B - C Flow cytometric analysis of TAMs ( CD11b + F480 + ) in MC38 ( B ) and KPC ( C ) tumours receiving indicated treatments . Tumours were harvested 5 days following IR and disaggregated for analysis by flow cytometry . The left panels in B and C show the data derived from the flow cytometry with representative plots shown in the right panels . Data are presented as mean SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( n = 6 mice / group , 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Flow cytometric analysis of TAMs (CD11b+ F480+) in MC38 (B) and KPC (C) tumours receiving indicated treatments. Tumours were harvested 5 days following IR and disaggregated for analysis by flow cytometry. The left panels in B and C show the data derived from the flow cytometry with representative plots shown in the right panels. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group, 3 independent experiments).


D - E , Tumour growth kinetics of MC38 ( D ) and KPC ( E ) tumours receiving the indicated treatments . Data are presented as mean tumour volume ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment .

O O O O O O O O O O O O O O O O O O O O O O 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-E, Tumour growth kinetics of MC38 (D) and KPC (E) tumours receiving the indicated treatments. Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment.


F - G , shows flow cytometric analysis of CD206hi ( F ) TAMs in MC38 and KPC tumours 5 days following IR . Data are presented as mean ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( n = 6 mice / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, shows flow cytometric analysis of CD206hi (F) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).


F - G , shows flow cytometric analysis of iNOShi ( G ) TAMs in MC38 and KPC tumours 5 days following IR . Data are presented as mean ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( n = 6 mice / group ) .

F-G, shows flow cytometric analysis of iNOShi (G) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).


H - I , CD11b + F480 + TAMs were isolated from MC38 ( H ) and KPC ( I ) tumours 5 days following irradiation ( + / - aCSF ) and expression of selected immune stimulatory and immunosuppressive genes was analysed by RT - qPCR ( n = 3 ) . Data are presented as mean ± SEM ( 10Gy vs 10Gy + aCSF n = 6 mice / group ) . Statistical significance was determined by Mann - Witney . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I, CD11b+ F480+ TAMs were isolated from MC38 (H) and KPC (I) tumours 5 days following irradiation (+/-aCSF) and expression of selected immune stimulatory and immunosuppressive genes was analysed by RT-qPCR (n=3). Data are presented as mean ± SEM (10Gy vs 10Gy + aCSF n=6 mice/group). Statistical significance was determined by Mann-Witney. *P <0.05 **P <0.01 ***P <0.001.


A - B MC38 ( A ) and KPC ( B ) tumours were harvested 5 days following 10Gy IR ± aCSF as indicated . The left panels show the percentage of CD45 + CD3 + CD8 + T cells in these tumours after the indicated treatments . Representative flow cytometry plots from the irradiated groups are shown in the right panels . Tumours that did not receive irradiation were harvested when tumours reached 500mm3 . Data are presented as mean SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( A ) and Kruskal - Wallis test with Dunn ' s multiple comparison ( B ) ( n = 6 mice / group , 3 independent experiments ) .

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A-B MC38 (A) and KPC (B) tumours were harvested 5 days following 10Gy IR ± aCSF as indicated. The left panels show the percentage of CD45+ CD3+ CD8+ T cells in these tumours after the indicated treatments. Representative flow cytometry plots from the irradiated groups are shown in the right panels. Tumours that did not receive irradiation were harvested when tumours reached 500mm3. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (A) and Kruskal-Wallis test with Dunn's multiple comparison (B) (n=6 mice/group, 3 independent experiments).


C , Flow cytometric analysis of Ki67 expression in the CD8 + T cells in MC38 tumours from A . Data are presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O 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, Flow cytometric analysis of Ki67 expression in the CD8+ T cells in MC38 tumours from A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group).


D , Flow cytometric analysis of PD - 1 expression on CD8 + T cells from MC38 in tumours as in A . Data are presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Flow cytometric analysis of PD-1 expression on CD8+ T cells from MC38 in tumours as in A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group).


E - F Flow cytometric analysis of IFN - γ expression in CD8 + T cells isolated from MC38 tumours in A . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 mice / group , 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Flow cytometric analysis of IFN-γ expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments).


E - F , Flow cytometric analysis of Granzyme B expression in CD8 + T cells isolated from MC38 tumours in A . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 mice / group , 3 independent experiments ) .

E-F, Flow cytometric analysis of Granzyme B expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments).


G - H , Immunofluorescent staining of MC38 ( G ) and KPC ( H ) tumour sections , blue = DAPI , green = CD8 .

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

G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections, blue=DAPI, green = CD8.


I - J , Tumour growth in CD8 depleted C57BL6 wild - type mice bearing MC38 ( I ) and KPC ( J ) tumours receiving treatment as indicated ( n = 6 mice / group ) .

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

I-J, Tumour growth in CD8 depleted C57BL6 wild-type mice bearing MC38 (I) and KPC (J) tumours receiving treatment as indicated (n=6 mice/group).


K , Flow cytometric quantification of intra - tumoural CD8 T cells following anti - CD8 treatment . Data presented as mean SEM and analysed by unpaired t - test ( n = 5 mice / group ) .

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

K, Flow cytometric quantification of intra-tumoural CD8 T cells following anti-CD8 treatment. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group).


L - M , Tumour growth in athymic nude mice bearing MC38 ( I ) and KPC ( J ) tumours receiving treatments as indicated ( n = 6 mice / group ) . Data are presented as mean tumour volume ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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

L-M, Tumour growth in athymic nude mice bearing MC38 (I) and KPC (J) tumours receiving treatments as indicated (n=6 mice/group). Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment. *P <0.05 **P <0.01 ***P <0.001.


A - B , CD8 + T cells were isolated from the spleens of MC38 tumour bearing mice . The tumours were radiated with 10Gy and cells were harvested 5 days later . Quantification ( A ) and representative images ( B ) of MC38 tumour cell specific tumour - derived CD8 T cell responses detected by IFN - γ ELISpot . The tumour specific CD8 + T cell response was evaluated after T cell incubation with naïve or irradiated MC38 cells for 24 hours . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 3 mice / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, CD8+ T cells were isolated from the spleens of MC38 tumour bearing mice. The tumours were radiated with 10Gy and cells were harvested 5 days later. Quantification (A) and representative images (B) of MC38 tumour cell specific tumour-derived CD8 T cell responses detected by IFN-γ ELISpot. The tumour specific CD8+ T cell response was evaluated after T cell incubation with naïve or irradiated MC38 cells for 24 hours. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3 mice/group).


C - D , Flow cytometric detection of major histocompatibility complex I ( MHCI ) expressed on MC38 ( C ) and KPC ( D ) tumour cells 48 hours following exposure to 10Gy IR . The left graph shows the overall data , with representative flow cytometry plots on the right . Data presented as mean ± SEM and analysed by Mann - Witney ( n = 3 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D, Flow cytometric detection of major histocompatibility complex I (MHCI) expressed on MC38 (C) and KPC (D) tumour cells 48 hours following exposure to 10Gy IR. The left graph shows the overall data, with representative flow cytometry plots on the right. Data presented as mean ± SEM and analysed by Mann-Witney (n=3/group).


E - F , Flow cytometric quantification of major histocompatibility complex I ( MHCI ) expression in vivo . Gated MC38 ( E ) and KPC ( F ) tumour cells were analysed 48 hours following exposure to 10Gy IR . Data presented as mean ± SEM and analysed by unpaired t - test ( n = 3 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F, Flow cytometric quantification of major histocompatibility complex I (MHCI) expression in vivo. Gated MC38 (E) and KPC (F) tumour cells were analysed 48 hours following exposure to 10Gy IR. Data presented as mean ± SEM and analysed by unpaired t-test (n=3/group).


G - H , Flow cytometric quantification of dendritic cells ( CD11b + CD11c + MHCII + ) in MC38 ( G ) and KPC ( H ) tumours receiving treatment as indicated and as in E - F . Data presented as mean SEM and analysed by unpaired t - test ( n = 5 mice / group ) .

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

G-H, Flow cytometric quantification of dendritic cells (CD11b+ CD11c+ MHCII+) in MC38 (G) and KPC (H) tumours receiving treatment as indicated and as in E-F. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group).


J - K , Tumour growth in mice bearing two MC38 tumours receiving 10Gy IR to the primary lesion ( J ) ± systemic aCSF therapy ( K ) . The differences in tumour volume 9 days following IR was analysed by unpaired t - test ( n = 5 mice / group ) . L - M , Tumour growth in mice bearing two KPC tumours receiving 10Gy IR to the primary lesion ( J ) ± systemic aCSF therapy ( M ) . The difference in mean tumour volume 10 days following IR was analysed by unpaired t - test ( n = 8 mice / group ) .

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

J-K, Tumour growth in mice bearing two MC38 tumours receiving 10Gy IR to the primary lesion (J) ± systemic aCSF therapy (K). The differences in tumour volume 9 days following IR was analysed by unpaired t-test (n=5 mice/group). L-M, Tumour growth in mice bearing two KPC tumours receiving 10Gy IR to the primary lesion (J) ± systemic aCSF therapy (M). The difference in mean tumour volume 10 days following IR was analysed by unpaired t-test (n=8 mice/group).


N - O , Flow cytometric analysis of macrophages ( N ) and CD8 T cells ( O ) in primary and secondary MC38 tumours . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 5 mice / group ) . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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

N-O, Flow cytometric analysis of macrophages (N) and CD8 T cells (O) in primary and secondary MC38 tumours. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=5 mice/group). *P <0.05 **P <0.01 ***P <0.001.


PD - L1 expression on MC38 and KPC cells 48 hours following 10Gy irradiation in tissue culture ( A , B ) or 10 Gy irradiation of tumours ( C , D ) analysed by flow cytometry . Data presented as mean SEM and analysed by Mann - Witney ( n = 3 , A , B ) . Data presented as mean SEM and analysed by unpaired t - test ( n = 5 mice / group , C , D ) .

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

PD-L1 expression on MC38 and KPC cells 48 hours following 10Gy irradiation in tissue culture (A,B) or 10 Gy irradiation of tumours (C,D) analysed by flow cytometry. Data presented as mean SEM and analysed by Mann-Witney (n=3, A,B). Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group, C,D).


E - F , Flow cytometric analysis of PD - L1 ( E ) and PD - L2 ( F ) on TAMs in MC38 tumours receiving treatment as indicated above . Data presented as mean SEM and analysed by Mann - Witney ( n = 5 mice / group )

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F, Flow cytometric analysis of PD-L1 (E) and PD-L2 (F) on TAMs in MC38 tumours receiving treatment as indicated above. Data presented as mean SEM and analysed by Mann-Witney (n=5 mice/group)


G - H , Tumour growth in mice bearing MC38 ( G ) and KPC ( H ) tumours receiving the indicated treatments . Data presented for individual mice . Pie charts indicate the number of regressions observed .

G-H, Tumour growth in mice bearing MC38 (G) and KPC (H) tumours receiving the indicated treatments. Data presented for individual mice. Pie charts indicate the number of regressions observed.


I - J , Tumour growth in mice bearing KPC tumours mice receiving 10Gy IR + systemic aPD - L1 to the primary lesion ( I ) ± systemic aPD - L1 + aCSF therapy ( J ) . The difference in tumour volume 8 ( I ) or 10 ( J ) days following IR was analysed by unpaired t - test ( n = 8 mice / group ) . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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

I-J, Tumour growth in mice bearing KPC tumours mice receiving 10Gy IR + systemic aPD-L1 to the primary lesion (I) ± systemic aPD-L1 + aCSF therapy (J). The difference in tumour volume 8 (I) or 10 (J) days following IR was analysed by unpaired t-test (n=8 mice/group). *P <0.05 **P <0.01 ***P <0.001.


A - D Progression of microglial infiltration in the ONL . Early in the course of rod degeneration at P18 , few isolated apoptotic TUNEL - positive nuclei ( red , inset ) were present in the ONL , with Iba1 + microglialsomata and processes ( green ) confined to the inner retina ( A ) . At P21 , microglia in the outer plexiform layer ( OPL ) extended radial processes into the ONL ( inset ) , with a concurrent increase in the number of TUNEL + nuclei ( B ) . At P30 , retinalmicroglia infiltrated the entire depth of the markedly thinned ONL and the subretinal space ( SRS ) ( inset ) ( C ) . At P46 , microglia became less dense in the ONL and reverted to a more ramified morphology ( D ) . Scale bar , 50 μm . E - G Quantitative analyses demonstrate that ONL atrophy progression , characterized by ONL thickness ( empty symbols ) and number of rows of ONLnuclei ( filled symbols ) ( E ) , occurred concurrently with the progression of ONL apoptosis , followed as the density of TUNEL + nuclei in the ONL ( F ) , and microglia infiltration , followed as microglial density in the ONL ( G ) ( n = 3 animals at each time point ) . Data points and error bars indicate mean ± SEM . H - K Higher magnification images showing microglial process infiltration ( H ) , phagosome formation in infiltrating processes ( I ) , and microglial transformation into amoeboid morphologies containing multiple phagosomes ( J , K ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Progression of microglial infiltration in the ONL. Early in the course of rod degeneration at P18, few isolated apoptotic TUNEL-positive nuclei (red, inset) were present in the ONL, with Iba1+microglialsomata and processes (green) confined to the inner retina (A). At P21, microglia in the outer plexiform layer (OPL) extended radial processes into the ONL (inset), with a concurrent increase in the number of TUNEL+nuclei (B). At P30, retinalmicroglia infiltrated the entire depth of the markedly thinned ONL and the subretinal space (SRS) (inset) (C). At P46, microglia became less dense in the ONL and reverted to a more ramified morphology (D). Scale bar, 50 μm.E-G Quantitative analyses demonstrate that ONL atrophy progression, characterized by ONL thickness (empty symbols) and number of rows of ONLnuclei (filled symbols) (E), occurred concurrently with the progression of ONL apoptosis, followed as the density of TUNEL+nuclei in the ONL (F), and microglia infiltration, followed as microglial density in the ONL (G) (n = 3 animals at each time point). Data points and error bars indicate mean ± SEM.H-K Higher magnification images showing microglial process infiltration (H), phagosome formation in infiltrating processes (I), and microglial transformation into amoeboid morphologies containing multiple phagosomes (J,K).


A - C Microglia infiltrating the ONL demonstrate upregulation of the phagocytic molecule , CD68 . Top images in ( A ) show composites of CD68 ( red ) , Iba1 ( green ) , and DAPI ( blue ) staining ; bottom images show the same field with CD68 staining only . At P18 , non - infiltrating microglia expressed low or undetectable levels of CD68 , a lysosome - associated membrane protein . At P21 - 23 , microglia infiltrating the ONL strongly upregulated CD68 ( arrowheads ) . At P30 , CD68 immunopositivity in ONL microglia decreased and was mostly confined to amoeboid cells in the subretinal space . ( B ) Magnified view of inset in ( A ) demonstrates localization of CD68 to microglia phagosomes . ( C ) CD68 expression ( area of immunopositivity as a fraction of the ONL ) demonstrated a peaked at P22 and decreased by P30 . Scale bar , 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Microglia infiltrating the ONL demonstrate upregulation of the phagocytic molecule, CD68. Top images in (A) show composites of CD68 (red), Iba1 (green), and DAPI (blue) staining; bottom images show the same field with CD68 staining only. At P18, non-infiltrating microglia expressed low or undetectable levels of CD68, a lysosome-associated membrane protein. At P21-23, microglia infiltrating the ONL strongly upregulated CD68 (arrowheads). At P30, CD68 immunopositivity in ONL microglia decreased and was mostly confined to amoeboid cells in the subretinal space. (B) Magnified view of inset in (A) demonstrates localization of CD68 to microglia phagosomes. (C) CD68 expression (area of immunopositivity as a fraction of the ONL) demonstrated a peaked at P22 and decreased by P30. Scale bar, 20 μm.


D - F Upregulation of MFG - E8 , a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors , in the ONL . ( D ) MFG - E8 was low or absent in the ONL at P18 , but emerged at P21 - 23 , localizing to photoreceptor cell bodies in a column - like pattern ( arrow ) and as a punctate signal within infiltrating microglia ( arrowhead ) , before decreasing throughout the ONL at P30 . ( E ) A magnified orthogonal view of the inset from ( D ) demonstrating punctate MFG - E8 immunopositivity within ONL microglia . ( F ) Quantification of MFG - E8 expression demonstrated a prominent emergence at P22 and a subsequent decrement by P30 . Scale bar , 20 μm .

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D-F Upregulation of MFG-E8, a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors, in the ONL. (D) MFG-E8 was low or absent in the ONL at P18, but emerged at P21-23, localizing to photoreceptor cell bodies in a column-like pattern (arrow) and as a punctate signal within infiltrating microglia (arrowhead), before decreasing throughout the ONL at P30. (E) A magnified orthogonal view of the inset from (D) demonstrating punctate MFG-E8 immunopositivity within ONL microglia. (F) Quantification of MFG-E8 expression demonstrated a prominent emergence at P22 and a subsequent decrement by P30. Scale bar, 20 μm.


G - K Increase in phosphatidylserine ( PS ) exposure in the ONL during photoreceptor degeneration . ( G ) At P18 , PS immunopositivity is near absent in the ONL , but increased significantly in ONLsomata at P21 - 23 , before decreasing at P30 . ( H ) Quantitation of PS exposure by image analysis ( by fractional area of PS immunopositivity within the ONL ( top ) , and the mean intensity of PS staining in the ONL ( bottom ) ) demonstrated a transient increase at P22 . ( I ) Co - immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods ( inset shows at high magnification the co - labeling of PS and rhodopsin in multiple rodsomata ) . ( J ) Conversely , immunolabeling of cones with conearrestin demonstrates the sparse co - localization of PS in cones ( inset shows close juxtaposition but no colocalization of PS and arrestin labeling ) . ( K ) Scoring of rhodopsin + rods and arrestin + cones for PS co - labeling demonstrates that a large majority of rods , but only a small minority of cones , showed PS exposure ( two - sided unpaired t - test , n = 3 animals at P22 ) . Scale bars = 20 μm .

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G-K Increase in phosphatidylserine (PS) exposure in the ONL during photoreceptor degeneration. (G) At P18, PS immunopositivity is near absent in the ONL, but increased significantly in ONLsomata at P21-23, before decreasing at P30. (H) Quantitation of PS exposure by image analysis (by fractional area of PS immunopositivity within the ONL (top), and the mean intensity of PS staining in the ONL (bottom)) demonstrated a transient increase at P22. (I) Co-immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods (inset shows at high magnification the co-labeling of PS and rhodopsin in multiple rodsomata). (J) Conversely, immunolabeling of cones with conearrestin demonstrates the sparse co-localization of PS in cones (inset shows close juxtaposition but no colocalization of PS and arrestin labeling). (K) Scoring of rhodopsin+rods and arrestin+cones for PS co-labeling demonstrates that a large majority of rods, but only a small minority of cones, showed PS exposure (two-sided unpaired t-test, n = 3 animals at P22). Scale bars = 20 μm.


A - C Phagocytosis of rods by infiltrating microglia . ( A ) Representative example of a Iba1 + microglial process extending into the ONL with a phagosome at its terminal end . Each phagosome contained a photoreceptornucleus ( labeled with DAPI , arrow ) that was identified as a rodphotoreceptor by rhodopsin immunopositivity ( superposition of Iba1 + phagosome with rhodopsin + soma in orthogonal views ) . ( B ) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin - positive ( arrowhead ) and rhodopsin - negative ( arrow ) nuclei . ( C ) Rhodopsin + nuclei can be localized within CD68 - positive phagosomes in infiltrating microglia , indicating phagocytic engulfment of rods . Scale bar , 10 μm . D , E The peak of microglial phagocytic activity occurred around P21 and declined subsequently , as measured by the number of rods phagocytosed ( D ) and the number of photoreceptors phagocytosed per microglial cell ( E ) ( n = 3 animals at each time point ) .

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A-C Phagocytosis of rods by infiltrating microglia. (A) Representative example of a Iba1+microglial process extending into the ONL with a phagosome at its terminal end. Each phagosome contained a photoreceptornucleus (labeled with DAPI, arrow) that was identified as a rodphotoreceptor by rhodopsin immunopositivity (superposition of Iba1+phagosome with rhodopsin+soma in orthogonal views). (B) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin-positive (arrowhead) and rhodopsin-negative (arrow) nuclei. (C) Rhodopsin+nuclei can be localized within CD68-positive phagosomes in infiltrating microglia, indicating phagocytic engulfment of rods. Scale bar, 10 μm.D, E The peak of microglial phagocytic activity occurred around P21 and declined subsequently, as measured by the number of rods phagocytosed (D) and the number of photoreceptors phagocytosed per microglial cell (E) (n = 3 animals at each time point).


F - J Infiltrating microglia phagocytose TUNEL - negative rods in the ONL . ( F ) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently , the majority of rodphotoreceptors undergoing microglial phagocytosis were non - apoptotic ( arrows ) , while many apoptotic photoreceptors were not phagocytosed by microglia ( arrowheads ) ; representative example of a P21 retina demonstrates non - overlapping patterns of TUNEL positivity ( red ) and microglial phagocytosis ( Iba1 , green ) . ( G ) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptornuclei in phagosomes were rhodopsin - positive ( white ) , they were mostly TUNEL - negative . Only a small fraction of microglialphagosomes contained TUNEL + , rhodopsin + rods ( example shown in H ) . Scale bars , 20 μm . ( I ) Quantitative analysis of photoreceptornuclei within microglialphagosomes according to rhodopsin ( black symbols ) , and TUNEL labeling ( red symbols ) at different times during rod degeneration . ( J ) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21 - 23 demonstrates that TUNEL + apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations , indicating separate but parallel mechanisms of rod degeneration . Quantitative analyses involved retinal sections from n = 3 animals at each age .

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F-J Infiltrating microglia phagocytose TUNEL-negative rods in the ONL. (F) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently, the majority of rodphotoreceptors undergoing microglial phagocytosis were non-apoptotic (arrows), while many apoptotic photoreceptors were not phagocytosed by microglia (arrowheads); representative example of a P21 retina demonstrates non-overlapping patterns of TUNEL positivity (red) and microglial phagocytosis (Iba1, green). (G) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptornuclei in phagosomes were rhodopsin-positive (white), they were mostly TUNEL-negative. Only a small fraction of microglialphagosomes contained TUNEL+, rhodopsin+rods (example shown in H). Scale bars, 20 μm. (I) Quantitative analysis of photoreceptornuclei within microglialphagosomes according to rhodopsin (black symbols), and TUNEL labeling (red symbols) at different times during rod degeneration. (J) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21-23 demonstrates that TUNEL+ apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations, indicating separate but parallel mechanisms of rod degeneration. Quantitative analyses involved retinal sections from n = 3 animals at each age.


A - C Evidence of microglial phagocytosis of photoreceptors in other mouse models of RP . Histological analysis of a rd1mouseretina ( A ) demonstrates microglial phagocytosis of photoreceptornuclei in vibratome sections ( left ) and in flat - mounted retina ( right ) . Phagocytosed nuclei were predominantly negative for TUNEL staining ( arrows ) . Similar findings were found in the retinas of the rd16mouse ( loss - of - function mutation in the photoreceptor - expressed CEP290 gene , 1 month old ) ( B ) and the RPGRIP - deficient mouse ( 6 months old ) ( C ) . Scale bar , 20 μm .

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A-C Evidence of microglial phagocytosis of photoreceptors in other mouse models of RP. Histological analysis of a rd1mouseretina (A) demonstrates microglial phagocytosis of photoreceptornuclei in vibratome sections (left) and in flat-mounted retina (right). Phagocytosed nuclei were predominantly negative for TUNEL staining (arrows). Similar findings were found in the retinas of the rd16mouse (loss-of-function mutation in the photoreceptor-expressed CEP290 gene, 1 month old) (B) and the RPGRIP-deficient mouse (6 months old) (C). Scale bar, 20 μm.


D - G Evidence of microglial phagocytosis of photoreceptors in other human histopathological specimens of RP . ( D ) Retinal section from a 30 - year - old male donor with autosomal recessive RP ( AR RP ) showing extensive microglial infiltration of the ONL ; expanded inset ( right ) shows multiple photoreceptor nuclei in phagosomes that were predominantly negative for TUNEL staining . ( E , F ) Retinal sections from two separate donors with autosomal dominant RP ( AD RP1 = 68 - year - old man , T17M rhodopsin mutation , AD RP1 = 50 - year - old woman , Q - 64 - ter rhodopsin mutation ) showing similar evidence of microglial phagocytosis . ( G ) Retinal section from a 46 - year - old male donor with X - linked RP ( XL RP ) . Arrowheads indicate phagocytosed photoreceptor nuclei . Scale bars , 20 μm .

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D-G Evidence of microglial phagocytosis of photoreceptors in other human histopathological specimens of RP. (D) Retinal section from a 30-year-old male donor with autosomal recessive RP (AR RP) showing extensive microglial infiltration of the ONL; expanded inset (right) shows multiple photoreceptor nuclei in phagosomes that were predominantly negative for TUNEL staining. (E, F) Retinal sections from two separate donors with autosomal dominant RP (AD RP1 = 68-year-old man, T17M rhodopsin mutation, AD RP1 = 50-year-old woman, Q-64-ter rhodopsin mutation) showing similar evidence of microglial phagocytosis. (G) Retinal section from a 46-year-old male donor with X-linked RP (XL RP). Arrowheads indicate phagocytosed photoreceptor nuclei. Scale bars, 20 μm.


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21 - 24 . Infiltrating microglia established rapid and transient physical contacts with nearby photoreceptorsomata ( red and yellow circles ) via their processes ; these processes often terminated in a " cup " - like structure ( arrow ) that upon soma contact proceeded to extend across the entire soma ( arrowhead , and red circle and * ) to engulf it completely . The majority of microglia - photoreceptor contacts were transient with the microglial processes dynamically contacting and releasing photoreceptorsomata in repetitive cycles ( e . g . yellow circle and * ) ( period ≅ 10 - 15 min ) .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24.Infiltrating microglia established rapid and transient physical contacts with nearby photoreceptorsomata (red and yellow circles) via their processes; these processes often terminated in a "cup"-like structure (arrow) that upon soma contact proceeded to extend across the entire soma (arrowhead, and red circle and *) to engulf it completely. The majority of microglia-photoreceptor contacts were transient with the microglial processes dynamically contacting and releasing photoreceptorsomata in repetitive cycles (e.g. yellow circle and *) (period ≅10-15 min).


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONL microglia in rd10 retinal explants at ages P21 - 24 . Example in which the engulfment of photoreceptorsomata by microglial phagocytic cup was followed up by actual phagocytosis in which the engulfed cell ( red * ) is translocated intracellularly within the microglial cell toward the cell body . Phagocytosis of soma occurred simultaneously with " probing " of other somata by processes of the same microglial cell ( yellow * ) .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONL microglia in rd10 retinal explants at ages P21-24. Example in which the engulfment of photoreceptorsomata by microglial phagocytic cup was followed up by actual phagocytosis in which the engulfed cell (red *) is translocated intracellularly within the microglial cell toward the cell body. Phagocytosis of soma occurred simultaneously with "probing" of other somata by processes of the same microglial cell (yellow *).


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21 - 24 . Phagocytosis of photoreceptorsomata occurred via flattened microgliallamellipodialmicroglial processes ( arrow ) that extended across somata ( yellow * ) to engulf them . Nuclei of phagocytosed photoreceptors within microglialphagosomes occasionally developed staining for propidium iodide ( red arrow ) ; these subsequently faded and disappeared over ≅ 10 min , possibly representing intraphagosomal breakdown .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24. Phagocytosis of photoreceptorsomata occurred via flattened microgliallamellipodialmicroglial processes (arrow) that extended across somata (yellow *) to engulf them. Nuclei of phagocytosed photoreceptors within microglialphagosomes occasionally developed staining for propidium iodide (red arrow); these subsequently faded and disappeared over ≅10 min, possibly representing intraphagosomal breakdown.


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21 - 24 . Amoeboid infiltrating microglia lacking extended processes were also observed to phagocytose photoreceptors via phagocytic cups formed at their cell body ( red * ) .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24. Amoeboid infiltrating microglia lacking extended processes were also observed to phagocytose photoreceptors via phagocytic cups formed at their cell body (red *).


Retinalmicroglia were depleted in rd10 / CreDTAmice by the oral administration of tamoxifen ( in corn oil ) to activate microglia - specific Cre - mediated recombination and diphtheria toxin expression ; control CreDTA littermates were administered corn oil without tamoxifen . A - D Depletion of retinalmicroglia in the rd10 retina . Representative retinal section from a P28 control animal demonstrates Iba1 + microglia in the retina , including those infiltrating the ONL ( A ) , while a tamoxifen - administered littermate ( B ) was substantially depleted of retinalmicroglia . Scale bar , 40 μm . Microglial cell counts in the entire retina ( C ) and in the ONL only ( D ) confirmed efficient depletion of infiltrating microglia following tamoxifen administration ( n = 8 control and 11 depleted animals from four litters , two - sided unpaired t - test ) . E - J Effect of microglial depletion on retinal degeneration at P28 - 29 . ONL atrophy and thinning in control animals ( E ) was significantly more advanced relative to microglia - depleted littermates ( F ) . Scale bar , 40 μm . Quantification of mean ONL thickness ( G ) and mean number of layers of ONLnuclei ( H ) at P28 - 29 demonstrate significantly greater ONL preservation in depleted retinas ; the degree of ONL preservation correlated with the extent of microglia depletion ( I ) . ( J ) The mean density of TUNEL + nuclei in the ONL was not significantly decreased in depleted vs . control animals ( n = 8 control and 11 depleted animals from four litters , two - sided unpaired t - test ) . K - N Continuation of microglial depletion until P37 - 39 resulted in the persistence of morphological rescue ( K - M ) , with a significant reduction in TUNEL + nuclei density ( N ) ( n = 6 control and nine depleted animals from two litters , two - sided unpaired t - test ) . O - R Similar rescue effects as in ( K - N ) remained apparent when depletion was sustained until P50 ( O - R ) , a time when rod degeneration in the rd10 model is relatively complete ( n = 9 control and 10 depleted animals from three litters , two - sided unpaired t - test ) . S , T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark - ( S ) and light - adapted ( T ) responses in ERG testing in depleted animals ( green lines ) relative to control animals ( black lines ) , in both a - and b - wave amplitudes across multiple flash intensities ( n = 9 control and nine depleted animals , * P < 0 . 05 in one - way ANOVA with Sidak ' s multiple comparison test ) .

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Retinalmicroglia were depleted in rd10/CreDTAmice by the oral administration of tamoxifen (in corn oil) to activate microglia-specific Cre-mediated recombination and diphtheria toxin expression; control CreDTA littermates were administered corn oil without tamoxifen.A-D Depletion of retinalmicroglia in the rd10 retina. Representative retinal section from a P28 control animal demonstrates Iba1+microglia in the retina, including those infiltrating the ONL (A), while a tamoxifen-administered littermate (B) was substantially depleted of retinalmicroglia. Scale bar, 40 μm. Microglial cell counts in the entire retina (C) and in the ONL only (D) confirmed efficient depletion of infiltrating microglia following tamoxifen administration (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).E-J Effect of microglial depletion on retinal degeneration at P28-29. ONL atrophy and thinning in control animals (E) was significantly more advanced relative to microglia-depleted littermates (F). Scale bar, 40 μm. Quantification of mean ONL thickness (G) and mean number of layers of ONLnuclei (H) at P28-29 demonstrate significantly greater ONL preservation in depleted retinas; the degree of ONL preservation correlated with the extent of microglia depletion (I). (J) The mean density of TUNEL+nuclei in the ONL was not significantly decreased in depleted vs. control animals (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).K-N Continuation of microglial depletion until P37-39 resulted in the persistence of morphological rescue (K-M), with a significant reduction in TUNEL+nuclei density (N) (n = 6 control and nine depleted animals from two litters, two-sided unpaired t-test).O-R Similar rescue effects as in (K-N) remained apparent when depletion was sustained until P50 (O-R), a time when rod degeneration in the rd10 model is relatively complete (n = 9 control and 10 depleted animals from three litters, two-sided unpaired t-test).S, T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark- (S) and light-adapted (T) responses in ERG testing in depleted animals (green lines) relative to control animals (black lines), in both a- and b-wave amplitudes across multiple flash intensities (n = 9 control and nine depleted animals, *P < 0.05 in one-way ANOVA with Sidak's multiple comparison test).


A , B Effect of phagocytosis inhibition on retinal microglia ex vivo . Retinal explants acutely isolated from P21 - 23 rd10 / CX3CR1GFP / + mice were incubated in Ringer ' s solution ( control ) , or Ringer ' s solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD ( 400 μM ) for 1 h . GFP - labeled ( green ) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes . No morphological changes were detected in microglia incubated in cRAD ( insets show morphologies at higher magnification ) . Scale bar , 40 μm . Quantifications of total number of phagosomes per 40 × field ( left ) and the mean number of phagosomes per microglia ( right ) demonstrate significant reductions in phagocytic activity of cRGD , relative to cRAD , exposure ( n = 8 imaging fields from in each condition ) .

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A, B Effect of phagocytosis inhibition on retinal microglia ex vivo. Retinal explants acutely isolated from P21-23 rd10/CX3CR1GFP/+ mice were incubated in Ringer's solution (control), or Ringer's solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD (400 μM) for 1 h. GFP-labeled (green) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes. No morphological changes were detected in microglia incubated in cRAD (insets show morphologies at higher magnification). Scale bar, 40 μm. Quantifications of total number of phagosomes per 40× field (left) and the mean number of phagosomes per microglia (right) demonstrate significant reductions in phagocytic activity of cRGD, relative to cRAD, exposure (n = 8 imaging fields from in each condition).


C - N Structural effects of in vivo inhibition of microglial phagocytosis . P20 somatamice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD , and in the control contralateral eye with the inactive analogue , cRAD . At P23 , prominent microglia infiltration in the ONL observed in control - injected eyes ( C ) was decreased in the contralateral cRGD - injected eyes ( D ) . ONLmicroglia in control eyes demonstrated more numerous phagosomes ( inset , arrows ) ( E ) compared with cRGD - injected eyes ( F ) . Pairwise comparisons of control - vs . cRGD - injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia ( G ) and microglialphagosomes ( H ) , and mean phagosome number per microglia ( I ) . ONL atrophy in control eyes ( J ) was more advanced compared to cRGD - injected eyes ( K ) , with significantly greater mean ONL thickness ( L ) , and ONLnuclear layers ( M ) in cRGD - injected eyes . Mean density of TUNEL + ONLnuclei was lower , with marginal significance ( N ) ( n = 13 animals ; measurements in cRGD - injected eyes normalized to contralateral control eyes , two - sided paired t - test ) . Scale bar , 40 μm .

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C-N Structural effects of in vivo inhibition of microglial phagocytosis. P20 somatamice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD, and in the control contralateral eye with the inactive analogue, cRAD. At P23, prominent microglia infiltration in the ONL observed in control-injected eyes (C) was decreased in the contralateral cRGD-injected eyes (D). ONLmicroglia in control eyes demonstrated more numerous phagosomes (inset, arrows) (E) compared with cRGD-injected eyes (F). Pairwise comparisons of control- vs. cRGD-injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia (G) and microglialphagosomes (H), and mean phagosome number per microglia (I). ONL atrophy in control eyes (J) was more advanced compared to cRGD-injected eyes (K), with significantly greater mean ONL thickness (L), and ONLnuclear layers (M) in cRGD-injected eyes. Mean density of TUNEL+ONLnuclei was lower, with marginal significance (N) (n = 13 animals; measurements in cRGD-injected eyes normalized to contralateral control eyes, two-sided paired t-test). Scale bar, 40 μm.


O - T Functional effects of in vivo inhibition of microglial phagocytosis . Representative dark - adapted ERG recordings at P26 ( following intravitreal injections at P20 and P23 ) in a cRGD - injected eye ( red ) relative to the contralateral control eye ( black ) ( O ) showing that mean a - ( P ) and b - wave ( Q ) amplitudes were significantly greater in the cRGD - injected vs . control eyes at higher flash intensities . Representative light - adapted responses ( R ) illustrating that while mean light - adapted a - wave amplitudes ( S ) were slightly but not significantly greater in cRGD - injected eyes , mean b - wave amplitudes ( T ) were significantly greater at higher flash intensities ( n = 13 animals , two - sided paired t - test , * P < 0 . 05 , with exact values alongside ) .

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O-T Functional effects of in vivo inhibition of microglial phagocytosis. Representative dark-adapted ERG recordings at P26 (following intravitreal injections at P20 and P23) in a cRGD-injected eye (red) relative to the contralateral control eye (black) (O) showing that mean a- (P) and b-wave (Q) amplitudes were significantly greater in the cRGD-injected vs. control eyes at higher flash intensities. Representative light-adapted responses (R) illustrating that while mean light-adapted a-wave amplitudes (S) were slightly but not significantly greater in cRGD-injected eyes, mean b-wave amplitudes (T) were significantly greater at higher flash intensities (n = 13 animals, two-sided paired t-test, *P < 0.05, with exact values alongside).


Microglia infiltrating the rd10 retina upregulate IL - 1β expression during degeneration . Iba1 + microglia ( green ) were immunonegative for IL - 1β ( red ) at P18 but become prominently IL - 1β immunopositive at P22 ( arrows ) . Scale bars , 25 μm .

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Microglia infiltrating the rd10 retina upregulate IL-1β expression during degeneration. Iba1+ microglia (green) were immunonegative for IL-1β (red) at P18 but become prominently IL-1β immunopositive at P22 (arrows). Scale bars, 25 μm.


Depletion of infiltrating microglia in the rd10 retina decreases IL - 1β levels . Cytokine levels in rd10 / CreDTA mouse retinas were assayed following tamoxifen - induced microglial depletion ( from P21 to P37 - 50 , green bars ; n = 9 animals ) and compared with their untreated littermate controls ( white bars , n = 8 animals ; values normalized to control animals in the same litter ) . IL - 1β protein levels were significantly lowered following microglial depletion , but IL - 6 , CCL2 , or TNFα were not significantly changed .

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Depletion of infiltrating microglia in the rd10 retina decreases IL-1β levels. Cytokine levels in rd10/CreDTA mouse retinas were assayed following tamoxifen-induced microglial depletion (from P21 to P37-50, green bars; n = 9 animals) and compared with their untreated littermate controls (white bars, n = 8 animals; values normalized to control animals in the same litter). IL-1β protein levels were significantly lowered following microglial depletion, but IL-6, CCL2, or TNFα were not significantly changed.


Inhibition of microglial phagocytosis results in decreased microglial IL - 1β expression . Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20 , and analyzed at P23 by immunohistochemistry for Iba1 ( green ) and IL - 1β ( red ) , and compared to uninjected eyes and with eyes injected with inactive analog , cRAD ( n = 3 eyes in each category ) . Immunopositivity was high in infiltrating microglia ( indicated by arrows ) for untreated and cRAD - injected control eyes and significantly reduced in microglia in cRGD - injected eyes ( upper panel ) , as demonstrated by the quantification of IL - 1β expression ( area of immunopositivity as a fraction of the ONL ) ( lower left panel ) . ONL thickness correlated negatively with the extent of IL - 1β immunopositivity ( lower right panel ) . Scale bar , 25 μm .

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Inhibition of microglial phagocytosis results in decreased microglial IL-1β expression. Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20, and analyzed at P23 by immunohistochemistry for Iba1 (green) and IL-1β (red), and compared to uninjected eyes and with eyes injected with inactive analog, cRAD (n = 3 eyes in each category). Immunopositivity was high in infiltrating microglia (indicated by arrows) for untreated and cRAD-injected control eyes and significantly reduced in microglia in cRGD-injected eyes (upper panel), as demonstrated by the quantification of IL-1β expression (area of immunopositivity as a fraction of the ONL) (lower left panel). ONL thickness correlated negatively with the extent of IL-1β immunopositivity (lower right panel). Scale bar, 25 μm.


IL - 1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL - 1 receptor antagonist , anakinra ( from P20 - 22 to P26 - 27 ) in one eye of each rd10 animal , while the contralateral eye was injected with PBS as a control . Upper panels show representative retinal sections from control PBS - injected and IL - 1RA - injected eyes from the same animal . ONL atrophy in control eyes was more advanced compared to IL - 1RA - injected eyes , with greater mean ONL thickness and lower mean density of TUNEL + ONL nuclei in IL - 1RA - injected eyes ( lower panels ) ( n = 15 animals ; measurements in IL - 1RA - injected eyes normalized to contralateral control eyes , paired t - test ) . Scale bar , 25 μm .

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IL-1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL-1 receptor antagonist, anakinra (from P20-22 to P26-27) in one eye of each rd10 animal, while the contralateral eye was injected with PBS as a control. Upper panels show representative retinal sections from control PBS-injected and IL-1RA-injected eyes from the same animal. ONL atrophy in control eyes was more advanced compared to IL-1RA-injected eyes, with greater mean ONL thickness and lower mean density of TUNEL+ ONL nuclei in IL-1RA-injected eyes (lower panels) (n = 15 animals; measurements in IL-1RA-injected eyes normalized to contralateral control eyes, paired t-test). Scale bar, 25 μm.


( D - F ) Box plots showing diastolic ( D ) and systolic ( E ) diameters and cardiac contractility analyses ( percent fractional shortening ) ( F ) performed using SOHA approach for controls ( Hand - Gal4 and UAS - dmiR - 1 sponge ) and Hand > sponge dmiR - 1 contexts at ages one and five weeks . n = 20 hearts . Central band corresponds to median . Whiskers correspond to Min to Max . Box corresponds to interquartile range from 25th to 75th percentile . Data information : Scale bar = 20 µm . p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * ) p - value = 0 . 021 , ( * * * ) p - value = 0 . 0002 . Non - parametric Mann Whitney test was performed to compare control samples and samples of interest .

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(D-F) Box plots showing diastolic (D) and systolic (E) diameters and cardiac contractility analyses (percent fractional shortening) (F) performed using SOHA approach for controls (Hand-Gal4 and UAS-dmiR-1 sponge) and Hand>sponge dmiR-1 contexts at ages one and five weeks. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: Scale bar = 20 µm. p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.


( G , G ' ) Representative spot views generated using Imaris from in situ hybridization with miRCURY LNA probe for dmiR - 1 and used for quantification of dmiR - 1 levels . Spot views of dmiR - 1 in hearts of one - week - old control ( UAS - mblRNAi ) ( G ) and DM1 flies ( Hand > mblRNAi ) ( G ' ) are shown . Each spot represents a pool of dmiR - 1 transcripts detected in the same area . The zoom region in G corresponds to area in which FISH signal was quantified . Data information : Scale bar = 40 µm .

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(G,G') Representative spot views generated using Imaris from in situ hybridization with miRCURY LNA probe for dmiR-1 and used for quantification of dmiR-1 levels. Spot views of dmiR-1 in hearts of one-week-old control (UAS-mblRNAi) (G) and DM1 flies (Hand>mblRNAi) (G') are shown. Each spot represents a pool of dmiR-1 transcripts detected in the same area. The zoom region in G corresponds to area in which FISH signal was quantified. Data information: Scale bar = 40 µm.


( H , I ) Scatter plot graph showing the signal intensity quantified in cardioblasts of one - and five - week - old flies for controls ( UAS - Bru3 , UAS - mblRNAi ) and DM1 contexts ( Hand > Bru3 , Hand > mblRNAi ) . n = 9 hearts . Bars corresponds to mean ± SD . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * ) p - value = 0 . 021 , ( * * * ) p - value = 0 . 0002 . ( * * * * ) p - value < 0 . 0001 . Non - parametric Mann Whitney test was performed to compare control samples and samples of interest .

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(H, I) Scatter plot graph showing the signal intensity quantified in cardioblasts of one- and five-week-old flies for controls (UAS-Bru3, UAS-mblRNAi) and DM1 contexts (Hand>Bru3, Hand>mblRNAi). n = 9 hearts. Bars corresponds to mean ± SD. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.


( C - F ' ) dmiR - 1 binding site in 3 ' UTR - Mp region is required to negatively regulate Mp expression in vivo . Adult hearts from transgenic GFP - sensor lines carrying 3 ' UTRMp region with ( UAS - GFP 3 ' UTRMp ) or without dmiR - 1 seed site ( UAS - GFP ∆ 3 ' UTRMp ) . These lines were combined with the UAS - dmiR1 line to generate double transgenic lines and tested for GFP expression in non - targeted context ( crossed with w1118 ) ( C , E ) or in heart - targeted context ( crossed with heart specific driver Hand - Gal4 ) ( D , F ) . When crossed with Hand - Gal4 , GFP expression in Hand > GFP 3 ' UTRMp ; dmiR1 line ( carrying dmiR - 1 seed site ) ( D ' ) is attenuated in cardiac tube and pericardial cells compared to Hand > GFP ∆ 3 ' UTRMp ; dmiR - 1 line ( lacking dmiR - 1 seed site ) ( F ' ) . Scale bar = 40 µm .

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(C-F') dmiR-1 binding site in 3'UTR-Mp region is required to negatively regulate Mp expression in vivo. Adult hearts from transgenic GFP-sensor lines carrying 3'UTRMp region with (UAS-GFP 3'UTRMp) or without dmiR-1 seed site (UAS-GFP ∆3'UTRMp). These lines were combined with the UAS-dmiR1 line to generate double transgenic lines and tested for GFP expression in non-targeted context (crossed with w1118) (C,E) or in heart-targeted context (crossed with heart specific driver Hand-Gal4) (D,F). When crossed with Hand-Gal4, GFP expression in Hand>GFP 3'UTRMp; dmiR1 line (carrying dmiR-1 seed site) (D') is attenuated in cardiac tube and pericardial cells compared to Hand>GFP ∆3'UTRMp; dmiR-1 line (lacking dmiR-1 seed site) (F'). Scale bar = 40 µm.


( K , L ) Graphs of Mp signal quantification in cardioblasts from adult flies aged one and five weeks for controls ( UAS - mblRNAi , UAS - Bru3 ) and DM1 contexts ( Hand > mblRNAi , Hand > Bru3 ) using CTCF method . For each genotype 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4 . Bars correspond to mean ± SD . Data information : p - value < 0 . 05 considered statistically significant ; ( * ) p - value = 0 . 033 ; ( * * ) p - value = 0 . 021 ; ( * * * ) p - value = 0 . 0002 ; ( * * * * ) p - value < 0 . 0001 . Non - parametric test Mann Whitney test was performed to compare control samples and samples of interest .

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(K,L) Graphs of Mp signal quantification in cardioblasts from adult flies aged one and five weeks for controls (UAS-mblRNAi, UAS-Bru3) and DM1 contexts (Hand>mblRNAi, Hand>Bru3) using CTCF method. For each genotype 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4. Bars correspond to mean ± SD. Data information: p-value<0.05 considered statistically significant; (*) p-value = 0.033; (**) p-value = 0.021; (***) p-value = 0.0002; (****) p-value<0.0001. Non-parametric test Mann Whitney test was performed to compare control samples and samples of interest.


( E - G ) Box plots showing cardiac variables ( diastolic ( E ) and systolic ( F ) diameters ) and percent fractional shortening ( G ) for controls ( Hand - Gal4 and UAS - Mp ) and Mp overexpression ( Hand > Mp ) conditions at one and five weeks . Notice an increase in diastolic and systolic diameters with reduced cardiac contractility in Hand > Mp in comparison to controls . n = 20 hearts . Central band corresponds to median . Whiskers correspond to Min to Max . Box corresponds to interquartile range from 25th to 75th percentile . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * ) p - value = 0 . 021 , ( * * * ) p - value = 0 . 0002 . Non - parametric Mann Whitney test was performed to compare control samples and samples of interest .

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(E-G) Box plots showing cardiac variables (diastolic (E) and systolic (F) diameters) and percent fractional shortening (G) for controls (Hand-Gal4 and UAS-Mp) and Mp overexpression (Hand>Mp) conditions at one and five weeks. Notice an increase in diastolic and systolic diameters with reduced cardiac contractility in Hand>Mp in comparison to controls. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.


( A - B ' ) Adult heart of 1 week of age labeled for Mp ( green ) and actin ( red ) for control ( UAS - Mp RNAi ) ( A , A ' ) and mutant ( Hand > Mp RNAi ) contexts ( B , B ' ) . Data information : Scale bar = 20µm .

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(A-B') Adult heart of 1 week of age labeled for Mp (green) and actin (red) for control (UAS-Mp RNAi) (A,A') and mutant (Hand>Mp RNAi) contexts (B,B'). Data information: Scale bar = 20µm.


( C ) Fluorescence signal intensity quantification for Mp expression in cardioblasts in adult heart of 1 week for control ( UAS - Mp RNAi ) and mutant ( Hand > Mp RNAi ) contexts using CTCF method . For each genotype 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4 . Bars correspond to mean ± SD . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * ) p - value = 0 . 021 , ( * * * ) p - value = 0 . 0002 . ( * * * * ) p - value < 0 . 0001 . Non - parametric Mann Whitney test was performed to compare control samples and samples of interest .

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(C) Fluorescence signal intensity quantification for Mp expression in cardioblasts in adult heart of 1 week for control (UAS-Mp RNAi) and mutant (Hand>Mp RNAi) contexts using CTCF method. For each genotype 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4. Bars correspond to mean ± SD. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.


( D - F ) Box plots showing cardiac size analyzes ( diastolic ( D ) and systolic diameters ( E ) ) and percent fractional shortening ( F ) performed by SOHA approach for controls ( Hand - Gal4 and UAS - Mp RNAi ) and mutant ( Hand > Mp RNAi ) contexts at 1 and 5 weeks of age , showing reduced size of cardiac tube in Hand > Mp RNAi context in comparison to the Hand - Gal4 control . n = 20 hearts . Central band corresponds to median . Whiskers correspond to Min to Max . Box corresponds to interquartile range from 25th to 75th percentile . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * ) p - value = 0 . 021 , ( * * * ) p - value = 0 . 0002 . ( * * * * ) p - value < 0 . 0001 . Non - parametric Mann Whitney test was performed to compare control samples and samples of interest .

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(D-F) Box plots showing cardiac size analyzes (diastolic (D) and systolic diameters (E)) and percent fractional shortening (F) performed by SOHA approach for controls (Hand-Gal4 and UAS-Mp RNAi) and mutant (Hand>Mp RNAi) contexts at 1 and 5 weeks of age, showing reduced size of cardiac tube in Hand>Mp RNAi context in comparison to the Hand-Gal4 control. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.


( A , B ) ( A ) Col15A1 transcript levels tested by RT - qPCR and ( B ) Col15A1 protein levels tested by Western blot in cardiac samples from healthy donors and from DM1 patients with ( DCM + ) and without DCM ( DCM - ) . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * * ) p - value = 0 . 0002 . ( * * * * ) p - value < 0 . 0001 . Parametric t - test was performed to compare control samples and samples of interest in A ,

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(A, B) (A) Col15A1 transcript levels tested by RT-qPCR and (B) Col15A1 protein levels tested by Western blot in cardiac samples from healthy donors and from DM1 patients with (DCM+) and without DCM (DCM-). Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (***) p-value =0.0002. (****) p-value<0.0001. Parametric t-test was performed to compare control samples and samples of interest in A, B


( C ) miR - 1 levels tested by RT - qPCR in cardiac samples from healthy donors and from DM1 patients . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * * ) p - value = 0 . 0002 . ( * * * * ) p - value < 0 . 0001 . Parametric t - test was performed to compare control samples and samples of interest in C .

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(C) miR-1 levels tested by RT-qPCR in cardiac samples from healthy donors and from DM1 patients. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (***) p-value =0.0002. (****) p-value<0.0001. Parametric t-test was performed to compare control samples and samples of interest in C.


( D , F ) Box plots showing cardiac variables ( diastolic ( D ) and systolic ( E ) diameters and percent fractional shortening ( F ) ) assessed by SOHA approach for controls ( UAS - Bru3 ; Mp RNAi and UAS - UPRT ; Bru3 ) , Mp rescue ( Hand > Bru3 ; Mp RANi ) and DM1 context ( Hand > UPRT ; Bru3 ) at five weeks of age showing rescue of diastolic and systolic diameters in Hand > Bru3 ; Mp RNAi in comparison to controls ( D , E ) and rescue of cardiac contractility ( F ) n = 20 hearts . Central band corresponds to median . Whiskers correspond to Min to Max . Box corresponds to interquartile range from 25th to 75th percentile . Data information : p - value < 0 . 05 considered statistically significant . ( * ) p - value = 0 . 033 , ( * * * ) p - value = 0 . 0002 . ( * * * * ) p - value < 0 . 0001 . Non - parametric Mann Whitney test was performed to compare control samples and samples of interest in D , E , F .

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(D, F) Box plots showing cardiac variables (diastolic (D) and systolic (E) diameters and percent fractional shortening (F)) assessed by SOHA approach for controls (UAS-Bru3; Mp RNAi and UAS-UPRT; Bru3), Mp rescue (Hand>Bru3; Mp RANi) and DM1 context (Hand>UPRT; Bru3) at five weeks of age showing rescue of diastolic and systolic diameters in Hand>Bru3; Mp RNAi in comparison to controls (D,E) and rescue of cardiac contractility (F) n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest in D,E,F.


A . Images of P4 ( top ) and P21 ( bottom ) Mpdz + / + and Mpdz - / - mice . Arrowheads point to the domed foreheads of the latter .

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A. Images of P4 (top) and P21 (bottom) Mpdz+/+ and Mpdz-/- mice. Arrowheads point to the domed foreheads of the latter.


B . Mean weights of P18 - P21 Mpdz + / + and Mpdz - / - mice ( n = 8 ÷ 12 , mean ± SD ) .

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B. Mean weights of P18-P21 Mpdz+/+ and Mpdz-/- mice (n=8÷12, mean ± SD).


C . Coronal , axial , and sagittal ( top to bottom ) PET images of P18 - P21 Mpdz + / + and Mpdz - / - mice . The emission intensity is shown as a 7 - point temperature scale from black ( 0 ) to white ( 7 ) .

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C. Coronal, axial, and sagittal (top to bottom) PET images of P18-P21 Mpdz+/+ and Mpdz-/- mice. The emission intensity is shown as a 7-point temperature scale from black (0) to white (7).


D . Mean PET emission intensities in the indicated brain sections ( n = 4 , mean ± SD ) .

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D. Mean PET emission intensities in the indicated brain sections (n=4, mean ± SD).


A . Coronal , sagittal , and axial ( clockwise from top left corner image of each genotype ) MR images of Mpdz + / + and Mpdz - / - P18 - P21 mice . Ventricles are pseudo - colored in red and 3D - reconstructed in the lower left panels .

A. Coronal, sagittal, and axial (clockwise from top left corner image of each genotype) MR images of Mpdz+/+ and Mpdz-/- P18-P21 mice. Ventricles are pseudo-colored in red and 3D-reconstructed in the lower left panels.


B . Means of total ventricle volumes and total brain volumes of Mpdz + / + and Mpdz - / - mice ( n = 8 , mean ± SD ) .

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B. Means of total ventricle volumes and total brain volumes of Mpdz+/+ and Mpdz-/- mice (n=8, mean ± SD).


C . A coronal HE - stained section ( center ) flanked by anatomically - corresponding coronal T2 - weighted MR images of Mpdz - / - P18 - P21 mice . Arrows or arrowheads show the match between the lateral ventricle CP in the HE section and in the MR images .

C. A coronal HE-stained section (center) flanked by anatomically-corresponding coronal T2-weighted MR images of Mpdz-/- P18-P21 mice. Arrows or arrowheads show the match between the lateral ventricle CP in the HE section and in the MR images.


D . Duplicate rows of T2 - weighted coronal images and anatomically - corresponding T1 - weighted coronal images at 1 min and at the peak - signal time point after contrast - medium injection ; the bottom row shows a similar set of axial images . Areas surrounded by squares are magnified in the insets ; arrows mark the contrast - medium signal in the T1 - weighted images . Each row corresponds to one mouse aged 18 - 21 - days . E . Time courses of the normalized T1 - weighted image intensities corresponding to the areas surrounded by numbered squares on the T2 - weighted images . Scale bars , 1 mm ; insets , 0 . 5 mm .

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D. Duplicate rows of T2-weighted coronal images and anatomically-corresponding T1-weighted coronal images at 1 min and at the peak-signal time point after contrast-medium injection; the bottom row shows a similar set of axial images. Areas surrounded by squares are magnified in the insets; arrows mark the contrast-medium signal in the T1-weighted images. Each row corresponds to one mouse aged 18-21-days. E. Time courses of the normalized T1-weighted image intensities corresponding to the areas surrounded by numbered squares on the T2-weighted images. Scale bars, 1 mm; insets, 0.5 mm.


A . Coronal HE - stained brain sections of a P18 Mpdz + / + mouse ( out of a total of three ) showing the 3rd ventricle , and the proximal and distal sections of the aqueduct of Sylvius ; these features are marked by arrows in the insets . B . The same , for P18 Mpdz - / - mice .

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A. Coronal HE-stained brain sections of a P18 Mpdz+/+ mouse (out of a total of three) showing the 3rd ventricle, and the proximal and distal sections of the aqueduct of Sylvius; these features are marked by arrows in the insets. B. The same, for P18 Mpdz-/- mice.


C . Top views of brains of P7 Mpdz + / + and Mpdz - / - mice that show the sites of Evans blue injection , and the midline planes ( dashed line ) sectioned to produce the sagittal images below . They show the extent of Evans blue spread in the ventricles and surrounding tissue ( one out of three experiments ) . In , injection ; LV , lateral ventricle ; SA , Sylvian Aqueduct ; V3 , third ventricle ; V4 , fourth ventricle . Scale bars , 1 mm ; insets , 0 . 25 mm .

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C. Top views of brains of P7 Mpdz+/+ and Mpdz-/- mice that show the sites of Evans blue injection, and the midline planes (dashed line) sectioned to produce the sagittal images below. They show the extent of Evans blue spread in the ventricles and surrounding tissue (one out of three experiments). In, injection; LV, lateral ventricle; SA, Sylvian Aqueduct; V3, third ventricle; V4, fourth ventricle. Scale bars, 1 mm; insets, 0.25 mm.


A . Immunofluorescence images of 10 μm - thick sections of CP from third ventricle villi of Mpdz + / + and Mpdz - / - mice were immunolabeled as shown . The areas in the square frames are magnified in the bottom panels .

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A. Immunofluorescence images of 10 μm-thick sections of CP from third ventricle villi of Mpdz+/+ and Mpdz-/- mice were immunolabeled as shown. The areas in the square frames are magnified in the bottom panels.


CP sections from lateral ventricle villi immunolabeled for ZO1 ( B ) The images are representative of 2 Mpdz + / + mice and 3 Mpdz - / - mice . Scale bars , top panels , 100 μm ; bottom panels , 10 μm .

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CP sections from lateral ventricle villi immunolabeled for ZO1 (B) The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm.


CP sections from lateral ventricle villi immunolabeled for E - cadherin ( E - cad ; C ) . The images are representative of 2 Mpdz + / + mice and 3 Mpdz - / - mice . Scale bars , top panels , 100 μm ; bottom panels , 10 μm .

CP sections from lateral ventricle villi immunolabeled for E-cadherin (E-cad; C). The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm.


D . Immunoblots with the indicated antibodies ( top ) and their quantifications . The densitometry measurements were normalized relative to the signal of the samples transduced by non - targeting shRNA ( Ctrl ) . Note that the same β - actin immunoblot was used twice because the Jam - C and E - cadherin samples were immunoblotted on the same membrane ( mean ± SD , n = 3 ) .

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D. Immunoblots with the indicated antibodies (top) and their quantifications. The densitometry measurements were normalized relative to the signal of the samples transduced by non-targeting shRNA (Ctrl). Note that the same β-actin immunoblot was used twice because the Jam-C and E-cadherin samples were immunoblotted on the same membrane (mean ± SD, n=3).


A . TEM images of longitudinal sections of lateral ventricle CP villi of P18 - P21 Mpdz + / + and Mpdz - / - mice . Scale bar , 2 μm . B . A large number of voids of varying sizes was evident in the CPECs of Mpdz - / - mice . Higher magnification images of the areas in the rectangles are shown underneath the panels . Adherens junctions are denoted by horizontal lines and arrows ; voids are indicated by arrows . Scale bar , 1 μm ; insets , 200 nm .

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A. TEM images of longitudinal sections of lateral ventricle CP villi of P18-P21 Mpdz+/+ and Mpdz-/- mice. Scale bar, 2 μm. B. A large number of voids of varying sizes was evident in the CPECs of Mpdz-/- mice. Higher magnification images of the areas in the rectangles are shown underneath the panels. Adherens junctions are denoted by horizontal lines and arrows; voids are indicated by arrows. Scale bar, 1 μm; insets, 200 nm.


C . Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz + / + and Mpdz - / - mice . Scale bar , 100

C. Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nm


D . Mitochondria were smaller and frequently lacked large portions of their cristae . Autophagosomes are present in some of them ( arrows ) . Scale bar , 200 nm . The images are representative of 2 Mpdz + / + mice and 2 Mpdz - / - mice .

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D. Mitochondria were smaller and frequently lacked large portions of their cristae. Autophagosomes are present in some of them (arrows). Scale bar, 200 nm. The images are representative of 2 Mpdz+/+ mice and 2 Mpdz-/- mice.


E . Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non - targeting ( Ctrl ) shRNA . Each record represents 4 wells . MPDZ immunoblot of each cell group is shown in the inset .

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E. Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non-targeting (Ctrl) shRNA. Each record represents 4 wells. MPDZ immunoblot of each cell group is shown in the inset.


A . TEM images of CPEC sections from lateral ventricle villi of two Mpdz + / + and two Mpdz - / - P14 - P16 mice injected with HRP . The CPs were reacted with hydrogen peroxide and DAB ex - vivo . The dark particles are DAB deposits internalized by micropinocytosis . The magnified fields to the right show individual particles engulfed in macropinosomes . Note the layered structure of the particles , and the macropinosome that is open to the ventricular space in the Mpdz - / - section . Scale bars , 1 μm ; insets , 100 nm . B . Mean number of engulfed DAB particles per cell in Mpdz + / + and Mpdz - / - mice ( mean ± SD , n = 22 ) .

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A. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22).


C . A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field . Scale bars in panels C and D , 1 μm ; insets , 200 nm . D . A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell , and a magnified field that contains several macropinosomes . E . Mean numbers of DAB - containing macropinosomes close to the apical or basal sides of CPECs from Mpdz + / + or Mpdz - / - mice ( mean ± SD , n = 20 ) .

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C. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).


A . Immunofluorescence images of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + and Mpdz - / - P14 - P16 mice immunolabeled by anti - LDLR . Scale bars , 50 ( top ) and 25 ( bottom ) μm . B . Mean fluorescence intensities ( normalized relative to the highest recorded intensity ) per cell in several LDLR - immunolabeled CP sections ( mean ± SD , n = 101 ) .

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A. Immunofluorescence images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- P14-P16 mice immunolabeled by anti-LDLR. Scale bars, 50 (top) and 25 (bottom) μm. B. Mean fluorescence intensities (normalized relative to the highest recorded intensity) per cell in several LDLR-immunolabeled CP sections (mean ± SD, n=101).


C . Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ - targeting or non - targeting shRNA . Mean abundances are quantified by densitometry of the LDLR bands , normalized relative to the β - actin bands ( mean ± SD , n = 3 ) .

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C. Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ-targeting or non-targeting shRNA. Mean abundances are quantified by densitometry of the LDLR bands, normalized relative to the β-actin bands (mean ± SD, n=3).


D . Fluorescence images of hCPECs transduced by either MPDZ or non - targeting ( Ctrl ) shRNA and immunolabeled by anti - LDLR either before ( 0 min ) or after 8 min of constitutive endocytosis of LDLR . The histograms below show the mean fluorescence intensities per hCPEC in each cell group , normalized relative to the highest recorded intensity ( mean ± SD , n = 41 ÷ 53 ) .

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D. Fluorescence images of hCPECs transduced by either MPDZ or non-targeting (Ctrl) shRNA and immunolabeled by anti-LDLR either before (0 min) or after 8 min of constitutive endocytosis of LDLR. The histograms below show the mean fluorescence intensities per hCPEC in each cell group, normalized relative to the highest recorded intensity (mean ± SD, n=41÷53).


A . Mean protein concentration in the CSF of Mpdz + / + and Mpdz - / - P14 - P21 mice ( mean ± SD , n = 2 ÷ 3 ) .

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A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3).


B . Volcano plot showing that the protein contents in sera of P15 - P21 Mpdz + / + and Mpdz - / - mice are similar to each other .

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B. Volcano plot showing that the protein contents in sera of P15-P21 Mpdz+/+ and Mpdz-/- mice are similar to each other.


C . A volcano plot showing that 23 proteins are at least two - fold significantly more abundant in the CSF of P15 - 21 Mpdz - / - mice ( red circles , protein names are indicated ) whereas only two proteins were more abundant in the CSF of Mpdz + / + mice ( green circles ) . The CSF composition was analyzed in 3 Mpdz - / - and 3 Mpdz + / + mice .

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C. A volcano plot showing that 23 proteins are at least two-fold significantly more abundant in the CSF of P15-21 Mpdz-/- mice (red circles, protein names are indicated) whereas only two proteins were more abundant in the CSF of Mpdz+/+ mice (green circles). The CSF composition was analyzed in 3 Mpdz-/- and 3 Mpdz+/+ mice.


D . Images of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + and Mpdz - / - mice , immunolabeled for Nkcc1 . The areas in the marked squares are magnified below . Scale bars , top , 50 μm , bottom 20 μm .

D. Images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- mice, immunolabeled for Nkcc1. The areas in the marked squares are magnified below. Scale bars, top, 50 μm, bottom 20 μm.


E . Immunoblots of LDLR and Mpdz , with β - actin as loading control . The MPDZ immunoblot shows the efficiency of the shRNA - mediated knockdown . Bands from three immunoblots were quantified by normalization to β - actin bands ( mean ± SD ) .

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E. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD).


B . Western blots of MDI protein in wild type ( wt ) and mdi1 flies , showing that mdi1 is a protein - null mutation . Tubulin was used as loading control .

B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control.


C . Ovarioles expressing MDI - GFP stained with a mitochondrial marker , ATP synthase ( ATP - S ) . Overlapping signals ( merge ) indicate that MDI localizes to mitochondria . Note that MDI is highly expressed in germ cells at late germarium stages ( left panels ) and in egg chambers ( right panels ) . Bars , 10 m .

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C. Ovarioles expressing MDI-GFP stained with a mitochondrial marker, ATP synthase (ATP-S). Overlapping signals (merge) indicate that MDI localizes to mitochondria. Note that MDI is highly expressed in germ cells at late germarium stages (left panels) and in egg chambers (right panels). Bars, 10 m.


A - C . mtDNA replication in wt ( A ) and mdi1 ovarioles ( B , C ) as illustrated by EdU incorporation . Arrows point to mitochondrial DNA , arrowheads to nuclei . In wt ovaries ( A ) , mtDNA replication starts at germarium stage 2B and lasts into egg - chamber stage 2 ( S2 ) . In most mdi1 ovarioles ( B ) , mtDNA replication is undetectable at these stages , whereas in some ( C ) it appears delayed until germarium stage 3 . Bars , 10 m .

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A-C. mtDNA replication in wt (A) and mdi1 ovarioles (B, C) as illustrated by EdU incorporation. Arrows point to mitochondrial DNA, arrowheads to nuclei. In wt ovaries (A), mtDNA replication starts at germarium stage 2B and lasts into egg-chamber stage 2 (S2). In most mdi1 ovarioles (B), mtDNA replication is undetectable at these stages, whereas in some (C) it appears delayed until germarium stage 3. Bars, 10 m.


D . Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls . Each data point represents the mean of 3 independent replicates . Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . The relative mtDNA level was determined as the average of 3 biological repeats . P values of comparing mdi1 ; nos > mdi to mdi1 : hatching rate , P = 1 . 1995E - 05 ; mtDNA , P = 0 . 0078 . P values of comparing mdi1 ; nos > hAKAP1 to mdi1 : hatching rate , P = 1 . 6998E - 05 ; mtDNA level , P = 0 . 0009 .

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D. Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls. Each data point represents the mean of 3 independent replicates. Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was determined as the average of 3 biological repeats. P values of comparing mdi1; nos> mdi to mdi1: hatching rate, P= 1.1995E-05; mtDNA, P= 0.0078. P values of comparing mdi1; nos> hAKAP1 to mdi1: hatching rate, P= 1.6998E-05; mtDNA level, P= 0.0009.


B . Representative images of S2 cells expressing GFP fusions ( green ) of the MDI proteins diagramed in ( A ) . Staining with MitoTracker Red ( red ) to label mitochondria shows an overlapping signal ( yellow ) , indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS .

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B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS.