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rabbit anti prox1  (Millipore)
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Millipore rabbit anti prox1
(a) Expression of lens-specific markers ( Cry::ECFP , lens fiber cell (LFC) and <t>Prox1)</t> and retina-specific marker ( Atoh7::EGFP ) in medaka embryonic eyes at 3 dpf. Optical sections showing the expression of indicated markers detected by whole-mount immunohistochemistry with the use of anti-GFP (green), anti-LFC (magenta) and anti-Prox1(cyan) antibodies, co-labelled with DAPI nuclear stain (grey). (b) Organoid generation from crystallin reporter line ( Cry::ECFP ). Expression of lens-specific markers ( Cry::ECFP and LFC) in day 4 organoids detected by anti-GFP and anti-LFC antibodies. Represented by maximal projection for overview and single optical section for the detail of the lens. (c) Expression of Prox1 in day 4 organoids derived from wild-type medaka cells detected with anti-Prox1 antibody, co-stained with DAPI nuclear stain; displayed as maximal z-projection. (d ) Organoid generation from retina-specific reporter line ( Atoh7::EGFP ). Overlay of bright-field and wide-field images showing EGFP fluorescence in Atoh7 -expressing cells, lens indicated with asterisk. (e) Organoids generated from retina-specific reporter line ( Rx3::H2B-GFP ) labeled with anti-GFP and anti-LFC antibody displayed as maximal z-projection. (f) Schematic representation of distribution of retina– and lens-specific markers in day 4 organoids. (g) Optical section of day 4 organoid lens labeled with anti-Prox1 and anti-LFC antibodies, co-labeled with DAPI, showing elongated Prox1 + /LFC + cells on the surface of the lens. (h) Enlargement of indicated area in g. For Cry::ECFP , Atoh7::EGFP and Rx3::H2B-GFP . The percentage (%) indicates how many cells carry the indicated transgene. Arrow in b, c and e indicates position of the lens. Arrowheads in b and h indicate abnormally looking nuclei. le, lens; re, retina; wt, wild-type; LFC, lens fiber cell. Scale bar 50 μm in a, g and enlargement of b, 100 μm in b-e, 10 μm in h.
Rabbit Anti Prox1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti prox1  (Reliatech)
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Reliatech rabbit anti prox1
( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Rabbit Anti Prox1, supplied by Reliatech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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goat anti prox1  (R&D Systems)
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( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Goat Anti Prox1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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m0823 rrid ab 2114471 anti prox1 angiobio  (AngioBio Inc)
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AngioBio Inc m0823 rrid ab 2114471 anti prox1 angiobio
( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
M0823 Rrid Ab 2114471 Anti Prox1 Angiobio, supplied by AngioBio Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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prox1  (AngioBio Inc)
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AngioBio Inc prox1
( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Prox1, supplied by AngioBio Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti mouse prox1 antibody  (Danaher Inc)
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( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Anti Mouse Prox1 Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti prox1  (Danaher Inc)
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( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Rabbit Anti Prox1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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prox1  (Proteintech)
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Proteintech prox1
( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Prox1, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti prox1  (Proteintech)
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( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of <t>Prox1</t> mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).
Anti Prox1, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


(a) Expression of lens-specific markers ( Cry::ECFP , lens fiber cell (LFC) and Prox1) and retina-specific marker ( Atoh7::EGFP ) in medaka embryonic eyes at 3 dpf. Optical sections showing the expression of indicated markers detected by whole-mount immunohistochemistry with the use of anti-GFP (green), anti-LFC (magenta) and anti-Prox1(cyan) antibodies, co-labelled with DAPI nuclear stain (grey). (b) Organoid generation from crystallin reporter line ( Cry::ECFP ). Expression of lens-specific markers ( Cry::ECFP and LFC) in day 4 organoids detected by anti-GFP and anti-LFC antibodies. Represented by maximal projection for overview and single optical section for the detail of the lens. (c) Expression of Prox1 in day 4 organoids derived from wild-type medaka cells detected with anti-Prox1 antibody, co-stained with DAPI nuclear stain; displayed as maximal z-projection. (d ) Organoid generation from retina-specific reporter line ( Atoh7::EGFP ). Overlay of bright-field and wide-field images showing EGFP fluorescence in Atoh7 -expressing cells, lens indicated with asterisk. (e) Organoids generated from retina-specific reporter line ( Rx3::H2B-GFP ) labeled with anti-GFP and anti-LFC antibody displayed as maximal z-projection. (f) Schematic representation of distribution of retina– and lens-specific markers in day 4 organoids. (g) Optical section of day 4 organoid lens labeled with anti-Prox1 and anti-LFC antibodies, co-labeled with DAPI, showing elongated Prox1 + /LFC + cells on the surface of the lens. (h) Enlargement of indicated area in g. For Cry::ECFP , Atoh7::EGFP and Rx3::H2B-GFP . The percentage (%) indicates how many cells carry the indicated transgene. Arrow in b, c and e indicates position of the lens. Arrowheads in b and h indicate abnormally looking nuclei. le, lens; re, retina; wt, wild-type; LFC, lens fiber cell. Scale bar 50 μm in a, g and enlargement of b, 100 μm in b-e, 10 μm in h.

Journal: bioRxiv

Article Title: Inverted Assembly of the Lens Within Ocular Organoids Reveals Alternate Paths to Ocular Morphogenesis

doi: 10.1101/2025.04.17.649366

Figure Lengend Snippet: (a) Expression of lens-specific markers ( Cry::ECFP , lens fiber cell (LFC) and Prox1) and retina-specific marker ( Atoh7::EGFP ) in medaka embryonic eyes at 3 dpf. Optical sections showing the expression of indicated markers detected by whole-mount immunohistochemistry with the use of anti-GFP (green), anti-LFC (magenta) and anti-Prox1(cyan) antibodies, co-labelled with DAPI nuclear stain (grey). (b) Organoid generation from crystallin reporter line ( Cry::ECFP ). Expression of lens-specific markers ( Cry::ECFP and LFC) in day 4 organoids detected by anti-GFP and anti-LFC antibodies. Represented by maximal projection for overview and single optical section for the detail of the lens. (c) Expression of Prox1 in day 4 organoids derived from wild-type medaka cells detected with anti-Prox1 antibody, co-stained with DAPI nuclear stain; displayed as maximal z-projection. (d ) Organoid generation from retina-specific reporter line ( Atoh7::EGFP ). Overlay of bright-field and wide-field images showing EGFP fluorescence in Atoh7 -expressing cells, lens indicated with asterisk. (e) Organoids generated from retina-specific reporter line ( Rx3::H2B-GFP ) labeled with anti-GFP and anti-LFC antibody displayed as maximal z-projection. (f) Schematic representation of distribution of retina– and lens-specific markers in day 4 organoids. (g) Optical section of day 4 organoid lens labeled with anti-Prox1 and anti-LFC antibodies, co-labeled with DAPI, showing elongated Prox1 + /LFC + cells on the surface of the lens. (h) Enlargement of indicated area in g. For Cry::ECFP , Atoh7::EGFP and Rx3::H2B-GFP . The percentage (%) indicates how many cells carry the indicated transgene. Arrow in b, c and e indicates position of the lens. Arrowheads in b and h indicate abnormally looking nuclei. le, lens; re, retina; wt, wild-type; LFC, lens fiber cell. Scale bar 50 μm in a, g and enlargement of b, 100 μm in b-e, 10 μm in h.

Article Snippet: Primary antibodies used in this study: rabbit anti-Rx2 ( ) (1:500), chicken anti-GFP (Invitrogen, Cat#:A10262, 1:500), mouse anti-lens fiber cell (Abcam, Cat#:ab185979, ZL-1, 1:500), rabbit anti-Prox1 (Millipore, Cat#:AB5475, 1:2000), rabbit anti-RFP (Clontech, Cat#:632496, 1:500).

Techniques: Expressing, Marker, Immunohistochemistry, Staining, Derivative Assay, Fluorescence, Generated, Labeling

(See also Figure S2 and S3, Video S1, S3 and S4.) (a) Schematic representation of lens formation in medaka. The lens develops from lens-competent head surface ectoderm (SE, magenta), retinal cells from the OV (green). Surface ectoderm forms the lens placode (lp) that further invaginates together with the OV to form the optic cup with a lens sphere surrounded by the retina. (b) Optical sections through wild-type medaka embryos at indicated stages stained with the antibody against Prox1, a lens placode-specific marker and LFC, a lens fiber cell marker. (c) Optical sections through organoids derived from the Rx3::H2B-GFP reporter line stained with antibody against Prox1 (magenta) and LFC (cyan). (d) Lens specific GFP expression in embryos of the Foxe3::GFP reporter line (magenta). Optical section showing the overlay of bright-field and GFP fluorescence in the 1 dpf and 2 dpf medaka embryos (left and middle). Detail of the eye (right) showing the expression domain of Foxe3::GFP (magenta) and LFC (cyan) detected by immunohistochemistry. (e) Optical section through a day 1 organoid (26 hpa) derived from Foxe3::GFP reporter line showing the lens-specific expression of GFP (magenta) detected by anti-GFP antibody, co-stained with DAPI (blue). (f) Optical section through a day 2 organoid derived from 50 % Foxe3::GFP and 50 % Rx2::H2B-RFP reporter blastula cells. Expression of RFP (green) and GFP (magenta), detected by antibodies is mutually exclusive, co-stained with DAPI (blue). (g) 3D rendering of Rx2::H2B-RFP (green), Foxe3::GFP (magenta) organoid shown in f, DAPI stained nuclei are in cyan. n numbers in c and e indicate the number of analyzed organoids with described phenotype. LFC, lens fiber cell; le, lens; re, retina; lp, lens placode; SE, surface ectoderm; OV, optic vesicle; dpf, day post fertilization; hpa, hours post aggregation. Dashed line in c, e and f indicates the outline of the organoid. Dashed line in d indicates the outline of the retina. Scale bar 100 μm.

Journal: bioRxiv

Article Title: Inverted Assembly of the Lens Within Ocular Organoids Reveals Alternate Paths to Ocular Morphogenesis

doi: 10.1101/2025.04.17.649366

Figure Lengend Snippet: (See also Figure S2 and S3, Video S1, S3 and S4.) (a) Schematic representation of lens formation in medaka. The lens develops from lens-competent head surface ectoderm (SE, magenta), retinal cells from the OV (green). Surface ectoderm forms the lens placode (lp) that further invaginates together with the OV to form the optic cup with a lens sphere surrounded by the retina. (b) Optical sections through wild-type medaka embryos at indicated stages stained with the antibody against Prox1, a lens placode-specific marker and LFC, a lens fiber cell marker. (c) Optical sections through organoids derived from the Rx3::H2B-GFP reporter line stained with antibody against Prox1 (magenta) and LFC (cyan). (d) Lens specific GFP expression in embryos of the Foxe3::GFP reporter line (magenta). Optical section showing the overlay of bright-field and GFP fluorescence in the 1 dpf and 2 dpf medaka embryos (left and middle). Detail of the eye (right) showing the expression domain of Foxe3::GFP (magenta) and LFC (cyan) detected by immunohistochemistry. (e) Optical section through a day 1 organoid (26 hpa) derived from Foxe3::GFP reporter line showing the lens-specific expression of GFP (magenta) detected by anti-GFP antibody, co-stained with DAPI (blue). (f) Optical section through a day 2 organoid derived from 50 % Foxe3::GFP and 50 % Rx2::H2B-RFP reporter blastula cells. Expression of RFP (green) and GFP (magenta), detected by antibodies is mutually exclusive, co-stained with DAPI (blue). (g) 3D rendering of Rx2::H2B-RFP (green), Foxe3::GFP (magenta) organoid shown in f, DAPI stained nuclei are in cyan. n numbers in c and e indicate the number of analyzed organoids with described phenotype. LFC, lens fiber cell; le, lens; re, retina; lp, lens placode; SE, surface ectoderm; OV, optic vesicle; dpf, day post fertilization; hpa, hours post aggregation. Dashed line in c, e and f indicates the outline of the organoid. Dashed line in d indicates the outline of the retina. Scale bar 100 μm.

Article Snippet: Primary antibodies used in this study: rabbit anti-Rx2 ( ) (1:500), chicken anti-GFP (Invitrogen, Cat#:A10262, 1:500), mouse anti-lens fiber cell (Abcam, Cat#:ab185979, ZL-1, 1:500), rabbit anti-Prox1 (Millipore, Cat#:AB5475, 1:2000), rabbit anti-RFP (Clontech, Cat#:632496, 1:500).

Techniques: Staining, Marker, Derivative Assay, Expressing, Fluorescence, Immunohistochemistry

( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of Prox1 mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of Prox1 mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Imaging, Mutagenesis, Functional Assay, Expressing, Labeling

( A ) Wholemount immunofluorescent labeling of PROX1 or mutant PROX1 in Emx2- Cre; Prox1 cKOs and littermate controls during neonatal and postnatal development. All images were taken from the middle turn. ( B ) The number of PROX1 immunolabeled nuclei per 100µm of cochlear length at neonatal stages and at three positions along the cochlear spiral (n=4 (E17.5), 6 (P0), 22 (P2) for controls and (n=3 (E17.5), 6 (P0), 22 (P2) for cKOs). ( C ) The ratio of supporting cells to hair cells at E17.5 does not differ between cKOs (n=4 cochleae, 29 sections) and controls (n=4 cochleae, 26 sections) when evaluated in sectioned cochleae. ( D ) The number of OHCs per 100µm of cochlear length does not differ between cKOs (n=6) and controls (n=5) at P0. ( E ) The overall length of the cochlear spiral at P2 does not change between cKOs (n=10) and controls (n=13). ( F ) ZO-1 immunolabeling of the reticular lamina shows the distribution of Deiters’ and OPC processes between OHCs. Higher magnification fields ( i, ii, iii ) from the Emx2 -Cre; Prox1 cKO where extra supporting cell processes are marked by colored shading. ( G ) Phalloidin stain of wholemount cochleae shows no difference in hair cell distribution, bundle morphology or polarity between cKOs (n=6) and littermate controls (n=5). Error bars show SEM, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Scale bars are 20µm.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Wholemount immunofluorescent labeling of PROX1 or mutant PROX1 in Emx2- Cre; Prox1 cKOs and littermate controls during neonatal and postnatal development. All images were taken from the middle turn. ( B ) The number of PROX1 immunolabeled nuclei per 100µm of cochlear length at neonatal stages and at three positions along the cochlear spiral (n=4 (E17.5), 6 (P0), 22 (P2) for controls and (n=3 (E17.5), 6 (P0), 22 (P2) for cKOs). ( C ) The ratio of supporting cells to hair cells at E17.5 does not differ between cKOs (n=4 cochleae, 29 sections) and controls (n=4 cochleae, 26 sections) when evaluated in sectioned cochleae. ( D ) The number of OHCs per 100µm of cochlear length does not differ between cKOs (n=6) and controls (n=5) at P0. ( E ) The overall length of the cochlear spiral at P2 does not change between cKOs (n=10) and controls (n=13). ( F ) ZO-1 immunolabeling of the reticular lamina shows the distribution of Deiters’ and OPC processes between OHCs. Higher magnification fields ( i, ii, iii ) from the Emx2 -Cre; Prox1 cKO where extra supporting cell processes are marked by colored shading. ( G ) Phalloidin stain of wholemount cochleae shows no difference in hair cell distribution, bundle morphology or polarity between cKOs (n=6) and littermate controls (n=5). Error bars show SEM, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Scale bars are 20µm.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Labeling, Mutagenesis, Immunolabeling, Staining

( A ) Experimental timecourse of EdU delivery to timed pregnant dams and wholemount immunolabeling of P0 cochleae for EdU-labeled and PROX1 or mutant PROX1 expressing cells in Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=3). ( B ) Immunolabeling for the cell division marker Ki-67 shows no overlap with PROX1 or mutant PROX1 expressing cells in sectioned cochleae at E15.5 from Emx2- Cre; Prox1 cKOs (n=4) or littermate controls (n=5). ( C ) Wholemount immunolabeling for PROX1 in the Bax knockout ( Bax -/- , n=3) or heterozygous control ( Bax +/- , n=5) cochleae and ratios of PROX1-labeled cells to OHCs at three positions along the length of the cochlear spiral. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Brackets in A&B mark the boundaries of the PROX1-expressing domains. Scale bars are 20µm (A,C) and 10µm (B).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Experimental timecourse of EdU delivery to timed pregnant dams and wholemount immunolabeling of P0 cochleae for EdU-labeled and PROX1 or mutant PROX1 expressing cells in Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=3). ( B ) Immunolabeling for the cell division marker Ki-67 shows no overlap with PROX1 or mutant PROX1 expressing cells in sectioned cochleae at E15.5 from Emx2- Cre; Prox1 cKOs (n=4) or littermate controls (n=5). ( C ) Wholemount immunolabeling for PROX1 in the Bax knockout ( Bax -/- , n=3) or heterozygous control ( Bax +/- , n=5) cochleae and ratios of PROX1-labeled cells to OHCs at three positions along the length of the cochlear spiral. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Brackets in A&B mark the boundaries of the PROX1-expressing domains. Scale bars are 20µm (A,C) and 10µm (B).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Immunolabeling, Labeling, Mutagenesis, Expressing, Marker, Knock-Out, Control, Standard Deviation

( A ) Conventional histochemical ISH of Bhlhe22- Cre; Prox1 cKO (n=3) and littermate control (n=3) cochleae using probes against the targeted Prox1 exons to assess expression in the organ of Corti (OC) and spiral ganglion neurons (SGN). ( B ) Peripheral axons of Type II SGNs immunolabeled for Neurofilament (NF-H) in Emx2- Cre; Prox1 cKOs (n=6) or littermate controls (n=3), and Bhlhe22- Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Arrowheads indicate incorrectly turning axons. ( C ) Quantification of axonal turning errors for Type II SGNs located in the basal turn of the cochlea. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (****p<0.0001). Scale bars are 100µm (A) and 20µm (B).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Conventional histochemical ISH of Bhlhe22- Cre; Prox1 cKO (n=3) and littermate control (n=3) cochleae using probes against the targeted Prox1 exons to assess expression in the organ of Corti (OC) and spiral ganglion neurons (SGN). ( B ) Peripheral axons of Type II SGNs immunolabeled for Neurofilament (NF-H) in Emx2- Cre; Prox1 cKOs (n=6) or littermate controls (n=3), and Bhlhe22- Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Arrowheads indicate incorrectly turning axons. ( C ) Quantification of axonal turning errors for Type II SGNs located in the basal turn of the cochlea. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (****p<0.0001). Scale bars are 100µm (A) and 20µm (B).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Control, Expressing, Immunolabeling, Standard Deviation

( A ) Differentially expressed genes (DEG) between Emx2 -Cre; Prox1 cKO and littermate control organ of Corti at P0 identified using DESeq2 with genes significantly downregulated in the cKO in red and those upregulated in blue. ( B ) Volcano plots restricted to the DEGs found in different groups of cochlear cell types with color density indicating the predicted contribution of that gene to the group’s overall transcriptome. Cell type groups and levels of gene expression are derived from public scRNAseq datasets generated from wild type tissue . ( C ) Average values of normalized Prox1 transcript alignments against all Prox1 exons compared to alignments against the two exons targeted in Emx2 -Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Error bars show standard deviation, and asterisks indicate statistical significance using Student’s t -test (****p<0.0001). ( D ) Changes in gene expression determined by DSeq2 for PCP, Nectin and Rac genes. ( E ) Changes in expression for genes signifying medial compartment and lateral compartment cell types. Red shading marks DEGs with statistically significant changes between genotypes (see also Supp. Table1&3). ( F ) Metascape Gene Ontology (GO) analysis of downregulated genes in the organ of Corti of Emx2- Cre; Prox1 cKOs.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Differentially expressed genes (DEG) between Emx2 -Cre; Prox1 cKO and littermate control organ of Corti at P0 identified using DESeq2 with genes significantly downregulated in the cKO in red and those upregulated in blue. ( B ) Volcano plots restricted to the DEGs found in different groups of cochlear cell types with color density indicating the predicted contribution of that gene to the group’s overall transcriptome. Cell type groups and levels of gene expression are derived from public scRNAseq datasets generated from wild type tissue . ( C ) Average values of normalized Prox1 transcript alignments against all Prox1 exons compared to alignments against the two exons targeted in Emx2 -Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Error bars show standard deviation, and asterisks indicate statistical significance using Student’s t -test (****p<0.0001). ( D ) Changes in gene expression determined by DSeq2 for PCP, Nectin and Rac genes. ( E ) Changes in expression for genes signifying medial compartment and lateral compartment cell types. Red shading marks DEGs with statistically significant changes between genotypes (see also Supp. Table1&3). ( F ) Metascape Gene Ontology (GO) analysis of downregulated genes in the organ of Corti of Emx2- Cre; Prox1 cKOs.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Control, Gene Expression, Derivative Assay, Generated, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet:

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Expressing

( A ) Evaluation of peripheral axon turning in Sema5a KO mice compared to littermate controls at P0 by wholemount immunolabeling against NF-H. ( B ) Immunofluorescent images of VANGL2 wholemount organ of Corti viewed as 3D-reconstructions. Arrowheads mark examples of polarized enrichment of Vangl2 at the intercellular junctions between neighboring supporting cells in littermate controls (n=4) while asterisks mark examples of cells lacking this polarity in Emx2- Cre; Prox1 cKOs (n=3) at P0. Prox1 immunolabeling marks cell nuclei. Images are collected from the basal turn of the cochlea. Scale bars are 20µm.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Evaluation of peripheral axon turning in Sema5a KO mice compared to littermate controls at P0 by wholemount immunolabeling against NF-H. ( B ) Immunofluorescent images of VANGL2 wholemount organ of Corti viewed as 3D-reconstructions. Arrowheads mark examples of polarized enrichment of Vangl2 at the intercellular junctions between neighboring supporting cells in littermate controls (n=4) while asterisks mark examples of cells lacking this polarity in Emx2- Cre; Prox1 cKOs (n=3) at P0. Prox1 immunolabeling marks cell nuclei. Images are collected from the basal turn of the cochlea. Scale bars are 20µm.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Immunolabeling

( A ) Wholemount immunofluorescence images of PROX1 and the OPC protein CD44 in the developing organ of Corti shows the absence of CD44 expression in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) when evaluated at P2. ( B ) Immunolabeling for the IPC protein P75-NTR shows no changes in the apical projections of IPCs visualized at the reticular lamina or the number or distribution of IPCs at the nuclear layer. ( C ) Immunolabeling for the medial compartment supporting cell protein FABP7 shows an expansion of FABP7 into the lateral compartment in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) at P7. Bracket marks the boundaries of the lateral compartment based on the distribution of Phalloidin-stained OHCs. ( E ) fISH showing the distribution of the medial gene Trh relative to IHCs expressing fgf8 and expansion of Trh expression in Emx2- Cre; Prox1 cKOs (n=4) compared to littermate controls (n=5) at P0. Brackets mark the position of the IHC row. Scale bars are 20µm.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Wholemount immunofluorescence images of PROX1 and the OPC protein CD44 in the developing organ of Corti shows the absence of CD44 expression in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) when evaluated at P2. ( B ) Immunolabeling for the IPC protein P75-NTR shows no changes in the apical projections of IPCs visualized at the reticular lamina or the number or distribution of IPCs at the nuclear layer. ( C ) Immunolabeling for the medial compartment supporting cell protein FABP7 shows an expansion of FABP7 into the lateral compartment in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) at P7. Bracket marks the boundaries of the lateral compartment based on the distribution of Phalloidin-stained OHCs. ( E ) fISH showing the distribution of the medial gene Trh relative to IHCs expressing fgf8 and expansion of Trh expression in Emx2- Cre; Prox1 cKOs (n=4) compared to littermate controls (n=5) at P0. Brackets mark the position of the IHC row. Scale bars are 20µm.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Immunofluorescence, Expressing, Immunolabeling, Staining

( A ) Cochlear sections cut from P14 Emx2 -Cre; Prox1 cKOs and littermate controls labeled with the nuclear dye DAPI and imaged against background autofluorescence from a second channel. Asterisks mark the tunnel of Corti center and brackets the position of the three rows of OHC nuclei. ( B ) Area of the tunnel of Corti in cochlear sections for Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=4) at 3 positions along the length of P14 cochleae. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (***p<0.0001). ( C ) Deiters’ cell phalangeal processes immunolabeled with antibodies against β1β2 Tubulin reveals differences in their structural morphology and organization relative to OHCs in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=3) at P14. OHCs are labeled using antibodies against Oncomodulin. Scale bars are 10µm (A).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Cochlear sections cut from P14 Emx2 -Cre; Prox1 cKOs and littermate controls labeled with the nuclear dye DAPI and imaged against background autofluorescence from a second channel. Asterisks mark the tunnel of Corti center and brackets the position of the three rows of OHC nuclei. ( B ) Area of the tunnel of Corti in cochlear sections for Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=4) at 3 positions along the length of P14 cochleae. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (***p<0.0001). ( C ) Deiters’ cell phalangeal processes immunolabeled with antibodies against β1β2 Tubulin reveals differences in their structural morphology and organization relative to OHCs in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=3) at P14. OHCs are labeled using antibodies against Oncomodulin. Scale bars are 10µm (A).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Labeling, Standard Deviation, Immunolabeling

( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of Prox1 mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Schematic cross-section of the mouse organ of Corti identifying the medial and lateral compartments and cell types they contain. Arrows mark the position of two viewing planes used in subsequent wholemount imaging; the reticular lamina at the apical surface of hair cells and supporting cells, and a deeper plane where the supporting cell nuclei are located. ( B ) Schematic of Prox1 mRNA depicting targeted exons, wild type and mutant PROX1 protein with described functional domains , targets of Prox1 ISH probes and location of antigens recognized by PROX1 antibodies. Cre-mediated deletion of the targeted exons removes the homeodomain but does not prevent translation of mutant PROX1 protein or impact conserved prospero domains, nuclear receptor boxes (NR) or nuclear localization signal (NLS). ( C ) Histochemical ISH for Prox1 mRNA at P0 demonstrates restricted gene deletion from the organ of Corti in Emx2 -Cre; Prox1 cKO cochleae (n=3 controls, n=4 cKOs). ( D ) HCR fISH for Prox1 mRNA confirms exon deletion from organ of Corti in Emx2 -Cre; Prox1 cKOs in the vicinity of Gfi1- expressing hair cells (n=3 controls, n=4 cKOs). ( E ) Immunofluorescent labeling of PROX1 marks Inner Pillar Cells (IPC), Outer Pillar Cells (OPC) and three rows of Deiters cells (DC1, DC2, DC3) in littermate controls and detects mutant PROX1 protein in the cKO (n=3 controls, n=6 cKOs). Scale bars are 100 µm (C) and 20µm (D,E).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Imaging, Mutagenesis, Functional Assay, Expressing, Labeling

( A ) Wholemount immunofluorescent labeling of PROX1 or mutant PROX1 in Emx2- Cre; Prox1 cKOs and littermate controls during neonatal and postnatal development. All images were taken from the middle turn. ( B ) The number of PROX1 immunolabeled nuclei per 100µm of cochlear length at neonatal stages and at three positions along the cochlear spiral (n=4 (E17.5), 6 (P0), 22 (P2) for controls and (n=3 (E17.5), 6 (P0), 22 (P2) for cKOs). ( C ) The ratio of supporting cells to hair cells at E17.5 does not differ between cKOs (n=4 cochleae, 29 sections) and controls (n=4 cochleae, 26 sections) when evaluated in sectioned cochleae. ( D ) The number of OHCs per 100µm of cochlear length does not differ between cKOs (n=6) and controls (n=5) at P0. ( E ) The overall length of the cochlear spiral at P2 does not change between cKOs (n=10) and controls (n=13). ( F ) ZO-1 immunolabeling of the reticular lamina shows the distribution of Deiters’ and OPC processes between OHCs. Higher magnification fields ( i, ii, iii ) from the Emx2 -Cre; Prox1 cKO where extra supporting cell processes are marked by colored shading. ( G ) Phalloidin stain of wholemount cochleae shows no difference in hair cell distribution, bundle morphology or polarity between cKOs (n=6) and littermate controls (n=5). Error bars show SEM, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Scale bars are 20µm.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Wholemount immunofluorescent labeling of PROX1 or mutant PROX1 in Emx2- Cre; Prox1 cKOs and littermate controls during neonatal and postnatal development. All images were taken from the middle turn. ( B ) The number of PROX1 immunolabeled nuclei per 100µm of cochlear length at neonatal stages and at three positions along the cochlear spiral (n=4 (E17.5), 6 (P0), 22 (P2) for controls and (n=3 (E17.5), 6 (P0), 22 (P2) for cKOs). ( C ) The ratio of supporting cells to hair cells at E17.5 does not differ between cKOs (n=4 cochleae, 29 sections) and controls (n=4 cochleae, 26 sections) when evaluated in sectioned cochleae. ( D ) The number of OHCs per 100µm of cochlear length does not differ between cKOs (n=6) and controls (n=5) at P0. ( E ) The overall length of the cochlear spiral at P2 does not change between cKOs (n=10) and controls (n=13). ( F ) ZO-1 immunolabeling of the reticular lamina shows the distribution of Deiters’ and OPC processes between OHCs. Higher magnification fields ( i, ii, iii ) from the Emx2 -Cre; Prox1 cKO where extra supporting cell processes are marked by colored shading. ( G ) Phalloidin stain of wholemount cochleae shows no difference in hair cell distribution, bundle morphology or polarity between cKOs (n=6) and littermate controls (n=5). Error bars show SEM, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Scale bars are 20µm.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Labeling, Mutagenesis, Immunolabeling, Staining

( A ) Experimental timecourse of EdU delivery to timed pregnant dams and wholemount immunolabeling of P0 cochleae for EdU-labeled and PROX1 or mutant PROX1 expressing cells in Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=3). ( B ) Immunolabeling for the cell division marker Ki-67 shows no overlap with PROX1 or mutant PROX1 expressing cells in sectioned cochleae at E15.5 from Emx2- Cre; Prox1 cKOs (n=4) or littermate controls (n=5). ( C ) Wholemount immunolabeling for PROX1 in the Bax knockout ( Bax -/- , n=3) or heterozygous control ( Bax +/- , n=5) cochleae and ratios of PROX1-labeled cells to OHCs at three positions along the length of the cochlear spiral. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Brackets in A&B mark the boundaries of the PROX1-expressing domains. Scale bars are 20µm (A,C) and 10µm (B).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Experimental timecourse of EdU delivery to timed pregnant dams and wholemount immunolabeling of P0 cochleae for EdU-labeled and PROX1 or mutant PROX1 expressing cells in Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=3). ( B ) Immunolabeling for the cell division marker Ki-67 shows no overlap with PROX1 or mutant PROX1 expressing cells in sectioned cochleae at E15.5 from Emx2- Cre; Prox1 cKOs (n=4) or littermate controls (n=5). ( C ) Wholemount immunolabeling for PROX1 in the Bax knockout ( Bax -/- , n=3) or heterozygous control ( Bax +/- , n=5) cochleae and ratios of PROX1-labeled cells to OHCs at three positions along the length of the cochlear spiral. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (**p<0.01, ***p<0.001). Brackets in A&B mark the boundaries of the PROX1-expressing domains. Scale bars are 20µm (A,C) and 10µm (B).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Immunolabeling, Labeling, Mutagenesis, Expressing, Marker, Knock-Out, Control, Standard Deviation

( A ) Conventional histochemical ISH of Bhlhe22- Cre; Prox1 cKO (n=3) and littermate control (n=3) cochleae using probes against the targeted Prox1 exons to assess expression in the organ of Corti (OC) and spiral ganglion neurons (SGN). ( B ) Peripheral axons of Type II SGNs immunolabeled for Neurofilament (NF-H) in Emx2- Cre; Prox1 cKOs (n=6) or littermate controls (n=3), and Bhlhe22- Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Arrowheads indicate incorrectly turning axons. ( C ) Quantification of axonal turning errors for Type II SGNs located in the basal turn of the cochlea. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (****p<0.0001). Scale bars are 100µm (A) and 20µm (B).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Conventional histochemical ISH of Bhlhe22- Cre; Prox1 cKO (n=3) and littermate control (n=3) cochleae using probes against the targeted Prox1 exons to assess expression in the organ of Corti (OC) and spiral ganglion neurons (SGN). ( B ) Peripheral axons of Type II SGNs immunolabeled for Neurofilament (NF-H) in Emx2- Cre; Prox1 cKOs (n=6) or littermate controls (n=3), and Bhlhe22- Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Arrowheads indicate incorrectly turning axons. ( C ) Quantification of axonal turning errors for Type II SGNs located in the basal turn of the cochlea. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (****p<0.0001). Scale bars are 100µm (A) and 20µm (B).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Control, Expressing, Immunolabeling, Standard Deviation

( A ) Differentially expressed genes (DEG) between Emx2 -Cre; Prox1 cKO and littermate control organ of Corti at P0 identified using DESeq2 with genes significantly downregulated in the cKO in red and those upregulated in blue. ( B ) Volcano plots restricted to the DEGs found in different groups of cochlear cell types with color density indicating the predicted contribution of that gene to the group’s overall transcriptome. Cell type groups and levels of gene expression are derived from public scRNAseq datasets generated from wild type tissue . ( C ) Average values of normalized Prox1 transcript alignments against all Prox1 exons compared to alignments against the two exons targeted in Emx2 -Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Error bars show standard deviation, and asterisks indicate statistical significance using Student’s t -test (****p<0.0001). ( D ) Changes in gene expression determined by DSeq2 for PCP, Nectin and Rac genes. ( E ) Changes in expression for genes signifying medial compartment and lateral compartment cell types. Red shading marks DEGs with statistically significant changes between genotypes (see also Supp. Table1&3). ( F ) Metascape Gene Ontology (GO) analysis of downregulated genes in the organ of Corti of Emx2- Cre; Prox1 cKOs.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Differentially expressed genes (DEG) between Emx2 -Cre; Prox1 cKO and littermate control organ of Corti at P0 identified using DESeq2 with genes significantly downregulated in the cKO in red and those upregulated in blue. ( B ) Volcano plots restricted to the DEGs found in different groups of cochlear cell types with color density indicating the predicted contribution of that gene to the group’s overall transcriptome. Cell type groups and levels of gene expression are derived from public scRNAseq datasets generated from wild type tissue . ( C ) Average values of normalized Prox1 transcript alignments against all Prox1 exons compared to alignments against the two exons targeted in Emx2 -Cre; Prox1 cKOs (n=4) and littermate controls (n=4). Error bars show standard deviation, and asterisks indicate statistical significance using Student’s t -test (****p<0.0001). ( D ) Changes in gene expression determined by DSeq2 for PCP, Nectin and Rac genes. ( E ) Changes in expression for genes signifying medial compartment and lateral compartment cell types. Red shading marks DEGs with statistically significant changes between genotypes (see also Supp. Table1&3). ( F ) Metascape Gene Ontology (GO) analysis of downregulated genes in the organ of Corti of Emx2- Cre; Prox1 cKOs.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Control, Gene Expression, Derivative Assay, Generated, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet:

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Expressing

( A ) Evaluation of peripheral axon turning in Sema5a KO mice compared to littermate controls at P0 by wholemount immunolabeling against NF-H. ( B ) Immunofluorescent images of VANGL2 wholemount organ of Corti viewed as 3D-reconstructions. Arrowheads mark examples of polarized enrichment of Vangl2 at the intercellular junctions between neighboring supporting cells in littermate controls (n=4) while asterisks mark examples of cells lacking this polarity in Emx2- Cre; Prox1 cKOs (n=3) at P0. Prox1 immunolabeling marks cell nuclei. Images are collected from the basal turn of the cochlea. Scale bars are 20µm.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Evaluation of peripheral axon turning in Sema5a KO mice compared to littermate controls at P0 by wholemount immunolabeling against NF-H. ( B ) Immunofluorescent images of VANGL2 wholemount organ of Corti viewed as 3D-reconstructions. Arrowheads mark examples of polarized enrichment of Vangl2 at the intercellular junctions between neighboring supporting cells in littermate controls (n=4) while asterisks mark examples of cells lacking this polarity in Emx2- Cre; Prox1 cKOs (n=3) at P0. Prox1 immunolabeling marks cell nuclei. Images are collected from the basal turn of the cochlea. Scale bars are 20µm.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Immunolabeling

( A ) Wholemount immunofluorescence images of PROX1 and the OPC protein CD44 in the developing organ of Corti shows the absence of CD44 expression in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) when evaluated at P2. ( B ) Immunolabeling for the IPC protein P75-NTR shows no changes in the apical projections of IPCs visualized at the reticular lamina or the number or distribution of IPCs at the nuclear layer. ( C ) Immunolabeling for the medial compartment supporting cell protein FABP7 shows an expansion of FABP7 into the lateral compartment in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) at P7. Bracket marks the boundaries of the lateral compartment based on the distribution of Phalloidin-stained OHCs. ( E ) fISH showing the distribution of the medial gene Trh relative to IHCs expressing fgf8 and expansion of Trh expression in Emx2- Cre; Prox1 cKOs (n=4) compared to littermate controls (n=5) at P0. Brackets mark the position of the IHC row. Scale bars are 20µm.

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Wholemount immunofluorescence images of PROX1 and the OPC protein CD44 in the developing organ of Corti shows the absence of CD44 expression in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) when evaluated at P2. ( B ) Immunolabeling for the IPC protein P75-NTR shows no changes in the apical projections of IPCs visualized at the reticular lamina or the number or distribution of IPCs at the nuclear layer. ( C ) Immunolabeling for the medial compartment supporting cell protein FABP7 shows an expansion of FABP7 into the lateral compartment in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=2) at P7. Bracket marks the boundaries of the lateral compartment based on the distribution of Phalloidin-stained OHCs. ( E ) fISH showing the distribution of the medial gene Trh relative to IHCs expressing fgf8 and expansion of Trh expression in Emx2- Cre; Prox1 cKOs (n=4) compared to littermate controls (n=5) at P0. Brackets mark the position of the IHC row. Scale bars are 20µm.

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Immunofluorescence, Expressing, Immunolabeling, Staining

( A ) Cochlear sections cut from P14 Emx2 -Cre; Prox1 cKOs and littermate controls labeled with the nuclear dye DAPI and imaged against background autofluorescence from a second channel. Asterisks mark the tunnel of Corti center and brackets the position of the three rows of OHC nuclei. ( B ) Area of the tunnel of Corti in cochlear sections for Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=4) at 3 positions along the length of P14 cochleae. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (***p<0.0001). ( C ) Deiters’ cell phalangeal processes immunolabeled with antibodies against β1β2 Tubulin reveals differences in their structural morphology and organization relative to OHCs in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=3) at P14. OHCs are labeled using antibodies against Oncomodulin. Scale bars are 10µm (A).

Journal: bioRxiv

Article Title: PROX1 Controls Cellular Patterning, Innervation and Differentiation in the Mouse Organ of Corti

doi: 10.1101/2025.04.15.649013

Figure Lengend Snippet: ( A ) Cochlear sections cut from P14 Emx2 -Cre; Prox1 cKOs and littermate controls labeled with the nuclear dye DAPI and imaged against background autofluorescence from a second channel. Asterisks mark the tunnel of Corti center and brackets the position of the three rows of OHC nuclei. ( B ) Area of the tunnel of Corti in cochlear sections for Emx2- Cre; Prox1 cKOs (n=4) and littermate controls (n=4) at 3 positions along the length of P14 cochleae. Error bars show standard deviation, and asterisks indicate statistical significance for differences between genotypes using Student’s t -test (***p<0.0001). ( C ) Deiters’ cell phalangeal processes immunolabeled with antibodies against β1β2 Tubulin reveals differences in their structural morphology and organization relative to OHCs in Emx2- Cre; Prox1 cKOs (n=3) compared to littermate controls (n=3) at P14. OHCs are labeled using antibodies against Oncomodulin. Scale bars are 10µm (A).

Article Snippet: Primary antibodies used in this study were: Rat anti-CD44 (1:1000, PFA, DSHB 5D2-27), Goat anti-FABP7 (1:1000, PFA, R&D Systems AF3166), Rat anti-Ki-67 (1:1000, PFA, Invitrogen 740008T), Rabbit anti-NFH (1:1000, PFA, Millipore AB1989), Chicken anti-NFH (1:500, PFA, Aves NFH), Goat anti-OCM (N-19) (1:250, PFA, Santa Cruz sc-7466), Rabbit anti-P75 (1:900, PFA, Millipore Sigma 07-476), Goat anti-PROX1 (1:300, PFA, R&D Systems AF2727), Rabbit anti-PROX1 (1:1000, PFA, Reliatech 102-PA32AG), Goat anti-SOX2 (1:200, PFA, Santa Cruz, 17320), Rabbit anti-SOX2 (1:1000, PFA, Millipore AB5603), Mouse anti-β1β2 TUBULIN (1:500, PFA, Sigma T8535), Rabbit anti-VANGL2 (1:1000, EMD Millipore ABN2242).

Techniques: Labeling, Standard Deviation, Immunolabeling