Journal: eLife

Article Title: A single-cell transcriptomic atlas reveals resident dendritic-like cells in the zebrafish brain parenchyma

doi: 10.7554/eLife.91427

Figure Lengend Snippet: ( A–F ) Immunofluorescence on transversal brain sections (14 µm) from Tg(mpeg1:GFP ) ( A–C ) or Tg(p2ry12:p2ry12- GFP ) ( D–F ) transgenic adult fish co-immunostained with anti-GFP (green) and anti-Lcp1 (magenta) antibodies. ( A–C ) All mpeg1 :GFP + mononuclear phagocytes in the brain parenchyma display Lcp1 immunostaining, as expected. ( D–F ) Similarly, all microglial cells, identified by GFP expression in the brain parenchyma of Tg(pr2y12:p2ry12-GFP ) fish, are Lcp1 + , as expected. ( G–J ) In sections of adult Tg(p2ry12:p2ry12-GFP; mpeg1:mCherry ) double transgenic animals, GFP labeling is not observed in all mCherry + cells. GFP (green), mCherry (gray), Lcp1 (magenta), and merge of the three channels. All images were taken using a 20 X objective and correspond to orthogonal projections. White arrowheads point to microglial cells (GFP + ; Lcp1 + or GFP + ; mCherry + ; Lcp1 + ) and yellow arrowheads to DC-like cells (GFP - ; Lcp1 + or GFP - ; mCherry + ; Lcp1 + ). Scale bars: 50 µm. ( K–N ) Confocal imaging of a midbrain vibratome section (100 µm) from an adult Tg(mhc2dab:GFP; cd45:DsRed ) brain. GFP (green), DsRed (magenta) and merge of the two channels are shown. Images correspond to orthogonal projections, white arrowheads point to GFP + ; DsRed + cells, and yellow arrowheads to GFP - ; DsRed high . Scale bar in ( K ): 500 µm, scale bar in ( L–N ): 50 µm. Images are representative of brain tissue sections from 2 to 3 fish.

Article Snippet: The following primary and secondary antibodies were used: chicken anti-GFP polyclonal antibody (1:500; Abcam, Cat# ab13970), rabbit anti-Lcp1 (1:1000), mouse anti-mCherry monoclonal antibody (1:500; Takara Bio Cat# 632543), Alexa Fluor 488-conjugated anti-chicken IgG antibody (1:500; Abcam Cat# ab150169), Alexa Fluor 594-conjugated anti-rabbit IgG (1:500; Abcam Cat# ab150076), Alexa Fluor 647-conjugated anti-mouse IgG (1:500; Abcam Cat# ab150107).

Techniques: Immunofluorescence, Transgenic Assay, Immunostaining, Expressing, Labeling, Imaging

Journal: eLife

Article Title: A single-cell transcriptomic atlas reveals resident dendritic-like cells in the zebrafish brain parenchyma

doi: 10.7554/eLife.91427

Figure Lengend Snippet: ( A–J ) Immunofluorescence on transverse brain sections (14 µm) from adult wild-type ( A–E ) and batf3 -/- mutant ( F–J ) fish carrying the Tg(p2ry12:p2ry12-GFP; mpeg1:mCherry ) double transgene and immunostained for GFP (green), mCherry (gray), and Lcp1 (magenta). Illustrative case of the merge of the three channels ( A, F ) allowing to identify GFP + ; mCherry + ; Lcp1 + microglia (white arrowheads) versus GFP - ; mCherry + ; Lcp1 + DC-like cells (yellow arrowheads). While DC-like cells are found in high numbers within the ventral part of control parenchyma ( A ), these are dramatically decreased following genetic loss of batf3 ( F ). Scale bars: 100 µm. ( B–E, G–J ). Single channels high magnification of the insets in A ( B–E ) and F ( G–J ). Scale bars: 50 µm. Images were taken using a 20 X objective and correspond to orthogonal projections. ( K–M ) Quantification of cell density for GFP + ; mCherry + ; Lcp1 + microglia and GFP - ; mCherry + ; Lcp1 + DC-like cells in the dorsal midbrain area or optic tectum ( K ), ventral midbrain area ( L ), and the entire section ( M ) of control (gray bars) and batf3 -/- (green bars) fish. Each dot represents a single fish and data are mean ± SEM. * p <0.05 (Mann-Whitney test), *** p <0.0001 (Two-tailed unpaired t-test). ( N ) Flow cytometry analysis of brain cell suspensions from wild-type and batf3 -/- adult fish carrying the Tg(p2ry12:p2ry12-GFP; mpeg1:mCherry ) reporter. The GFP + ; mCherry + fraction identifies microglia (green circle), whereas the GFP - ; mCherry + fraction contains mainly DC-like cells (blue frame). ( O ) Percentage of microglia and DC-like cells in brain cell suspensions for each genotype, relative to the whole living brain population, as shown in ( N ) ( wild-type , n=6; batf3 -/- , n=10). *** p <0.001 (Two-tailed unpaired t-test). n refers to number of biological replicates.

Article Snippet: The following primary and secondary antibodies were used: chicken anti-GFP polyclonal antibody (1:500; Abcam, Cat# ab13970), rabbit anti-Lcp1 (1:1000), mouse anti-mCherry monoclonal antibody (1:500; Takara Bio Cat# 632543), Alexa Fluor 488-conjugated anti-chicken IgG antibody (1:500; Abcam Cat# ab150169), Alexa Fluor 594-conjugated anti-rabbit IgG (1:500; Abcam Cat# ab150076), Alexa Fluor 647-conjugated anti-mouse IgG (1:500; Abcam Cat# ab150107).

Techniques: Immunofluorescence, Mutagenesis, Control, MANN-WHITNEY, Two Tailed Test, Flow Cytometry

Journal: eLife

Article Title: A single-cell transcriptomic atlas reveals resident dendritic-like cells in the zebrafish brain parenchyma

doi: 10.7554/eLife.91427

Figure Lengend Snippet: ( A–C ) Cell density quantification of non-myeloid cells (GFP - ; mCherry - ; Lcp1 + ) and total leukocyte (all Lcp1 + ) in the dorsal midbrain area or optic tectum ( A ), ventral midbrain area ( B ) and the whole section ( C ) of wild-type and batf3 -/- fish from . Data represent mean ± SEM (n=4 fish). ( D, E ) Q-PCR expression for dendritic cell (DC)-like markers siglec15l and ccl19a.1 in p2ry12:GFP - ; mpeg1:mCherry + DC like cells ( D ) and for microglia-specific genes p2ry12 , apoeb and ccl34b.1 in p2ry12:GFP + ; mpeg1:mCherry + microglia ( E ) sorted from wild-type (gray bars) and batf3 -/- (colored bars) adult brains. * p <0.05, *** p <0.001 (Two-tailed unpaired t-test). Data are represented as mean ± SEM. n refers to the number of biological replicates. ( F ) Number of cells (y-axis) versus mCherry fluorescence intensity (x-axis) histogram plot of brain cell suspensions from controls (gray) and batf3 -/- (blue) fish carrying the Tg(p2ry12:GFP; mpeg1:mCherry ) double transgene. It shows that all residual mpeg1:mCherry + cells in the batf3 mutant display lower fluorescence intensity as compared to their wild-type counterparts. ( G ) Proportion of mpeg1:mCherry + cells according to their low or high fluorescence intensity as shown in F. Data represent mean ± SEM (n=4). n refers to the number of biological replicates. **** p <0.0001 (Two-tailed unpaired t-test). ( H–K ) Illustrative case of vibratome midbrain sections of Tg(cd45:DsRed ) transgenic wild-type ( H,I ) and batf3 -/- ( J,K ) fish (n=3). The endogenous fluorescence of DsRed high DC-like cells permits their identification in the ventral part of wild-type brains (n=3). However, these cells are not found in the batf3 -/- mutant (n=3). Scale bar: 500 µm. ( I, K ) High magnification of the insets (white frame) in H ( I ) and J ( K ). Scale bar: 100 µm. Note that cells with lower fluorescence intensity (e.g. cd45 low microglia) are not detectable using this approach due to signal loss (quenching) following fixation.

Article Snippet: The following primary and secondary antibodies were used: chicken anti-GFP polyclonal antibody (1:500; Abcam, Cat# ab13970), rabbit anti-Lcp1 (1:1000), mouse anti-mCherry monoclonal antibody (1:500; Takara Bio Cat# 632543), Alexa Fluor 488-conjugated anti-chicken IgG antibody (1:500; Abcam Cat# ab150169), Alexa Fluor 594-conjugated anti-rabbit IgG (1:500; Abcam Cat# ab150076), Alexa Fluor 647-conjugated anti-mouse IgG (1:500; Abcam Cat# ab150107).

Techniques: Expressing, Two Tailed Test, Fluorescence, Mutagenesis, Transgenic Assay