Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Representation of the strategy used for transcriptomic analysis in time and space in the dorsal and lateral OB lineages. pCX-GFP plasmid was introduced into neural stem cells (NSCs) residing within the dorsal or lateral V-SVZ and GFP-positive cells were isolated by FACS at different time points after electroporation (Elpo). The mRNA content was analyzed by micro-array . ( B ) Quantification of Vax1 mRNA expression detected by micro-array analysis in dorsal (brown) and lateral (purple) progenies during neurogenesis. ( C–H ) In situ hybridization revealing Vax1 mRNA (in blue) combined with immuno-histochemistry using antibodies detecting (in brown) PAX6 ( C, C’, G ), ASCL1 ( D, D’ ), DLX2 ( E, E’, H ) or KI67 ( F, F’ ) proteins in the V-SVZ ( C–F ) or RMS ( G, H ) at postnatal day 3 ( P3 ). ( C’–F’ ) High magnification of cellular staining in the V-SVZ (area indicated by the yellow bracket in C-F). Arrows ( C’ ): examples of strong PAX6 only positive cell in the dorso-lateral SVZ; blue staining underneath labels cells from a distinct plane. Arrow heads ( E’, F’ ): double positive cells for DLX2 and KI67, respectively. High magnification of the RMS highlights the differential expression of Vax1 and Pax6 along the dorso-ventral axis ( G’,G” ) and the co-localization with Dlx2 ( H’,H” ). ( I ) Schematic representation of gene expression profile in different cell types of the neurogenic sequence. Circular arrow indicates proliferating cells. LV: lateral ventricle, RG: radial glia, TAP: transit amplified precursor, VZ: ventricular zone, SVZ: sub-ventricular zone. D: dorsal, L: lateral, S: septal, V: ventral. Scale bars: 100 µm ( C–F ), 20 µm ( C’–F’ ), 50 µm ( G–H ).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Plasmid Preparation, Isolation, Electroporation, Microarray, Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Quantitative Proteomics, Gene Expression, Sequencing, Amplification

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: Cells were analyzed 15 days after electroporation of lateral V-SVZ progenitors by Cre mRNA in WT or Vax1cKO mice. Data are shown as means ± SD, dots represent individual animals. WT: n = 12, Vax1cKO: n = 17. Figure 2—figure supplement 2—source data 1. Quantification of tdTomato+ PGC in Vax1 mutant.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Electroporation, Mutagenesis

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to determine the expression of microRNAs in Vax1-overexpressing progenitors. PCAG-Vax1 and pCX-GFP were simultaneously introduced into NSCs by electroporation of the lateral wall of postnatal P1 brains. Lateral V-SVZ was dissected out 2 days after electroporation and GFP+ cells were isolated by flow cytometry (FACS) to perform quantitative RT-PCR analysis. ( B ) Quantification of Vax1 mRNA level by qRT-PCR in control and Vax1OE conditions, normalized to beta-actin and reported in Vax1 condition as relative level to control, validating the overexpression of Vax1 after electroporation. ( C ) Quantification of miR-7 expression in both conditions. Expression level of miR-7 was normalized by invariant expression of microRNA let-7a and reported in Vax1 condition as relative level to control. Experiments in B and C were performed in triplicate, and data were obtained from ( B ) two independent biological replications or ( C ) three technical replications. ( D ) Genome browser images representing the chromosomal portions encoding the three MiR-7 loci (depicted in pink). Mir-7–1 lies within an intronic sequence of the Hnrnpk gene whereas MiR-7–2 and MiR-7b reside within intergenic sequences. Vax1-binding sites found in the upstream regulatory region of the three MiR-7 are represented by red boxes. ( E ) Model of cross-regulatory interaction between Vax1 , miR-7, and Pax6 in the lateral V-SVZ to control the number of dopaminergic neurons generated by the neural stem cells regionalized in this aspect. This model is supported by our present data and previous work where it was shown that miR-7 was required to inhibit PAX6 expression in lateral NSCs to produce the correct number of dopaminergic neurons in the postnatal OB. Here, we propose that Vax1 acts upstream of miR-7 by positively regulating its expression and consequently inhibiting PAX6. However, it is also possible that Vax1 directly represses the expression of Pax6 mRNA (dashed line) by acting on its promoter . Additionally, Vax1 is required to generate Calbindin neurons from the ventral aspect of the lateral V-SVZ. Figure 5—source data 1. Quantification of expression level of Vax1 and miR-7 in V-SVZ cells.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Expressing, Electroporation, Isolation, Flow Cytometry, Quantitative RT-PCR, Control, Over Expression, Sequencing, Binding Assay, Generated

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to target lateral V-SVZ NSCs with Cre -mRNA. Tomato+ recombined cells were isolated 2 days after electroporation. ( B ) Vax1 mRNA level quantified by RT-PCR was normalized to beta-actin and reported in Vax1cKO condition as relative level to control (WT).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Isolation, Electroporation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet:

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Sequencing, Recombinant, Plasmid Preparation, SYBR Green Assay, Software, Imaging

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and endothelial cell marker CD31 in the blood vessels of human nasal turbinates using six different anti-ACE2 antibodies and anti-CD31. b Double immunofluorescence staining of ACE2 and type II pneumocyte marker mucin 1 (MUC1) in the human lung using six different anti-ACE2 antibodies and anti-MUC1. Abcam ab15348 clone yielded the most robust staining of pneumocytes, while the other clones showed negligible or less specific membrane staining. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Marker, Staining, Clone Assay, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: Representative images of human tissues on a tissue microarray (TMA) stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348, and less specifically with Sigma HPA000288 (Fig. 2b; Supplementary Table 1 ). Scale bars: 100 μm.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Microarray, Staining, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and acetylated α-tubulin (ACTUB) on normal human nasal turbinate, ethmoid sinus, uncinate process (sinus), trachea, and bronchus, using anti-ACE2 and anti-ACTUB antibodies, respectively. b Representative double immunofluorescence staining of ACE2 and ACTUB on normal C57BL/6J mouse nasal turbinate and trachea. c Immunofluorescent staining of (top panel) ACE2, cilia marker ADP-ribosylation factor-like protein 13B (ARL13B), and cilia centrosome marker FGFR1 oncogene partner (FOP); (bottom panel) ACE2, and cilia markers ACTUB and ARL13B in a ciliated mouse cell line, IMCD3. d Immunofluorescent staining of ACE2 in the primary cilia of IMCD3 cells transiently transfected with human ACE2 (yellow outline) compared to endogenous mouse ACE2 (blue outline). e Quantified percentages of endogenous ACE2-positive cilia (34.67 ± 13.58%; control (Ctrl)) versus cilia with overexpressed human ACE2 (82.67 ± 4.73%). Ciliated cells were identified by staining of ARL13B. Error bars represent mean ± SD. ( n = 100 cells examined per experiment over three independent experiments). (Two-tailed Student’s t test, ** p = 0.004). f Representative multiplexed images of in situ hybridization against the SARS-CoV-2 Spike mRNA, in combination with immunofluorescence staining of ACE2 and the differentiated epithelial cell marker cytokeratin 8 (KRT8). SARS-CoV-2 Spike mRNA expression (red) was detected within ciliated epithelial cells containing motile cilia positive for ACE2 (green). The nuclei were stained using DAPI (blue) as a counterstain. Scale bars: 20 μm ( a , b top panels; f large panels); 5 μm ( a , b bottom panels; f small panels); 2 μm ( c , d ).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Staining, Marker, Transfection, Control, Two Tailed Test, In Situ Hybridization, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative immunofluorescence double staining of ACE2 and mucin 5AC (MUC5AC) reveals absence of co-localization of ACE2 within secretory goblet cells in the human nasal turbinate, uncinate process, and bronchus. b Representative in situ hybridization using an ACE2 probe in combination with an anti-MUC5AC antibody. ACE2 mRNA expression (red dots) was not detected within goblet cells marked by MUC5AC in the nasal turbinate, uncinate process, and trachea. Nuclei were stained using DAPI. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Immunofluorescence, Double Staining, In Situ Hybridization, Expressing, Staining

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a No statistically significant changes in ACE2 expression was detected among patients less than or greater than 65 years of age, males versus females, and patients with varying smoking history. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). b No statistically significant difference in ACE2 expression was observed between healthy controls and patients with chronic rhinosinusitis with polyps (CRSwNP) or without polyps (CRSsNP). (Kruskal–Wallis test, p > 0.05). c No statistically significant difference in ACE2 expression was noted between distinct human nasal tissue sites/regions. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). UNC uncinate process, Turb nasal turbinates, Eth ethmoid sinus, NP benign nasal polyps. The bottom and top of the box plots represent the 25th and 75th percentiles, respectively. The bands within the box show the median value, and the whiskers extending from both ends of the boxes are minimum and maximum values. Each dot represents one patient.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Quantification of ACE2 in controls and patients taking ARBs and ACEI. In the Stanford cohort, ACE2 is slightly but statistically significantly lower in patients taking ACEI (0.19 ± 0.02) compared to controls (0.26 ± 0.06). (Kruskal–Wallis test p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). There were no statistically significant differences in ACE2 expression between patients taking ARBs and controls in the Stanford, National Taiwan University Hospital (NTUH), and China Medical University Hospital (CMUH) cohorts. b In the Stanford cohort, when including only controls with hypertension (HTN) on other medications (“HTN w/o ARBs/ACEI”), ACE2 expression was statistically different between the groups (Kruskal–Wallis test, p = 0.044) but Dunn’s multiple comparison post-hoc test did not reveal any statistical significance between the three groups. No statistically significant differences were seen among patients taking ARBs compared to controls. c When cohorts from all three institutions were normalized by Z -score and integrated, patients taking ACEI (−0.72 ± 0.42) had a lower ACE2 expression compared to controls with hypertension (0.41 ± 1.07). (Kruskal–Wallis test, p = 0.032; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). Patients taking ARBs (−0.15 ± 0.95) showed a trend towards lower ACE2 compared to controls with hypertension, but this was not statistically significant. d ACE2 expression among patients of older (≥65 years) and younger (<65 years) age taking ARBs or ACEI was not statistically divergent from control patients of the same age group. (Kruskal–Wallis test, p > 0.05). e ACE2 expression among male and female patients on ARBs or ACEI trended comparably or lower than same-sex controls except for males taking ARBs in the CMUH group who showed a trend towards higher ACE2 expression. No statistically significant differences were observed. (Kruskal–Wallis test, p > 0.05). f Among non-smokers, there was a statistically significant trend towards lower ACE2 expression in patients taking ACEI compared to controls in the Stanford group (Kruskal–Wallis test, p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.035). No statistical significance was observed with the non-smokers on ARBs. All data are noted as mean ± SD. Kruskal–Wallis test was used for three group comparisons and two-tailed Mann–Whitney test was used for two-group comparisons. Box plots are similar in format to Fig. .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Comparison, Expressing, Medications, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: The luminal differentiated airway epithelial cells consist of ciliated columnar cells (~80%) and secretory goblet cells (~20%). Club cells are infrequently found in the human upper airway. The basal cell layer, which faces the lamina propria, is comprised of both basal and suprabasal cells, which are considered multipotent progenitors capable of renewing the airway epithelium. This schematic depicts how SARS-CoV-2 may bind to ACE2 expressed on the cilia of the nasal cavity following exposure to respiratory droplets or airborne particles.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques:

Journal: PLoS ONE

Article Title: GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

doi: 10.1371/journal.pone.0025279

Figure Lengend Snippet: ( A ) Comparison of estimated log 2 -fold changes from analysis of microarrays (x-axis) and RNA-Seq using GENE-counter running NBPSeq (y-axis). Only induced genes measurable by both platforms are presented. Differentially induced genes are colored to highlight genes uniquely identified using microarrays (open red down triangles) or RNA-Seq (open blue up triangles) and found common between the two methods (purple crosses). ( B ) Three-way Venn comparing differentially expressed genes identified from GENE-counter's assessment of RNA-Seq data and analysis of microarrays. Only genes measurable using both methods were included in the comparison.

Article Snippet: We hybridized the same mRNA samples to Affymetrix ATH1 microarrays and identified 366 induced genes ( ; GSE25818; http://www.ncbi.nlm.nih.gov/geo/ ).

Techniques: RNA Sequencing Assay