Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation

Abstract

The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair.

Keywords: cones; fusion; neurodegeneration; retina; retinal dystrophy; transplantation.

Figure 1

Progressive Restriction of Chrnb4-EGFP Expression in Developing Photoreceptors throughout Retinogenesis (A–FII) Transversal sections of retinas at different developmental stages were collected from Chrnb4-EGFP reporter mice. (A–BII) EGFP detection is shown in round and elongated cells adjacent to the basal and to the most apical retinal region in E12 and E14 retinas. (B) White arrows indicate spindled cells in the central part of the retina expressing weak EGFP (probably progenitors). (C–CII) The EGFP expression is more restricted at the edge of the retinal apical side of E15.5 retinas. Few round cells at the basal retinal side still show EGFP expression (white arrows). (D–EII) In E18 and PN0 retinas, EGFP expression is exclusively restricted to the developing outer nuclear layer (dONL) in which photoreceptors differentiate. (F–FII) EGFP is expressed in a subset of photoreceptor cells at PN6, not in all photoreceptors. (F) The white arrow indicates the margin of the dONL. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; PN, post-natal day; E, embryonic day. Scale bars, 20 μm.

Figure 2

Investigation of the Chrnb4-EGFP-Positive Cell Proliferation during the Cone Generation Wave Pregnant females were labeled for 24 hr with EdU at E12, E13, E15, or E17. (A–DII) Transversal sections are shown of retinas at E13, E14, E16, and E18 stained for EGPF and EdU. (A–D) Merged pictures show EGFP (green) and DAPI nuclear staining (in blue). (AI–DI) EdU labelings (red) at different developmental stages are shown. (AII–DII) Merged pictures show EdU-positive cells (red) and EGFP-positive cells (in green). Dashed lines delimit the apical (Ap) from the basal (Ba) retinal regions used for counting in (F) and (G). (E–G) Percentage of double EdU-/EGFP -positive cells were counted over the EGFP-positive cells detected in the whole (E), apical (F), and basal (G) retina. (H–J) Percentage of double Ki67- and EGFP-positive cells counted over the total number of EGFP-positive cells confirms the results achieved with the EdU labeling. DAPI, nuclear staining (blue); Ba, basal; Ap, apical. (E–G and J) ANOVA with Tukey’s correction, ***p < 0.001; n = 4. Error bars correspond to SEM. Scale bars, 25 μm (A–D, H, AI–DI, and HI) and 15 μm (AII–DII and HII).

Figure 3

EGFP Co-expression with the Post-mitotic Cone Marker RXRγ at E12 (A–CII) Transversal sections of E12 retinas from Chrnb4-EGFP reporter mice. (A–AII) OTX2, essential for photoreceptor differentiation, is detected in elongated EGFP-positive cells adjacent to the apical retinal region. (B–BII) Double RXRγ- EGFP-positive cones localized at both the apical and basal side of the retina. (C–CII) EGFP co-expression with PAX6-positive cells adjacent to the basal retinal region is shown. (A–C) Dashed lines delimit the Ap from the Ba retinal region used for counting in (D). (D) Percentage of double EGFP- RXRγ-positive cells were counted over the EGFP-positive cells detected in the whole, apical-central, and basal retina. Error bars correspond to SEM. (AII, BII, and CII) High-magnification pictures show the corresponding white squares. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; n = 3. Scale bars, 50 μm (A–CI) and 15 μm (magnified insets).

Figure 4

E12-Derived EGFP-Positive Cells Generate Cones after Transplantation into Adult NOD/SCID Retinas (A) Flow cytometry analysis of EGFP-positive cells from dissociated E12 mouse retinas. Different EGFP intensities and cell complexities were detected and gated in blue, green, and yellow, respectively. All EGFP-positive cells were injected. (B–E) Example is shown of rosette-like structures formed in the sub-retinal space of NOD/SCID mice, 4 weeks after grafting (white arrows in B). (C) PAX6-positive (in red) and EGFP-negative cells in the sub-retinal space of adult retinas are shown. (D) Transplanted cells resulted to be KI67 negative. (E–EII) Double GNAT2- EGFP-positive cone cells. (EI–II) High magnification of cell graft is shown (E, white square). DAPI, nuclear staining (blue); ONL and INL, outer and inner nuclear layer; n = 5. Scale bars, 30 μm (B–E) and 15 μm (EI–II).

Figure 5

EGFP Expression in Newly Born Post-mitotic RXRγ-Positive Cones of E15.5 Retinas (A–CII) Transversal sections of E15.5 retinas from Chrnb4-EGFP mice. (A–AII) OTX2 detection is shown in EGFP-positive photoreceptors at the apical retinal side. (B–BII) Double RXRγ- EGFP-positive cones localized at both the apical and basal sides of the retina. (C–CII) EGFP co-expression with PAX6-positive cells adjacent to the basal retinal side is shown. (D) Percentage of double EGFP- RXRγ-positive cells was counted over the EGFP-positive cells detected in the whole, apical, and basal retina. (E) Percentage of RXRγ⁺-EGFP⁺ was counted over the RXRγ-positive cells detected in the basal and apical-central retina. Error bars correspond to SEM. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; n = 4. Scale bars, 20 μm (A–CII) and 8 μm (magnified pictures in insets).

Figure 6

Presumptive Cone Precursors and Newborn Cones from E15.5 Chrnb4-EGFP Retinas Generate the Appearance of EGFP-Positive Mature Cones after Transplantation into Adult NOD/SCID Retinas (A) Flow cytometry analysis of Chrnb4-EGFP-positive cell populations from dissociated E15.5 mouse retinas. Only the cells presenting the highest EGFP expression (P5 gated cells, in green) were transplanted. (B and C) Examples show (B) mature and (C) immature cone photoreceptors detected in adult recipient retinas. Note the presence of outer segments (white arrow, B). (D) Example is shown of a 2-month-old Chrnb4-EGFP-positive mouse retina stained for GNAT2 as control staining. Mature GNAT2-positive (E) and GNAT1-negative cones (F) 4 weeks after transplantation into NOD/SCID mice are shown. (B, E, and F) White arrows indicate external segment or synapsis. (G) Quantification is shown of morphologically mature cells observed in the adult retinas defined by the presence of the external segment or synapsis-like morphology. DAPI, nuclear staining (blue); ONL and INL, outer and inner nuclear layer; ES, external segment; n = 4 per experiment. (D) No significant variations of cell integration were revealed with two different ages of mouse recipients, n = 3. Error bars correspond to SEM. Scale bars, 20 μm (B and C) and 5 μm (D–F insets).

Figure 7

Evidences of Donor and Host Cell Material Exchanges after the Transplantation of E15.5 Chrnb4-EGFP Cells into the Adult DsRed Retinas (A–BV) Transversal sections of adult DsRed retinas transplanted with E15.5 Chrnb4-EGFP positive cells. (A–AV) An example is shown of a transplanted cell present in the host retina expressing only EGFP analyzed by the Manders’overlap coefficient visualized in the image as white spots. (B–BV) The same analyses were performed in a transplanted cell expressing EGFP and the host marker DsRed pointed out by the white arrow (BIV–V). (C–DI) Quantification of single- and double-labeled cells reveals that the large majority of EGFP-positive cells also express the host DsRed transgene reporter marker (C) and are dispersed in the ONL with a preference for the first two rows of photoreceptors (DI). EGFP-positive cells not containing DsRed are usually isolated (CI) and are also mainly located in the two first rows of photoreceptors of the outer part of the ONL (D). Error bars correspond to SEM. DAPI, nuclear staining (blue); ONL and INL, outer and inner nuclear layer; n = 6 injected mice. Scale bars, 15 μm (A–AII and B–BIII) and 5 μm (AIV and BIV–BV).

Figure 8

Chrnb4-EGFP Is Expressed in All Cone Subtypes of Adult Mouse Retinas (A–DII) Transversal sections of Chrnb4-EGFP mouse adult retinas. EGFP co-localization is shown with cone-specific proteins, such as GNAT2 (AII), RXRγ (BII), S-OPSIN (CII), and M/L-OPSIN (DII) (red labelings). The column AII–DII is a magnification of pictures from A to DI. ONL and INL, outer and inner nuclear layer; DAPI, nuclear staining (blue). Scale bars, 10 μm (A–D and AI–DI) and 20 μm (AII–CII).