Journal: Science Advances

Article Title: A neural substrate of compulsive alcohol use

doi: 10.1126/sciadv.abg9045

Figure Lengend Snippet: ( A ) Representative images of CeA photomicrographs showing Fos (green) and PKCδ (red) immunoreactivity colabeling [scale bars, 200 μm (left) and 50 μm (right)] in punishment-resistant ( n = 10) and punishment-sensitive ( n = 9) rats. ( B to D ) Mean number of cells (±SEM) positive for Fos, PKCδ, and double-labeled cells/mm 2 . * P < 0.001, * P < 0.05, * P < 0.01. ( E ) Mean fold change in Prkcd mRNA levels in punishment-resistant ( n = 8) and punishment-sensitive ( n = 6) rats, measured by qPCR. * P < 0.01.

Article Snippet: Inventoried TaqMan gene expression assay probes ( Prkcd : Rn00440891 and Gapdh : Rn4308313; Life Technologies, Carlsbad, CA) were used to assess the expression of the target gene on an ABI 7900HT Fast Real-time PCR system (Life Technologies, Carlsbad, CA).

Techniques: Labeling

Journal: Science Advances

Article Title: A neural substrate of compulsive alcohol use

doi: 10.1126/sciadv.abg9045

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Virus injection site (scale bar, 2 mm) and mean fold change in Prkcd mRNA levels following a viral-mediated knockdown ( n = 8 per group). * P < 0.05. ( C ) Expression of Prkcd in the CeA measured by RNAscope. Brown dots represent the expression of Prkcd [scale bars, 2 mm (left), 50 μm (right), and 5 μm (inset)] ( n = 8 per group). * P < 0.05. ( D ) Representative images of CeA photomicrographs (scale bar, 50 μm) showing Fos (blue), PKCδ (magenta) immunoreactivity, and yellow fluorescent protein (YFP) colabeling. Mean number of cells positive (±SEM) for Fos, PKCδ, and double-labeled cells in punishment-resistant ( n = 6 per group) and punishment-sensitive ( n = 5 per group) rats/mm 2 . # P < 0.001, * P < 0.001, * P < 0.05, * P < 0.01. ( E ) Mean number of alcohol-reinforced lever presses (±SEM) during the 30-min punishment session control in punishment-resistant ( n = 19) and punishment-sensitive rats ( n = 19) receiving shCtrl, or shRNA knockdown of PKCδ ( n = 18; n = 19). # P < 0.001. ( F ) Mean resistance score (±SEM). # P < 0.001.

Article Snippet: Inventoried TaqMan gene expression assay probes ( Prkcd : Rn00440891 and Gapdh : Rn4308313; Life Technologies, Carlsbad, CA) were used to assess the expression of the target gene on an ABI 7900HT Fast Real-time PCR system (Life Technologies, Carlsbad, CA).

Techniques: Virus, Injection, Knockdown, Expressing, RNAscope, Labeling, Control, shRNA

Journal: medRxiv

Article Title: Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis

doi: 10.1101/2020.10.26.20219089

Figure Lengend Snippet: (a-c) (a) Vulnerabilities to infection by coronaviruses, influenza, and rhinovirus can be predicted based on entry factors expression and visualized using expression matrices. Epithelia appear especially at-risk for viral infection. (b) When focused on the 8 epithelial cell populations, vulnerabilities to SARS-CoV-2 were apparent in both glands and mucosa. These results strongly suggest that the oral cavity may be vulnerable to viral infection, especially for SARS-CoV-2. Expression matrices, including low frequency ACE2/TMPRSS2 co-expressing cells in Basal 1, ducts, mucous acini, and myoepithelial clusters, further support broad SARS-CoV-2 vulnerabilities. (c) UMAPS demonstrate distinct cluster vulnerabilities with ACE2 highest in most oral epithelia; however, expression of proteases demonstrated tissue-specific expression patterns with TMPRSS2 (enriched for SGs) and TMPRSS11D (enriched for mucosal cells). Endosomal proteases, CTSB and CTSL exhibited broad expression across vulnerable cell types. (d-f) (d) Like the human integrated atlas, by using the cell type expression of known host entry factors mouse atlas support the viral vulnerabilities of the lining mucosa and SGs (14 total populations including tissue-resident Langerhans cells; LC) as high risk sites for infection by coronaviruses (SARS-CoVs, MERS, HCoVs), influenza, and rhinovirus. (e) These results can be further underscored by looking at co-expression of Ace2 and Tmprss2 is restricted to filiform and fungiform differentiated epithelial cells and SG ducts and acini. (f) UMAPS of SARS-CoV-2 entry factors demonstrate distinct cluster vulnerabilities with Ace2 highest expressed in suprabasal tissues, Tmprss2 expressed in SGs ducts and acini.

Article Snippet: Positive control probe: 320861; Negative control probe: 320871; ACE2 antibody: Mouse Monoclonal (protein tech #66699-1-IG), (ACE2 diluted 1/2000, PBS 1% BSA, 1% donkey serum PBS); SARS-CoV-2 probe: ACD, 848561; ACE2 probe: #848151-C3; TMPRSS2 probe: 470341-C2; CSTL probe: 858611-C3; Secondary antibodies: Al secondary antibodies were in donkey (D-anti Goat, D-anti Rabbit, diluted 1/300 in 1/300 PBS 1% BSA, 1% donkey serum, PBS).

Techniques: Infection, Expressing

Journal: medRxiv

Article Title: Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis

doi: 10.1101/2020.10.26.20219089

Figure Lengend Snippet: (a , b) UMAPs overlay of SARS-CoV-2 entry factors ACE2 and TMPRSS2, 4 , and 11D in (a) minor saliva glands and (b) gingiva highlight ACE2 expression in the saliva gland ducts and acini, and that TMPRSS proteases exhibits abundant expression across epithelia. Interestingly, TMPRSS2 expression is most concentrated in the saliva glands and TMPRSS4 and TMPRSS11D are higher expressed in gingiva, with 11D displaying a defining difference between these two oral epithelial niches. (c , d) Using healthy volunteer (c) gland and (d) gingival tissue sections, mRNA expression was confirmed using RNAscope® in situ hybridization (ISH) for ACE2, TMPRSS2, −4 as well as -11D in gingiva; (see 11D in SG: ). Due to the known shedding/sloughing of suprabasal epithelial cells (d,e; illustrations), we examined both basal and suprabasal (SB) expression, revealing enrichment of all examined entry factors in suprabasal over basal cells. (e) Using ISH, we map ACE2 and TMPRSS2 (for matched TMPRSS4 and −11D, see ) in diverse oral tissues (buccal mucosa, ventral tongue, and the dorsal tongue) as well as examining the oropharynx for the first time (soft palate, tonsils). This again supported the heterogeneity that can be found in the oral cavity—not only considering basal versus suprabasal enrichment—but also across sites. This mapping also revealed all sites are vulnerable to infection in suprabasal cells that are sloughed into saliva, a striking finding. Arrowheads in ( c-e ) indicate high expression (red), Scale bars: (c) 50 μm, (d , e) 25 μm.

Article Snippet: Positive control probe: 320861; Negative control probe: 320871; ACE2 antibody: Mouse Monoclonal (protein tech #66699-1-IG), (ACE2 diluted 1/2000, PBS 1% BSA, 1% donkey serum PBS); SARS-CoV-2 probe: ACD, 848561; ACE2 probe: #848151-C3; TMPRSS2 probe: 470341-C2; CSTL probe: 858611-C3; Secondary antibodies: Al secondary antibodies were in donkey (D-anti Goat, D-anti Rabbit, diluted 1/300 in 1/300 PBS 1% BSA, 1% donkey serum, PBS).

Techniques: Expressing, RNAscope, In Situ Hybridization, Infection

Journal: medRxiv

Article Title: Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis

doi: 10.1101/2020.10.26.20219089

Figure Lengend Snippet: (a) Immunofluorescent (IF) confocal microscopy demonstrated AQP5 (red, acini) colocalization with ACE2 (green) reveal that ACE2 is enriched on the apical (luminal) and basolateral membranes and is concentrated in the SG acini/ducts (labeled ‘a’ or ‘d’). White arrows indicate colocalization between ACE2 and AQP5. ACE2 is also expressed to a similar degree in the saliva gland ducts but rarely expressed in myoepithelial cells circumscribing the acini (data not shown). (b-d) (b) Using healthy volunteer gland sections, the unique minimal TMPRSS11D expression in ducts and acini was confirmed using RNAscope® in situ hybridization. (c , d) To further confirm co-expression of ACE2 and TMPRSS2 , RNAscope® fluorescent in situ hybridization and immunohistochemistry for pan-cytokeratin (pCK), shows that acini and ducts co-express ACE2 and TMPRSS2 further highlighting their vulnerability to infection in (c) minor and (d) parotid saliva glands. (e) Analysis of available bulk RNA sequencing data suggests that the minor (minor) and parotid (PG) saliva glands are vulnerable to SARS-CoV-2 infection compared to the submandibular glands (SMG). The minor glands express ∼3x higher ACE2 compared to the major glands. For comparison, the gland express an equivalent amount of TMPRSS2 across all three glands samples. (f) Considering the unique niches of the oral cavity, we used ISH mapping for ACE2 and TMPRSS2 (for ACE2 and TMPRSS2 , see Figure 4) across the oral (buccal mucosa, ventral tongue, and the dorsal tongue) as well as examining the oropharynx for the first time (soft palate, tonsils). This again supports oral niche heterogeneity and that all sites are vulnerable to infection in suprabasal cells that are sloughed into saliva. Dotted black box in (b) represents zoomed-in area. Scale bars: ( b ) 100μm, ( a ) 50μm, ( c , d , f ) 25μm.

Article Snippet: Positive control probe: 320861; Negative control probe: 320871; ACE2 antibody: Mouse Monoclonal (protein tech #66699-1-IG), (ACE2 diluted 1/2000, PBS 1% BSA, 1% donkey serum PBS); SARS-CoV-2 probe: ACD, 848561; ACE2 probe: #848151-C3; TMPRSS2 probe: 470341-C2; CSTL probe: 858611-C3; Secondary antibodies: Al secondary antibodies were in donkey (D-anti Goat, D-anti Rabbit, diluted 1/300 in 1/300 PBS 1% BSA, 1% donkey serum, PBS).

Techniques: Confocal Microscopy, Labeling, Expressing, RNAscope, In Situ Hybridization, Immunohistochemistry, Infection, RNA Sequencing, Comparison

Journal: medRxiv

Article Title: Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis

doi: 10.1101/2020.10.26.20219089

Figure Lengend Snippet: (a , b) (a) In situ hybridization (ISH) demonstrates infection in the minor SGs (pan-cytokeratin; pCK). (b) H&E of COVID-19 saliva glands reveals variable histology, including variable chronic sialadenitis and some lymphocytic aggregates. (c) Infection was confirmed using digital droplet PCR on additional tissues recovered from 16 COVID-19 victims (12/24 in >1 SG). Minor SGs exhibited higher viral burden than parotid in paired samples (n=7). (d , e) Detection of SARS-CoV-2 infection and replication in the salivary glands using spike (V-nCoV2019-S) and sense (V-nCoV2019-orf1ab-sense) probes, respectively (arrows denote replication signal). (f-h) ISH of suprabasal cells confirm high concentration of ACE2;TMPRSS2 cells in cells just before sloughing/shedding. (g) Sloughed saliva cells were found to express all SARS-CoV-2 entry factors. We show that these cells can be infected by SARS-CoV-2. (f) This often occurs in highly expressing ACE2 cells. (h) Using 3D confocal microscopy, we demonstrate virus inside of these epithelial cells (see Supp. Movies 1-3). (i , j) Across saliva cells, there is infection heterogeneity of pCK cells). (j) Using 10 samples collected from COVID-19 outpatients (UNC OBS-C), we confirm pCK cells are the primary infected population. (k) SARS-CoV-2 can be found to be associated with the diverse oral microbiome on shed epithelial cells; Dotted black box in (b) represents zoomed-in regions; arrowheads in (d) replicating SARS-CoV-2 sense strands); in (f) indicate ACE2 (red), TMPRSS2 (green) or co-expressing (yellow) suprabasal cells; dotted white lines (h) highlight cell membranes; pink brackets in (g) represent high expressing cells; arrowheads in (i) indicate SARS-CoV-2+ (red) cells. Arrowheads in (k) indicate SARS-CoV-2 (red), universal 16S probe (green) that are co-expressing (yellow); statistical test in (i): paired Student’s t-test; * = p<0.05. Scale bars: ( a , b , d , e , h ) 25 μm, ( f , g ) 10μm.

Article Snippet: Positive control probe: 320861; Negative control probe: 320871; ACE2 antibody: Mouse Monoclonal (protein tech #66699-1-IG), (ACE2 diluted 1/2000, PBS 1% BSA, 1% donkey serum PBS); SARS-CoV-2 probe: ACD, 848561; ACE2 probe: #848151-C3; TMPRSS2 probe: 470341-C2; CSTL probe: 858611-C3; Secondary antibodies: Al secondary antibodies were in donkey (D-anti Goat, D-anti Rabbit, diluted 1/300 in 1/300 PBS 1% BSA, 1% donkey serum, PBS).

Techniques: In Situ Hybridization, Infection, Concentration Assay, Expressing, Confocal Microscopy, Virus

Journal: medRxiv

Article Title: Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis

doi: 10.1101/2020.10.26.20219089

Figure Lengend Snippet: (a) In situ hybridization (ISH) also reveals infection in the major parotid gland (PG). (b) Microscopic assessment of the COVID-19 saliva glands by H&E reveals variable histological presentations including atrophy and fibrosis. (c) Viral load in minor saliva glands is generally higher when compared to glands using ddPCR. (d) Shed cells in saliva display unique TMPRSS family heterogeneity, which we also observed in tissue-specific oral atlases for glands and mucosa. Saliva glands express the highest TMPRSS2 in mouse and human atlases, and some shed epithelial cells express similar patterns, suggesting they may be shed from the SG ducts. Others, express higher TMPRSS4, suggesting these cells may be from the suprabasal mucosa. (e-f) (e) ISH mapping validation pipeline for discovering epithelial expression of SARS-CoV-2 entry factor expression in shedding suprabasal cells, first starting with H&E histochemical staining. (f) ISH was used with negative and positive probe controls. (g) Ciliated cells in saliva are exceedingly rare (α-tubulin+) but can present and infected. Dotted black box in (b , e) represents zoom-in regions; white arrow in (f) represents sloughing trajectory; dotted white lines (g) highlight cell membranes; arrowheads in (g , h) indicate SARS-CoV-2+ (red) cells. Scale bars: (b) 50μm (a , d , e , h) 25μm, (g) 10μm.

Article Snippet: Positive control probe: 320861; Negative control probe: 320871; ACE2 antibody: Mouse Monoclonal (protein tech #66699-1-IG), (ACE2 diluted 1/2000, PBS 1% BSA, 1% donkey serum PBS); SARS-CoV-2 probe: ACD, 848561; ACE2 probe: #848151-C3; TMPRSS2 probe: 470341-C2; CSTL probe: 858611-C3; Secondary antibodies: Al secondary antibodies were in donkey (D-anti Goat, D-anti Rabbit, diluted 1/300 in 1/300 PBS 1% BSA, 1% donkey serum, PBS).

Techniques: In Situ Hybridization, Infection, Biomarker Discovery, Expressing, Staining

Journal: medRxiv

Article Title: Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis

doi: 10.1101/2020.10.26.20219089

Figure Lengend Snippet: (a) Using a recently pre-printed salivary neutrophil atlas from healthy patients50, we confirm that virtually no neutrophils express SARS-CoV-2 ACE2 or TMPRSS2 . Neutrophils constitute ∼50% of salivary cells and epithelial cells the other 50% with trace amounts of lymphocytes and other cell populations. (b) Using cell blocks of heathy saliva, we validate our findings that shed salivary epithelial (pan-cytokeratin positive; pCK+) cells express both ACE2 and TMPRSS2 . We see minimal to no expression of these SARS-CoV-2 entry factors in pCK-negative cells. (c) From the NIH Car Line study, the majority of asymptomatic subjects only displayed NP swab positivity, including two patients who developed symptoms during the prospective sampling (CoV12 and CoV13). (d) Taking this cohort of asymptomatic and symptomatic subjects, infectious saliva fraction was measured with masks and unmasked highlighting the need for masks to prevent the spread of SARS-CoV-2 via saliva droplets. While obvious, this measure highlights the ability to prevent the spread of the oral infection axis (see Cov01 saliva ejection with no mask ‘none’). (e , f) (e) We confirm our findings from the UNC OBS-C study and from NIH Car Line study that highest saliva viral load and reported taste alterations (dysgeusia in CoV19), we observe significant salivary epithelial (pCK+)cells; See also . (f) results of pol and spike quantification of salivary cell infection among 3 samples show consistency of the infection quantification method. Scale bars: (a , e) 25μm.

Article Snippet: Positive control probe: 320861; Negative control probe: 320871; ACE2 antibody: Mouse Monoclonal (protein tech #66699-1-IG), (ACE2 diluted 1/2000, PBS 1% BSA, 1% donkey serum PBS); SARS-CoV-2 probe: ACD, 848561; ACE2 probe: #848151-C3; TMPRSS2 probe: 470341-C2; CSTL probe: 858611-C3; Secondary antibodies: Al secondary antibodies were in donkey (D-anti Goat, D-anti Rabbit, diluted 1/300 in 1/300 PBS 1% BSA, 1% donkey serum, PBS).

Techniques: Expressing, Sampling, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: Photoreceptors are preserved in RPE-Abca4-Tg/Abca4 −/− vs. Abca4 −/− mice. ( A ) Representative retina images from 1-y-old albino mice acquired by light microscopy. (Scale bars, 20 μm.) ( B ) Total numbers of photoreceptor nuclei were counted per 100-μm 2 cell area. Note the increased number of cells in the ONL of RPE-Abca4-Tg/Abca4 −/− mice compared with Abca4 −/− mice indicating partial rescue of photoreceptor degeneration. Data are presented as mean ± SD; n = 5–9 mice per group; RPE-Abca4-Tg/Abca4 −/− vs. Abca4 −/− , * P = 0.0319; Abca4 −/− vs. BALB/c, ** P < 0.0001; and RPE-Abca4-Tg/Abca4 −/− vs. BALB/c, P = 0.0061.

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: Light Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: ABCA4 colocalizes with endolysosomal markers. ( A ) Representative merged confocal images of retina/RPE sections from 2-mo-old wild-type BALB/c ( Upper ) and albino Abca4 −/− ( Lower ) mice reacted with antibodies to ABCA4 (red) and LAMP1 (green). Note that ABCA4 and LAMP1 colocalize in wild-type RPE but not in the OS. LAMP1, but not ABCA4, immunoreactivity is also present in the Abca4 −/− RPE cells. ( B ) Representative merged confocal images of retina sections from 5-mo-old wild-type (129/Sv) ( Top ), Abca4 −/− ( Middle ), and Mertk −/− ( Bottom ) mice immunostained with ABCA4 (red) and Rab5 (green) antibodies. Colocalization of ABCA4 and Rab5 is observed in both 129/Sv and Mertk −/− RPE cells as indicated by the orange signal. In the RPE of Abca4 −/− retina section only Rab5 immunoreactivity is seen. White arrows indicate retinal detachment, and the white asterisk indicates the absence of OS in the Mertk −/− retina due to photoreceptor degeneration. ( C ) Representative confocal images of fixed hfRPE cells labeled with ABCA4 (red) ( Top ) or endosomal CAV1 (green) ( Middle ) antibodies. ( Bottom ) Merged confocal images of ABCA4 and CAV1. Note the colocalization of ABCA4 and CAV1. The green labeling of the filter in the CAV1 panel is due to nitrocellulose autofluorescence. Nuclei are stained with DAPI (blue). For murine RPE sections, n = 3 mice per group. For hfRPE cells, each experiment was repeated three times with three different donor cell lines. (Magnification: C , 60×.) (Scale bars, 10 μm.)

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: Proposed function of ABCA4 in the endolysosomal membranes of RPE. ( A ) Normal RPE cell. 11cRAL released during proteolysis of rhodopsin within a phagolysosome condenses with PE on the luminal surface to form 11- cis - N -retinylidene-phosphatidylethanolamine (11c- N -ret-PE), which undergoes isomerization to form a mixture of all- trans (at) and 11c- N- ret-PEs. Both N- ret-PE isomers are flipped by ABCA4 to the cytoplasmic surface, where hydrolysis of N- ret-PE is driven by mass action through binding of 11cRAL by cellular retinaldehyde-binding protein (CRALBP) or the reduction of atRAL to atROL by retinol dehydrogenase type 11 (RDH11). The atROL is processed by the RPE visual cycle through esterification by lecithin retinol acyltransferase (LRAT) to yield an all- trans -retinyl ester such as all- trans -retinyl palmitate (atRP), isomerization by RPE65 to yield 11- cis -retinol (11cROL), and oxidation by retinol dehydrogenase type 5 (RDH5) to yield 11cRAL, which binds to CRALBP. 11cRAL leaves the RPE cell to regenerate visual pigments in the adjacent photoreceptor OS. ( B ) Abca4 −/− mutant RPE cell. The lack of ABCA4 in RPE endolysosomes of Abca4 −/− mice or STGD1 patients causes delayed clearance of retinaldehydes and hence higher concentrations of both free retinaldehydes and N- ret-PE. This leads to secondary condensation of atRAL or 11cRAL with N- ret-PE to form bisretinoids.

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: Binding Assay, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: ( A – C ) Abca4 mRNA and protein is expressed in RPE cells. In situ hybridization using the RNAscope assay with an Abca4 -specific probe on human cadaveric ocular sections ( A ), mouse retina sections ( B ), and hfRPE cells in culture ( C ). Note the intense chromogenic reactivity (red punctate staining, indicated by the black arrows) for Abca4 mRNA in outer nuclear layer (ONL) and inner segments (IS) of the photoreceptor cells and in RPE cells of human ( A ) and wild-type BALB/c sections ( B , Left ). This reactivity is absent in Abca4 −/− tissue ( B , Right ). Red punctate staining (white arrows) corresponding to ABCA4 mRNA is also observed in hfRPE cultured cells ( C ). CC, choriocapillaris; INL, inner nuclear layer. (Scale bars, 20 μm.) ( D ) ABCA4 immunohistochemistry (red fluorescence) on retina sections from pigmented wild-type (129/Sv), Mertk −/− , and Abca4 −/− mice. Note that ABCA4 immunoreactivity is seen in the RPE and OS of 129/Sv mice and in the RPE but not in the OS (indicated by white asterisk) of Mertk −/− mice but is not seen in the retina section from an Abca4 −/− mouse. The white arrows indicate retinal detachment. Cell nuclei are stained with DAPI (blue). (Scale bars, 10 μm.) ( E ) Representative immunoblots for ABCA4 protein using neural retina and RPE/eyecup homogenates loaded as a fraction of one mouse eye per lane, as indicated. The RNAscope assay ( A – C ) was done with two human cadaveric eyes, three cultured hfRPE cells of different donor eyes, and n = 3 mice (5-mo-old) of each genotype; Immunohistochemistry experiments ( D ) were repeated three times with n = 3 5-mo-old mice per group. The immunoblotting experiment ( E ) was done in duplicates varying the fraction of the homogenate corresponding to one mouse eye ( n = 4 mice for each experiment).

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: In Situ Hybridization, RNAscope, Staining, Cell Culture, Immunohistochemistry, Fluorescence, Western Blot

Journal: eLife

Article Title: Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

doi: 10.7554/eLife.67436

Figure Lengend Snippet: ( A-C ) UMAP plots of Nkx2.1 ( A ), Gsx2 ( B ), and Emx1 ( C ) expression in the scRNA-Seq neurogenic lineage (see also ). ( D ) Schematic of the region identity prediction calculation, where anchor gene sets (dashed gray lines) are calculated between Ventral Dissection (magentas) and Dorsal Dissection (greens) sNucRNA-Seq B cell nuclei and scRNAseq B cells (blues) and each scRNAseq B cell is given a Dorsal or Ventral Dissection predicted identity score (see also ). ( E ) The net predicted identity scores of each scRNA-Seq B cell plotted in UMAP space, where strongly Dorsal Dissection predicted identity cells are dark green, and strongly Ventral Dissection predicted identities are dark magenta. Dark gray cells were not included in the analysis. ( F ) Dot plot of the average Dorsal or Ventral Dissection predicted identity scores for scRNA-Seq B cell clusters B(14) and B(5+22). ( G ) UMAP plot of B cell cluster identities used in the following analysis: B(14) (light blue) and B(5+22) (dark blue). ( H ) (i) Venn diagram summarizing differential gene expression analysis between clusters B(14) (light blue) and B(5+22) (dark blue). (ii) Numbers of candidate marker genes identified after selecting significantly upregulated genes expressed in no more than 40% of cells of the other cluster. ( I ) Heatmap depicting expression of the top 10 differentially expressed genes between clusters B(14) (left) and B(5+22) (right). ( J ) UMAP plot of Dio2 expression in the scRNA-Seq neurogenic lineage. ( K - L ) Confocal micrographs of Dio2 RNA (magenta), S100b (green), and GFAP (white) protein expression in the dorsal ( K ) and ventral ( L ) V-SVZ. ( M-N ). Quantification of DAPI+, S100ß- Dio2 RNAscope puncta along the length of the V-SVZ (0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge) with the median puncta distribution location plotted as a horizontal line for each sample (n=3, each indicated by a different shade, ~ bregma 1.18, 0.98 and 0.62 mm). N. Summary schematic of Dio2 expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98 and 0.14 mm); strong expression (magenta); sparse (light magenta). Boxed areas denote locations of dorsal ( K ) and ventral ( L ) high-magnification images. ( O ) UMAP plot of Urah expression in the scRNA-Seq neurogenic lineage. ( P - Q ) Confocal micrographs of Urah RNA (magenta), S100b (green), and GFAP (white) protein expression in the dorsal ( P ) and ventral ( Q ) V-SVZ. ( R-S ) Quantification of DAPI+, S100ß- Urah RNAscope puncta along the length of the V-SVZ (0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge) with the median puncta distribution location plotted as a horizontal line for each sample (n=3, each indicated by a different shade, bregma~ 1.34, 1.18 and 0.98 mm). S. Summary schematic of Urah expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98 and 0.14 mm); strong expression (magenta); sparse (light magenta). Boxed areas denote locations of dorsal ( P ) and ventral ( Q ) high-magnification images. T UMAP plot of Crym expression in the scRNA-Seq neurogenic lineage. ( U-V ) Confocal micrographs of Crym RNA (magenta), DCX (green), and GFAP (white) protein expression in the dorsal ( U ) and ventral ( V ) V-SVZ. ( W-X ). Quantification of DAPI+,S100B- Crym RNAscope puncta along the length of the V-SVZ (as in M: 0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge; n=3, bregma~ 1.42, 1.18 and 0.98 mm). X. Summary schematic of Crym expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98 and 0.14 mm); strong expression (magenta); low expression (white). Boxed areas denote locations of dorsal ( U ) and ventral ( V ) high-magnification images. DAPI: blue, LV: lateral ventricle, d: dorsal, v: ventral, CC: corpus callosum, Str: striatum. Scale bars: 10 μm (K, L, P, Q, U, and V). Figure 3—source data 1. Quantifications of Crym, Dio2 , and Urah RNAscope spots.

Article Snippet: Interestingly, density plots of RNAscope spots for Slit2+ DCX- cells showed higher numbers of spots ventrally, while Slit2+ DCX+ were equally distributed along the ventricular wall.

Techniques: Expressing, Dissection, Gene Expression, Marker, RNAscope

Journal: eLife

Article Title: Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

doi: 10.7554/eLife.67436

Figure Lengend Snippet: ( A ) UMAP plot highlighting the putative dorsal and ventral B and A cell clusters. ( B ) Schematic illustrating the approach to identify genes that are differentially enriched in dorsal B and A, and ventral B and A cells, comparing B(14) to A(1) and B(5) and B(22) to A(0). ( C ) Expression patterns of dorsal (top row) and ventral markers (bottom row) identified as differentially enriched throughout the B-C-A lineage. ( D ) Cell scores based on the combined expression of genes in the ventral lineage signature (ventral score; purple) and genes in the dorsal lineage signature (dorsal score; green) throughout the neurogenic lineage. Lineage scores, as the combined expression of genes of the dorsal and ventral signatures, remain relatively constant throughout all cells along the neurogenic lineage. ( E ) High Score (top quartile of each lineage score) dorsal (green) and ventral (purple) cells in the neurogenic lineage. ( F ) Expression of dorsal signature genes in High Score dorsal (left) and High Score ventral cells (right) along the neurogenic lineage progression, assessed by pseudotime (see also ). ( G ) Expression of ventral signature genes in High Score dorsal (left) and High Score ventral cells (right) along the neurogenic lineage progression, assessed by pseudotime. ( H ) Volcano plot of significantly differentially expressed genes between High Score dorsal and High Score ventral neurogenic lineages. ( I - J ) RNAscope validation of dorsal lineage marker Pax6 (magenta) with DCX (green) and GFAP (white) immunostaining. High-magnification images of the V-SVZ dorsal wedge ( I ). J. High-magnification image of the V-SVZ where Pax6 colocalizes with an A cell (arrow) and B cell (arrowhead) (see also ). ( K - L ) RNAscope validation of dorsal lineage marker Rlbp1 (magenta) in the dorsal wedge ( K ). L. High-magnification images of Rlbp1 in the dorsal V-SVZ, where puncta are visible in a DCX-positive A cell (arrow). ( M - O ) Quantifications of DAPI+,DCX- ( M ) or DAPI+,DCX+ ( N ) Rlbp1 RNAscope puncta along the length of the V-SVZ (0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge) with the median puncta distribution location plotted as a horizontal line for each sample (n=3, each indicated by a different shade, ~ bregma 1.34, 1.18, and 0.98 mm). ( O ) Summary schematic of Rlbp1 expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98, and 0.14 mm); strong expression (magenta); sparse (light magenta). Note that Pax6 showed a similar distribution. ( P-Q ) RNAscope validation of ventral lineage marker Adgrl3 (magenta) in the ventral V-SVZ. ( Q ) High-magnification image of Adgrl3 in the ventral V-SVZ, colocalizing with an A cell (arrow). ( R - S ). RNAscope validation of ventral lineage marker Slit2 (magenta) in the ventral V-SVZ. S. High-magnification image of the ventral V-SVZ, where Slit2 puncta colocalize with an A cell (arrow) and B cell (arrowhead). ( T-V ). Quantifications of DAPI+,DCX- ( T ) or DAPI+,DCX+ ( U ) Slit2 RNAscope puncta along the length of the V-SVZ (0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge) with the median puncta distribution location plotted as a horizontal line for each sample (n=3, each indicated by a different shade, ~ bregma 1.34, 1.18, and 0.62 mm). V. Summary schematic of Slit2 expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98, and 0.14 mm); strong expression (magenta); sparse (light magenta). Note that Adgrl3 showed a similar distribution. CC: corpus callosum, Str: striatum, LV: lateral ventricle. Scale bars: 15 μm (K, P, and R), 10 μm ( I ), and 5 μm (J, L, Q, and S). Figure 6—source data 1. Quantifications of Rlbp1 and Slit2 RNAscope spots.

Article Snippet: Interestingly, density plots of RNAscope spots for Slit2+ DCX- cells showed higher numbers of spots ventrally, while Slit2+ DCX+ were equally distributed along the ventricular wall.

Techniques: Expressing, RNAscope, Biomarker Discovery, Marker, Immunostaining

Journal: eLife

Article Title: Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

doi: 10.7554/eLife.67436

Figure Lengend Snippet: ( A ) Schematic illustrating the control comparison to identify genes enriched in both ventral B and dorsal A cells, or in dorsal B and ventral A cells. ( B ) UMAP plots showing expression of the three genes identified in the analysis in ( A ), including Efna5, Cntnap2 , and Flrt2 . ( C ) UMAP plot colored by position score, based on net ventral (purple) and dorsal (green) AUCell scores. ( D ) UMAP of the neurogenic lineage split by classification into the ventral (purple) or dorsal (green) lineage. ( E ) Violin plots of AUCell score distribution of dorsal and ventral cells shown in ( D ). NA: cells not assigned. ( F ) Expression of dorsal signature genes in all cells along the neurogenic lineage progression represented by pseudotime. ( G ) Expression of ventral signature genes in all cells along the neurogenic lineage progression represented by pseudotime. ( H ) Gene ontology analysis on differentially expressed genes between High Score dorsal and ventral B cells and High Score dorsal and ventral A cells reveal overrepresented gene categories. ( I - O ) Low-magnification images of the entire V-SVZ in a coronal section, labeled with RNAscope probes (magenta) against Pax6 ( J ); Rlbp1 ( L ); Adgrl3 ( N ), and Slit2 ( P ). ( J-L ) High magnification of the ventral V-SVZ from panels I ( J ) and K ( L ). ( N-P ) High magnification of the dorsal V-SVZ from panel M ( N ) and O ( P ). Scale bars: 100 μm (I, K, M, and O) and 15 μm (J, L, N, and P).

Article Snippet: Interestingly, density plots of RNAscope spots for Slit2+ DCX- cells showed higher numbers of spots ventrally, while Slit2+ DCX+ were equally distributed along the ventricular wall.

Techniques: Control, Comparison, Expressing, Labeling, RNAscope

Journal: eLife

Article Title: Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

doi: 10.7554/eLife.67436

Figure Lengend Snippet: ( A ) A summary of cell types in the neurogenic lineage identified by scRNA-Seq and their classification into dorsal and ventral transcriptional identities. ( B ) Schematic depicting the dorsal and ventral domains newly identified by scRNA-Seq and snucRNA-Seq, and confirmed by staining and RNAscope.

Article Snippet: Interestingly, density plots of RNAscope spots for Slit2+ DCX- cells showed higher numbers of spots ventrally, while Slit2+ DCX+ were equally distributed along the ventricular wall.

Techniques: Staining, RNAscope

Journal: eLife

Article Title: Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

doi: 10.7554/eLife.67436

Figure Lengend Snippet:

Article Snippet: Interestingly, density plots of RNAscope spots for Slit2+ DCX- cells showed higher numbers of spots ventrally, while Slit2+ DCX+ were equally distributed along the ventricular wall.

Techniques: Sequencing, Multiplex Assay, Red Blood Cell Lysis, Blocking Assay, RNAscope, Negative Control, Positive Control, Software