( n = 25 , 31 , 21 , 16 ) . pros NBs exhibit similar nucleolus / nuclear ratio ( white arrows , nucleolus marked by Fib ) compared to wild type and pros ; TorDN NBs , quantified in D ( n = 10 , 18 , 15 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

A-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16). pros NBs exhibit similar nucleolus /nuclear ratio (white arrows, nucleolus marked by Fib) compared to wild type and pros;TorDN NBs, quantified in D (n=10,18,15).


E - F ) Representative pictures showing pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / nuclear ratio ( E - F )

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

E-F) Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F)


pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / nuclear ratio ( E - F ) , nucleolus labelled with Fib ( red ) [ quantified in G ( n = 44 , 43 ) ] , clone size [ quantified in H ( n = 41 , 34 ) ] , or the proportion of Dpn + NBs and Elav + neurons per clone [ quantified in I ( n = 15 , 15 , 15 , 15 ) ] .

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pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F), nucleolus labelled with Fib (red) [quantified in G (n=44,43)], clone size [quantified in H (n=41,34)], or the proportion of Dpn+ NBs and Elav+ neurons per clone [quantified in I (n=15,15,15,15)].


J - K ) Representative pictures showing that nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K )

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J-K) Representative pictures showing that nerfin-1 clones (green) generated in myc hypomorphic background (J-K)


nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K ) exhibited significantly reduced nucleolus / nuclear ratio , [ nucleolus labelled with Fib ( red ) ] and reduced clone size , quantified in L ( n = 34 , 27 ) and M ( n = 106 , 75 ) , and displayed an increased proportion of differentiated Elav + neurons , and reduced proportion of Dpn + NBs per clone , quantified in N ( n = 20 , 20 , 20 , 20 ) . scale bar =

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

nerfin-1 clones (green) generated in myc hypomorphic background (J-K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in L (n=34,27) and M (n= 106,75), and displayed an increased proportion of differentiated Elav+ neurons, and reduced proportion of Dpn+ NBs per clone, quantified in N (n=20,20,20,20). scale bar =10μm


O ) Reducing Myc dosage with the hypomorphic Myc allele , mycP0 , abolished the increase in nerfin - 1 clone size mediated by 2x histidine dietary intake , 2x his ( n = 41 , 18 , 10 , 57 ) .

O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57).


P ) Overexpression of myc restored the growth of nerfin - 1 clones inhibited by histidine dietary reduction , 25 % his ( n = 51 , 36 , 39 , 29 ) .

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P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29).


Q , R ) pros ( Q ) and nerfin - 1 ( R ) clones are reduced upon Tor inhibition ( n = 22 , 63 ) compared to control ( n = 51 , 65 ) , but their growth differentially depend on amino acid transporter Slif ( n = 12 , 32 ) and downstream growth regulators eIF4E ( n = 21 , 47 ) and S6K ( n = 39 , 37 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

Q, R) pros (Q) and nerfin-1 (R) clones are reduced upon Tor inhibition (n=22,63) compared to control (n=51,65), but their growth differentially depend on amino acid transporter Slif ( n=12,32) and downstream growth regulators eIF4E (n= 21,47) and S6K (n=39,37).


, Maximum intensity projections of confocal z - stacks taken from the apical cochlear region of control ( A ) mice at postnatal day 11 . Inner hair cells ( IHCs ) were stained with antibodies against Kir2 . 1 ( green ) and the hair cell marker MYO7a ( blue ) . At least 3 mice for each genotype were used .

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, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of control (A) mice at postnatal day 11. Inner hair cells (IHCs) were stained with antibodies against Kir2.1 (green) and the hair cell marker MYO7a (blue). At least 3 mice for each genotype were used.


C , D , Currents from IHCs of control ( C , P9 ) and Kir2 . 1 - OE ( D , P10 ) pre - hearing mice . Currents were elicited by using depolarizing and hyperpolarizing voltage steps , with a nominal increment of 10 mV , from a holding potential of − 84 mV . Test potentials are shown next to some of the traces . Note that the large inward rectifier Kir2 . 1 current is only present in the IHC of the Kir2 . 1 - OE mouse ( D ) . The outward current is primarily carried by a delayed rectifier current IK . IK1 identifies the small inwardly rectifying K + current normally expressed in IHCs . E , Steady - state current - voltage curves obtained from IHCs of control ( P9 - P11 ) and Kir2 . 1 - OE ( P9 - P11 ) mice .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

C, D, Currents from IHCs of control (C, P9) and Kir2.1-OE (D, P10) pre-hearing mice. Currents were elicited by using depolarizing and hyperpolarizing voltage steps, with a nominal increment of 10 mV, from a holding potential of −84 mV. Test potentials are shown next to some of the traces. Note that the large inward rectifier Kir2.1 current is only present in the IHC of the Kir2.1-OE mouse (D). The outward current is primarily carried by a delayed rectifier current IK. IK1 identifies the small inwardly rectifying K+ current normally expressed in IHCs. E, Steady-state current-voltage curves obtained from IHCs of control (P9-P11) and Kir2.1-OE (P9-P11) mice.


F , G , Representative ∆ F / F0 traces from the IHCs and GER of P7 - P9 control ( F ) and Kir2 . 1 - OE ( G ) mice in the presence of 0 . 3 mM Ca2 + . Spontaneous ATP - dependent Ca2 + waves from the GER ( green traces ) were eliciting coordinated Ca2 + signals in the IHCs from both controls and Kir2 . 1 - OE mice . For each genotype , two separate sets of recordings from 2 mice are shown ( top and bottom right ) , with the top traces being linked to the images on the left : before [ 1 ] , during [ 2 ] and after [ 3 ] ) the generation of a large Ca2 + wave from the GER . For details about the frequency and duration of the Ca2 + waves , and the number of mice and recordings see Fig EV3 . All recordings were obtained at body temperature . Traces are computed as pixel averages of regions of interest centred on IHCs .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G, Representative ∆F/F0 traces from the IHCs and GER of P7-P9 control (F) and Kir2.1-OE (G) mice in the presence of 0.3 mM Ca2+. Spontaneous ATP-dependent Ca2+ waves from the GER (green traces) were eliciting coordinated Ca2+ signals in the IHCs from both controls and Kir2.1-OE mice. For each genotype, two separate sets of recordings from 2 mice are shown (top and bottom right), with the top traces being linked to the images on the left: before [1], during [2] and after [3]) the generation of a large Ca2+ wave from the GER. For details about the frequency and duration of the Ca2+ waves, and the number of mice and recordings see Fig EV3. All recordings were obtained at body temperature. Traces are computed as pixel averages of regions of interest centred on IHCs.


C , Peak - to - peak MET current - voltage curves from P6 - P7 apical - coil IHCs of 7 control ( 12 IHCs ) and 3 littermates Kir2 . 1 - OE mice ( 7 IHCs ) . Recordings were obtained by mechanically stimulating the hair bundles of IHCs at the same time as stepping their membrane potential from − 124 mV to + 96 mV in 20 mV increments . The two sets of data are not significantly different : P = 0 . 6320 , 2 - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Peak-to-peak MET current-voltage curves from P6-P7 apical-coil IHCs of 7 control (12 IHCs) and 3 littermates Kir2.1-OE mice (7 IHCs). Recordings were obtained by mechanically stimulating the hair bundles of IHCs at the same time as stepping their membrane potential from −124 mV to +96 mV in 20 mV increments. The two sets of data are not significantly different: P = 0.6320, 2-way ANOVA.


C , Peak - to - peak MET current - voltage curves from P8 - P9 apical - coil IHCs of 12 control ( 17 IHCs ) and 13 littermates Kir2 . 1 - OE mice ( 24 IHCs ) . The two sets of data are significantly different : P < 0 . 0001 , 2 - way ANOVA .

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C, Peak-to-peak MET current-voltage curves from P8-P9 apical-coil IHCs of 12 control (17 IHCs) and 13 littermates Kir2.1-OE mice (24 IHCs). The two sets of data are significantly different: P < 0.0001, 2-way ANOVA.


J , Example of FM1 - 43 uptake by IHCs from P11 control ( top ) and Kir2 . 1 - OE ( bottom ) mice , showing the lack of fluorescence labelling in the latter , which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2 . 1 channels . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

J, Example of FM1-43 uptake by IHCs from P11 control (top) and Kir2.1-OE (bottom) mice, showing the lack of fluorescence labelling in the latter, which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2.1 channels. At least 3 mice for each genotype were used.


A , B , Scanning electron microscope ( SEM ) images showing the IHC hair bundle structure in the apical coil of the cochlea of P11 control ( A ) and P8 - P11 Kir2 . 1 - OE ( B ) mice . Control IHCs and the large majority of P8 IHCs from Kir2 . 1 - OE mice show a normal hair bundle structure composed of three rows of stereocilia : tall , intermediate and short ( arrows ) . From about P9 in Kir2 . 1 - OE mice , some IHCs start to lose the third row of stereocilia ( arrowheads ) and some already exhibit some fusion of the stereocilia ( asterisk ) . These changes in hair - bundle structure became more prominent at P11 . At least 3 mice for each genotype were used . In these panels and those below , asterisks are used to define some of the abnormal hair bundles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B, Scanning electron microscope (SEM) images showing the IHC hair bundle structure in the apical coil of the cochlea of P11 control (A) and P8-P11 Kir2.1-OE (B) mice. Control IHCs and the large majority of P8 IHCs from Kir2.1-OE mice show a normal hair bundle structure composed of three rows of stereocilia: tall, intermediate and short (arrows). From about P9 in Kir2.1-OE mice, some IHCs start to lose the third row of stereocilia (arrowheads) and some already exhibit some fusion of the stereocilia (asterisk). These changes in hair-bundle structure became more prominent at P11. At least 3 mice for each genotype were used. In these panels and those below, asterisks are used to define some of the abnormal hair bundles.


C , D , SEM images of both IHCs and OHCs from the cochlea of P26 control ( C , upper panel ) and P26 Kir2 . 1 - OE ( D , upper panel ) mice . Lower panels show a higher - magnification view of the hair bundle of IHCs from both genotypes , highlighting the profound disruption of the stereocilia in IHCs overexpressing Kir2 . 1 channels . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

C, D, SEM images of both IHCs and OHCs from the cochlea of P26 control (C, upper panel) and P26 Kir2.1-OE (D, upper panel) mice. Lower panels show a higher-magnification view of the hair bundle of IHCs from both genotypes, highlighting the profound disruption of the stereocilia in IHCs overexpressing Kir2.1 channels. At least 3 mice for each genotype were used.


A , B , Maximum intensity projections of confocal z - stacks showing images of the hair bundles from apical - coil IHCs of P6 and P11 control ( A ) and Kir2 . 1 - OE ( B ) mice immunostained with antibodies against MYOSIN VI ( blue ) and EPS8 ( magenta ) . At least 3 mice for each genotype were used . In all panels ( A - D ) , stereocilia are labelled with phalloidin ( green ) . Note that despite the disrupted hair - bundle structure in the IHCs overexpressing Kir2 . 1 channels ; the stereocilia retained a normal distribution of these bundle proteins . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A,B, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P6 and P11 control (A) and Kir2.1-OE (B) mice immunostained with antibodies against MYOSIN VI (blue) and EPS8 (magenta). At least 3 mice for each genotype were used. In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.


C , D , Confocal images of the hair bundles of P11 IHCs from control ( C ) and Kir2 . 1 - OE ( D ) mice immunostained with antibodies against MYOSIN XV isoform 1 ( blue ) and WHIRLIN ( magenta ) . In all panels ( A - D ) , stereocilia are labelled with phalloidin ( green ) . Note that despite the disrupted hair - bundle structure in the IHCs overexpressing Kir2 . 1 channels ; the stereocilia retained a normal distribution of these bundle proteins . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

C,D, Confocal images of the hair bundles of P11 IHCs from control (C) and Kir2.1-OE (D) mice immunostained with antibodies against MYOSIN XV isoform 1 (blue) and WHIRLIN (magenta). In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.


A , B , Maximum intensity projections of confocal z - stacks showing the IHCs of the apical cochlear region from control ( A ) and Kir2 . 1 - OE ( B ) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 ( upper panels ) . Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control ( A ) and Kir2 . 1 - OE mice ( B ) . IHCs were stained with antibodies against the K + channel Kir2 . 1 ( green ) and Myosin 7a ( Myo7a , blue : cell marker ) . Note that after 2 days following the removal of DOX , Kir2 . 1overexpression was already largely downregulated . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

A,B, Maximum intensity projections of confocal z-stacks showing the IHCs of the apical cochlear region from control (A) and Kir2.1-OE (B) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 (upper panels). Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control (A) and Kir2.1-OE mice (B). IHCs were stained with antibodies against the K+ channel Kir2.1 (green) and Myosin 7a (Myo7a, blue: cell marker). Note that after 2 days following the removal of DOX, Kir2.1overexpression was already largely downregulated. At least 3 mice for each genotype were used.


H , I , Maximum intensity projections of confocal z - stacks showing images of the hair bundles from apical - coil IHCs of P14 control ( H ) and Kir2 . 1 - OE ( I ) mice stained with phalloidin . DOX was removed from the mother ' s drinking water when the pups were P5 . At least 3 mice for each genotype were used .

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H,I, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P14 control (H) and Kir2.1-OE (I) mice stained with phalloidin. DOX was removed from the mother's drinking water when the pups were P5. At least 3 mice for each genotype were used.