Differentiated horizontal interneurons clonally expand to form metastatic retinoblastoma in mice

Abstract

During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107, and p130) regulates cell-cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb(-/-);p107(+/-);p130(-/-) horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb(+/-);p107(-/-);p130(-/-) or Rb(-/-);p107(-/-);p130(+/-), retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. We hereby report that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.

Figure 1. Horizontal cells expand in the postnatal p107-single retina

(A) In Chx10-Cre;Rb^Lox/Lox;p107^+/−;p130^−/− mice, the entire retina lacked p130 and one copy of p107, and Rb was absent in mosaic stripes, which is consistent with the expression of Cre from the Chx10 promoter. (B,C) Immunofluorescent detection of horizontal cells in Rb;p130-deficient retinae with green nuclear counterstaining. Real-time RT-PCR (D), and immunoblot (E) analysis of p107 expression in p107-single (Chx10-Cre;Rb^Lox/Lox;p107^+/−;p130^−/−) and control (Rb^Lox/Lox;p107^+/−;p130^−/−) retinae. (F) Real time RT-PCR for p107 expression in p107-single and control retinae at P0 and P12. (G,H) Immunofluorescent detection of horizontal cells in P30 retinal sections and in dissociated cells (I,J) for p107-single and control littermates. Two other markers of horizontal cells (Prox1 and Lim1) were also increased at P30, as measured by dissociated cell scoring (K). Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 10 µm.

Figure 2. Horizontal cell proliferation in the postnatal mouse retina

P30 p107-single retinae were [³H]-thymidine labeled for 1 hour and then dissociated, immunostained for Lim1, calbindin, and Prox1 expression and overlaid with autoradiographic emulsion to detect the [³H]-thymidine (A). The proportion of immunopositive cells that incorporated [³H]-thymidine was scored for each antibody at 3 stages of development (B). 6 animals from each genotype were assessed at each stage, and each sample was scored in duplicate. The controls were Rb^Lox/Lox;p107^+/−;p130^−/− and Chx10-Cre;Rb^Lox/Lox;p130^−/− littermates. (C-E) Data on [³H]-thymidine labeling for individual animals at P30 is presented to illustrate the animal-to-animal variation with Chx10-Cre;Rb^Lox/Lox;p130^−/− littermates as a controls. (F) Co-immunolocalization of calbindin (green) and phospho-histone H3 (pH3) (purple) in P30 p107-single and control (Chx10-Cre;Rb^Lox/Lox;p130^−/−) littermates. Scale bars: 10 µm.

Figure 3. Proliferating horizontal cells are differentiated

(A-C) Representative images of the 50 nm serial sections for each horizontal cell imaged using transmission electron microscopy (TEM). Horizontal cell bodies are outlined in blue. A normal horizontal cell in a control littermate is shown in (A) and two dividing horizontal cells in M-phase are shown in (B,C) as indicated by the presence of condensed chromosomes (mitotic figures). The cell bodies were traced from images of 130-150 serial sections per horizontal cell and stacked to form a 3-d representation of the cells shown in (A-C) (D-F). The condensed chromosomes in B,C are outlined in pink in E,F. (G) Diagram of one type of conventional horizontal cell synaptic connection present on the cell body with input from a small diameter process from a neighboring horizontal cell. (H) TEM image of a typical horizontal cell body synapse on the surface of a dividing horizontal cell in the p107-single retina. The synaptic density (sd) is indicated by the bracket and the synaptic vesicles are shown by arrows. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer.

Figure 4. Dividing horizontal cells contribute to synaptic triads

Another type of conventional synapse made by horizontal cells is with rod and cone photoreceptor terminals via an invaginating process that is both presynaptic and postsynaptic. The horizontal cell neurite along with a bipolar cell dendrite invade the photoreceptor terminals as a bundle, termed a triad. (A-C) In the example shown here from the serial section analysis at increasing magnification, a dividing horizontal cell (blue) sends a thick process toward the outer plexiform layer consisting of photoreceptor terminals. (D) An illustration of a typical synaptic triad between horizontal neurites (blue), bipolar dendrites (yellow) and photoreceptor terminals such as a cone pedicle (shown). (E) In the section (#11) adjacent to that shown in (A-C) (#10) the neurite that extends to the outer plexiform layer forms a synaptic triad with a cone and bipolar cell. The diameter of the neurite is less than 100 nm and forms a synapse with a cone pedicle containing two synaptic ribbons in this section. The active zone of the synapse is marked by a pre-synaptic ribbon and a synaptic density. Synaptic vesicles in the horizontal cell neurite are indicated by arrows. Abbreviations: OPL, outer plexiform layer; sd, synaptic density; HC, horizontal cell; Bip, bipolar dendrite.

Figure 5. Clonal expansion of horizontal cells in the p107-single retina

(A) Adult p107-single mice received a single BrdU injection followed by an injection of [³H]-thymidine 48 hours later. Co-localization of BrdU, calbindin and [³H]-thymidine demonstrated that the horizontal neurons could progress through at least two rounds of S-phase. (B) P0 retinae from p107-single mice and their control littermates were infected with a replication-incompetent retrovirus that expressed nuclear LacZ (NIN-E) and cultured for 14 days. (C-F) Representative clones from control (C,D) and p107-single (E,F) retinae. (G,H) The proportion of large clones (≥4 cells) was increased in the p107-single retinae. (I) Co-immunolocalization of a horizontal cell marker (calbindin) and the nuclear β-gal reporter protein in large clones found in the p107-single retinae. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer;. Scale bars: 10 µm.

Figure 6. Live imaging of proliferating horizontal cells in p107-single retinae

2-photon live imaging of horizontal cells in P30 Gad67-GFP;Chx10-Cre;Rb^Lox/Lox;p107^+/−;p130^−/− retinae. The Gad67 promoter drives GFP expression in developing and mature horizontal cells but not progenitor or stem cells. (A) An example of 2 different horizontal cells viewed from the apical surface. One of these cells underwent cytokinesis during the 12 hour culture period (upper panels) and the other cell (lower panel) was unchanged (arrow) during the same time period. A tracing of the cell body at each timepoint is shown in the lower right of each panel in green. (B,C) In order to more clearly visualize the processes we used Imaris 5.0 to trace the processes and cell body in the dividing horizontal cell. The arrows indicate a clear separation of the two cell bodies shown with different colors (yellow and green) and different colored processes. Scale bars: 5 μm.

Figure 7. Metastatic retinoblastoma in p107-single mice

(A) By several months of age, p107-single mice developed bilateral retinoblastoma with ocular hypertrophy. (B-E) Histologic analysis revealed that the eyes were filled with tumor, and the tumor cells had invaded the ocular tissue, including the anterior chamber (D) and the optic nerve (E). The image shown in (C) is the boxed region in (B), showing classic retinoblastoma histopathology. When ocular rupture was imminent the mice were considered moribund and euthanized. (F) Plot of disease progression to moribund status in p107-single retinae and Rb;p130-deficient mice. (G,H) TEM analysis revealed that the p107-single retinoblastoma cells exhibited morphologic features of differentiated horizontal cells. (G) The tumor was filled with processes and synaptic densities (arrows), even adjacent to a proliferating tumor cell with a mitotic figure. A higher magnification view of the boxed region in (G) is shown in (H) to highlight the long, tapered process with relatively sparse synaptic vesicles; these traits are characteristic of horizontal cell processes. Open arrowheads in (H) indicate synaptic vesicles. Necropsy revealed that several of the p107-single mice had metastatic retinoblastoma (I-N). An example of metastatic retinoblastoma in the olfactory bulb of p107-single mice (J) compared to the littermate control (I). The metastatic retinoblastoma had invaded the surrounding bone marrow (M), which is a hallmark of human retinoblastoma. (N) A higher magnification view of the metastatic retinoblastoma showing that the tumor has the same histopathologic features as that of the original tumor in the eye (compare N to B and C). (O) PCR analysis of the knockout and wild type p107 allele in the p107-single retinoblastomas and the corresponding tail DNA from the same animals suggests that loss of heterozygosity (LOH) at the wild type p107 locus is not required for retinoblastoma progression. Scale bars in B,E: 50 μm. Scale bars in C,D: 25 μm.