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First published online October 30, 2006
doi: 10.1242/10.1242/dev.02598

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Ptf1a determines horizontal and amacrine cell fates during mouse retinal development

Yoshio Fujitani^1,*,,, Shuko Fujitani^1,2,*, Huijun Luo³, Feng Qiu³, Jared Burlison⁴, Qiaoming Long⁴, Yoshiya Kawaguchi⁵, Helena Edlund⁶, Raymond J. MacDonald⁷, Takahisa Furukawa⁸, Takashi Fujikado², Mark A. Magnuson⁴, Mengqing Xiang³ and Christopher V. E. Wright^1,

¹ Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University Medical School, Nashville, TN 37232-8240, USA.
² Department of Visual Science, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
³ Center for Advanced Biotechnology and Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA.
⁴ Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, USA.
⁵ Department of Surgery and Surgical Basic Science, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan.
⁶ Umeå Center for Molecular Medicine, University of Umeå, SE-901 87 Umeå, Sweden.
⁷ Department of Molecular Biology, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
⁸ Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.

View larger version (137K): [in a new window]   Fig. 1. Spatial and temporal expression patterns of Ptf1a protein during mouse retinogenesis. (A-G) Retinal sections from the indicated developmental stages were immunostained with an anti-Ptf1a antibody (red) and nuclei were counterstained with YoPro-1 (green). The expression of Ptf1a commences at E12.5 in the central retina (B), peaks around E14.5 in the outer neuroblastic layer (C) and continues to be strong around E18.5 (E). It begins to be downregulated significantly at P2 (F) and eventually disappears around P6-P7. At P6, Ptf1a expression is completely downregulated from the central retina. (H) Retinal sections from E18.5 Ptf1aCre/Cre embryos immunostained with an anti-Ptf1a antibody. (I) Retinal sections from BrdU-labeled E14.5 embryos were double immunostained with anti-Ptf1a (red) and anti-BrdU (green) antibodies. There was no colocalization between Ptf1a-immunoreactive nuclei and the S-phase nuclei immunoreactive for BrdU. Scale bar: 50 µm. Abbreviations: R, retina; L, lens; onbl, outer neuroblastic layer; inbl, inner neuroblastic layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer.

View larger version (127K): [in a new window]   Fig. 2. Lineage tracing analysis in the retina. All retinal sections were examined at 3 weeks of age. (A) X-gal staining of retinal sections from Ptf1aCre/+;R26R pups counterstained with Fast Red. No ß-gal activity was detected in the ONL. (B-L) Ptf1aCre/+;R26R-EYFP retinal sections double-labeled with anti-GFP (green) and the indicated antibodies (red). Brn3b (ganglion cells, B); Chx10 (bipolar cells, C); recoverin (rods and cones, D); calbindin (horizontal and amacrine cells, E); syntaxin (horizontal and amacrine cells, F); Gad65 (GABAergic amacrine cells, G); GABA (GABAergic amacrine cells, H); calretinin (amacrine subclass and ganglion cells, I); tyrosine hydroxylase (Th, dopaminergic amacrine cells, J); glycine transporter 1 (Glyt1, glycinergic amacrine cells, K); Cralbp (RPE and Müllar glia, L). Arrows in A,C,E indicate representative mature horizontal cells; arrowheads in E,H indicate non-colocalized cells. Scale bars: in A, 25 µm for A; in L, 25 µm for B-L. Abbreviations: INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; GCL, ganglion cell layer; IPL, inner plexiform layer.

View larger version (120K): [in a new window]   Fig. 3. Effect of targeted Ptf1a deletion on the formation of different retinal cell types. (A-P) Retinal explants were prepared from wild-type (A,C,E,G,I,K,M,O) and Ptf1aCre/Cre (B,D,F,H,J,L,N,P) embryos at 18.5 and then cultured for 2 weeks. The neuronal subtypes were examined by immunohistochemistry (red) for rhodopsin (rods; A,B), Chx10 (bipolar cells; C,D), calbindin (horizontal and amacrine cells; E,F), syntaxin (horizontal and amacrine cells; G,H), calretinin (amacrine cells and RGCs; I,J), GABA (GABAergic amacrine cells; K,L), tyrosine hydroxylase (Th, dopaminergic amacrine cells; M,N) and Cralbp (RPE and Müller glia; O,P). In retinal explants, there is postnatal degeneration of pre-existing RGCs, owing to RGC axon severance resulting from optic nerve transection during tissue preparation. During explant culture, folding of retinal tissue usually results in mirror-image-like duplication on the periphery (broken lines in L and P). Arrowheads in L,P indicate rosette-like structure formation. Scale bars: 25 µm. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer.

View larger version (101K): [in a new window]   Fig. 4. Defects in laminar structure and cell specification in Ptf1a mutant retinas. (A-F) X-gal staining of retinal sections from E18.5 Ptf1aCre/+;R26R embryos (A) and Ptf1aCre/Cre;R26R embryos (B) counterstained with Fast Red. (C,E) Higher-magnification images of the areas in the solid and broken rectangles in A, respectively. (D,F) Higher-magnification images of the areas in the solid and broken rectangles in B, respectively. Arrows in E indicate representative developing horizontal cells and the bracket in E indicates normal location of maturing amacrine cells in the E18.5 retina. The bracket in F indicates lacZ-positive nerve fiber layer, which is thicker than the control lacZ-negative nerve fiber layer (indicated by the small bracket in E). (G-J) Hematoxylin and Eosinstained sections of wild-type and mutant retinas at E18.5. Higher-magnification pictures of G and H are shown in I and J, respectively. Arrowheads in H indicate a front-line of cells in the outer neuroblastic layer invading into the GCL. (K,L) Wild type and Ptf1aCre/Cre E18.5 retinal sections were immunostained with an anti-Brn3b antibody. Scale bars: in B, 100 µm for A,B; in H, 50 µm for E-H; in D, 25 µm for C,D; in L, 25 µm for K,L; in J, 10 µm for I,J. (M) Top, increased ganglion cell number in Ptf1aCre/Cre retinas. Bottom, no significant change in central retinal thickness in the mutant. Histograms represent the mean±s.d. of measurements from five animals of each genotype. *P<0.001. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer.

View larger version (79K): [in a new window]   Fig. 5. Ptf1a lineage-labeled cells are transfated to RGCs in Ptf1a-deficient embryos. (A-F) An anti-GFP antibody was used to detect YFP in E18.5 Ptf1aCre;R26R-EYFP retinas and an anti-Brn3b antibody was used to detect differentiated ganglion cells. YFP acts as a lineage marker for cells that have expressed the Ptf1a locus. YFP (green) does not co-localize with Brn3b (red) in the presence of Ptf1a, but they do co-localize in Ptf1a-null embryos (compare E with F). (G-L) An anti-GFP antibody was used to detect citrine, a modified version of GFP, in E18.5 Ptf1aCitrine embryos. Citrine serves as a lineage marker for cells that are expressing or have expressed the Ptf1a locus during the past couple of days. Citrine (green) does not co-localize with Brn3b (red) in the presence of Ptf1a, but they do co-localize in Ptf1a-null embryos (compare K with L). Arrows in F indicate Brn3b+YFP- `newly specified RGCs' in the onbl. Arrows in L indicate Brn3b+citrine+ Ptf1a lineage-labeled RGCs. Scale bar: 50 µm. Abbreviations: onbl, outer neuroblastic layer; INL, inner nuclear layer; GCL, ganglion cell layer.

View larger version (51K): [in a new window]   Fig. 6. Downregulation of Ptf1a expression in the Foxn4-/- retina. (A) Real-time qRT-PCR analysis of Ptf1a, Foxn4 and Gapdh mRNA levels in E14.5 Foxn4+/- and Foxn4-/- retinas. Each histogram represents the mean±s.d. for four retinas. (B-E) Retinal sections of the indicated stages from wild-type and mutant mice were hybridized with a Ptf1a riboprobe. The absence of Ptf1a signals from Foxn4-/- retinas (C,E). Scale bar: 50 µm in B,C; 25 µm in D,E. Abbreviations: onbl, outer neuroblastic layer; inbl, inner neuroblastic layer.

View larger version (116K): [in a new window]   Fig. 7. Effect of targeted Ptf1a deletion on the expression of retinogenic factors. (A-J) Sections from E14.5 (A-H) or E18.5 (I,J) wild-type and Ptf1aCre/Cre retinas were hybridized with a Ptf1a (A,B), Foxn4 (C,D), Math3 (E,F), Neurod1 (G,H) or Math5 (I,J) riboprobes. Similar levels of Foxn4, Math3 and Neurod1 expression are seen in wild-type and null retinas. Math5 expression is upregulated in the null retina at E18.5. (K-N) Sections from E14.5 (K,L) or E18.5 (M,N) wild-type and null retinas were immunostained with antibodies against Prox1 (K,L) or Lim1 (M,N). Prox1 and Lim1 signals are absent from Ptf1aCre/Cre retinas (L,N). (O,P) Cells undergoing apoptosis (green) were TUNEL-labeled in wild-type and null retinas at E18.5. A moderate increase of apoptotic cells is seen in the Ptf1a-null retina. Sections in M-P were weakly counterstained with Yo-Pro-1 (M,N) or DAPI (O,P). Scale bar: 50 µm. Abbreviations: R, retina; L, lens; onbl, outer neuroblastic layer; GCL, ganglion cell layer; IPL, inner plexiform layer.

View larger version (29K): [in a new window]   Fig. 8. A model for directed differentiation of retinal progenitors towards horizontal and amacrine fates by the Foxn4-Ptf1a pathway. (A) Foxn4-expressing cells in the outer neuroblastic layer represent progenitors biased towards horizontal and amacrine fates (Li et al., 2004a). Subsets of Foxn4+ cells activate Ptf1a after exit from the cell cycle. Activation of Ptf1a depends on the Foxn4 function (Fig. 6). Ptf1a-expressing post-mitotic cells differentiate to horizontal or amacrine cells. (B) In the absence of Ptf1a, retinal precursors, which would normally activate Ptf1a, adopt ganglion cell fates or otherwise undergo apoptotic cell death (referred also in C). The cell fate switch to RGC takes place relatively quickly in the onbl after exit from the cell cycle. Green, horizontal cells; blue, amacrine cells; red, RGCs. Dark-red cells in B represent RGCs originating from Ptf1a-lineage-labeled precursor populations. (C) Foxn4 and Ptf1a control the genesis of amacrine and horizontal cells in cooperation with other retinogenic factors. Foxn4-expressing progenitors require sequential activation of Ptf1a and Prox1 for the genesis of horizontal cells. Foxn4 confers progenitors with the competence for the genesis of amacrine cells by activating the expression of Ptf1a, Math3 and Neurod1. Activation of Math3 or Neurod1 does not require Ptf1a function. The rod, cone, bipolar, ganglion and Müller cells are likely to be largely derived from the Pax6+ Foxn4- progenitors via activation of other retinogenic factors, which are not shown here for simplicity. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer.