1. Msx1-Positive Progenitors in the Retinal Ciliary Margin Give Rise to Both Neural and Non-neural Progenies in Mammals 
Marie-Claude Bélanger, Benoit Robert, Michel Cayouette 
Developmental Cell 
Volume 40, Issue 2, Pages (January 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 40, 137-150DOI: (10.1016/j.devcel.2016.11.020)
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3. Figure 1
The Mouse CMZ Is Spatially Patterned into a Proximal and Distal Domain
(A–O) Immunostaining (A–C; G–I; M–O) and in situ hybridization (D–F; J–L) for CMZ markers at E14, E16, and P0. Otx1 and Cdo are expressed in the entire CMZ (A–F; brackets), whereas Msx1 expression is restricted to the proximal (P) region (G–L) and Aqp1 expression is confined to the distal (D) region (M–O).
(P–R) Immunostaining of retinal sections at E16 for βIII-tubulin, Msx1, and Aqp1, showing spatial patterning of the CMZ into a retinal domain (βIII-tubulin-positive), a proximal CMZ (Msx1-positive), and a distal CMZ (Aqp1-positive).
(S–X) Immunostaining of retinal sections at E14 for markers of retinal progenitors (Sox2), newborn neurons (Brn3b, βIII-tubulin), and proliferating cells (Ki67). Brackets identify the CMZ, which is devoid of retinal marker expression.
Scale bars, 50 μm.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
The Msx1-CreERT2 Mouse Line Is a Reliable Tool for CMZ Cell Lineage Tracing
(A) Schematic of the mouse lines used to generate the Msx1CreERT2;RYFP line and the tamoxifen injection strategy used in experiments presented in this figure.
(B–E) Immunostaining of retinal sections for YFP (B and C) and Msx1 (D and E). Some non-specific YFP signal is observed in the sclera (asterisk). Some YFP-positive cells are positive for Msx1 (cyan arrowhead), while others are negative (white arrowhead). Brackets cover proximal (P) and distal (D) CMZ.
(F–Q) Co-immunostaining for YFP and markers of retinal progenitors (Sox2), newborn retinal neurons (Brn3b, βIII-tubulin) or the CMZ (Cdo). YFP reporter-positive cells localize specifically in the CMZ (brackets).
(R) Quantification of the proportion of cells expressing the marker Msx1 in the proximal domain of the CMZ at E14 and E16. Data represent mean ± SEM.
Scale bars, 50 μm.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
The Msx1 Lineage Generates Non-pigmented Ciliary Epithelium and Retinal Progenitors at P0
(A) Schematic of the tamoxifen injection strategy used in experiments presented in this figure.
(B–I) Immunostainings for YFP in retinal sections of the Msx1CreERT2;RYFP mouse line at P0. YFP reporter-positive cells are found in the developing CB (B and C, arrowheads) and iris (B and C, arrows), and more specifically in the non-pigmented CE of the CB (D and E, arrowhead), as shown by co-immunostaining of YFP-positive cells with Aqp1 (F and G, arrowheads). Some YFP-positive cell clusters are also detected in the neural retina (H and I, arrowheads).
(J) Distribution of YFP-positive cells in the retina and CE at P0 (n = 4; 1,466 cells analyzed; mean ± SD).
(K and L) Co-immunostaining for YFP and the retinal progenitor cell markers Sox2 and Chx10 in retinal sections of the Msx1CreERT2;RYFP mouse. Arrowheads point to enlarged region showing overlapping signal.
(M) Proportion of YFP-positive RPCs and neurons in the retina (n = 9 animals; mean ± SD).
(N–S) Immunostainings for Cdo and YFP on retinal explants prepared from E14 Msx1CreERT2;RYFP mice or Cre-negative littermates (control) and cultured for 6 days in presence of 4-hydroxytamoxifen. Cdo staining defines the CMZ domain (N and Q). Msx1-derived progenies (YFP+) were detected in the Msx1CreERT2;RYFP samples (O), but not in controls (R).
Scale bars, 25 μm. See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4 The Msx1 Lineage Generates Retinal Neurons
(A) Schematic of the tamoxifen injection strategy used in experiments presented in this figure.
(B–I) Immunostaining for YFP on retinal sections co-stained with Hoechst or imaged under bright-field illumination. YFP-positive cells are found in the developing CB (B and C, arrowheads) and iris (B and C, arrows), and more specifically in the superficial layer of the non-pigmented ciliary epithelium (D and E, arrowheads), which is identified by the expression of Aqp1 (F and G). YFP-positive cell clusters are also detected in the neural retina (H and I, arrowheads).
(J–M) Quantifications of the repartition of YFP-positive cells in the neural retina and CB (J, n = 3; 2,102 cells counted; mean ± SD), cluster size distribution (K, n = 3 animals, 97 clusters counted; mean ± SEM), cluster distance from the pars plana (L, n = 3 animals, 97 clusters analyzed; mean ± SEM), and cluster cell composition (M, n = 3 animals, 97 clusters and 936 cells analyzed; mean ± SEM).
(N–Ah) Co-immunostainings for YFP and retinal cell type-specific markers S-opsin (cones), recoverin (Recov., rods), PKCα (bipolars), calbindin (amacrines), glutamine synthetase (GS, Müller glia), Pax6 (amacrines, ganglion cells), and Brn3b (ganglion cells). Arrowheads point to double-positive cells enlarged at the bottom left of panels.
(Ai) A cartoon illustrating the cell lineage-tracing results obtained.
ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; RGC, retinal ganglion cells; Hz, horizontal cell; Am, amacrine; BP, bipolar; Mü, Müller glia. Scale bars, 50 μm (B–I) and 25 μm (N–Ah). See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
The Msx1 Lineage Does Not Give Rise to Retinal Progenies at Postnatal Stages
(A–D) Immunostaining for YFP in retinal sections of the Msx1CreERT2;RYFP mouse line 7 days after injection of tamoxifen at different stages, as indicated. YFP reporter-positive cells are found in the CB, but not in the iris or retina (P0, n = 7; P7, n = 5; P21–P30, n = 4).
(E and F) Immunostaining for YFP in retinal sections of the Msx1CreERT2;RYFP mouse line 1 week (E, n = 7) and 5 weeks (F, n = 11) after tamoxifen injection at P4. YFP reporter-positive cells are found in the CB but not in the retina or iris.
ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 50 μm.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6 Numb Inhibits the Neural Retina Fate in the Msx1 Lineage
(A) Schematic of the mouse lines used to generate the Numb/NbL cDKOMsx1CreERT2 line and the tamoxifen injection strategy used in experiments presented in this figure.
(B–E) Immunostaining for YFP in P0 retinal sections from control (Msx1CreERT2;RYFP;Numbflox/+;NbLdel/del) or cDKOMsx1-CreERT2 mouse lines. Arrowheads point to retinal cell clusters. Scale bars, 50 μm.
(F and G) Quantifications of the number of YFP-positive retinal cell clusters in controls and cDKOMsx1-CreERT2 mouse lines at P0 (F, mean ± SEM; ∗∗∗Student's t test, n = 13 controls, n = 6 cDKO), and overall distribution of the size of YFP-positive cell clusters (G, n = 9 controls, n = 6 cDKO; mean ± SEM).
(H–M) Immunostaining for markers of the CMZ (Cdo, Aqp1), and RPCs (Chx10) in retinal sections of controls and cDKOMsx1-CreERT2. Arrowheads point to overlapping signal. Scale bars, 25 μm.
(N) Graph showing the distribution of YFP-positive cells in the retina or CMZ at P0 (mean ± SD; ∗∗multiple Student's t test; n = 9 controls, n = 6 cDKO).
(O and P) Retinal sections of cDKOMsx1-CreERT2 mice injected with tamoxifen at P4 show no cell clusters in the retina after 1 week (O) or 5 weeks (P). Scale bars, 50 μm.
See also Figures S2–S4.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
Numb Is Asymmetrically Inherited in Some CMZ Progenitor Divisions
(A) Quantification of the number of YFP-positive cells with apico-basal polarity or unpolarized in controls or cDKOMsx1CreERT2 at P0 (n = 9 controls, 6 cDKO; mean ± SD).
(B) Distribution of YFP-positive cell cluster positions along the peripheral retina in controls and cDKO (n = 13 controls, 6 cDKO).
(C–F) Average length of the Msx1 or Cdo domain in controls and cDKO at E16 (C, n = 4 controls, n = 3 cDKO; D, n = 3 controls, n = 3 cDKO) and P0 (E and F, n = 3 controls, n = 3 cDKO). All graphs show mean ± SEM.
(G–I) Immunostaining for Numb on E14 retinal sections showing enrichment at the apical membrane in the CMZ (arrowheads). High-magnification views of Hoechst-labeled mitotic cells in the CMZ dividing with a horizontal (H) or vertical (I) mitotic spindle relative to the apical surface. Dotted line identifies the cleavage plane.
Scale bars, 50 μm. See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2017 Elsevier Inc. Terms and Conditions