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. 2004 Jan 7;24(1):229-37.
doi: 10.1523/JNEUROSCI.2980-03.2004.

Persistent progenitors at the retinal margin of ptc+/- mice

Ala Moshiri  1 , Thomas A Reh
Affiliations

Persistent progenitors at the retinal margin of ptc+/- mice

Ala Moshiri et al. J Neurosci. .

Abstract

The hedgehog signaling pathway is a key regulator of neural development, affecting both proliferation and differentiation of neural progenitors. Sonic hedgehog (Shh) is a mitogenic factor for retinal progenitors in vitro. To determine whether this signaling system is important in vivo for regulating retinal progenitor proliferation, we analyzed mice with a single functional allele of the Shh receptor patched (ptc). We found that ptc+/- mice had increased numbers of neural progenitors at every stage of retinal development that we examined. In addition, these mice had persistent progenitors at the retinal margin for up to 3 months of age, reminiscent of the ciliary marginal zone of lower vertebrates. To test whether the progenitors at the retinal margin of ptc+/- mice could be induced to regenerate retinal neurons in response to damage, we bred ptc+/- mice onto a retinal degeneration background (pro23his rhodopsin transgenic) and labeled newly generated cells with combined immunohistochemistry for bromodeoxyuridine and retinal neuron and photoreceptor-specific markers. We found newly generated neurons and photoreceptors at the retinal margin in ptc+/-;pro23his mice. We propose that the Shh pathway may act as a regulator of both prenatal and postnatal retinal growth.

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Figures

Figure 1.
Figure 1.
ptc+/- mice have a morphologically normal retina. A-C, P7 retinal sections from ptc+/- mice show typical retinal lamination indistinguishable from age-matched wild-type littermates (D-F). GC, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 2.
Figure 2.
ptc+/- mice have increased retinal proliferation during development. A, The number of M phase PH3+ cells was consistently greater in the ptc+/- mice when compared with wild-type littermates. The average number of PH3+ cells per retinal section is plotted. B, The percentage of increase in PH3+ cells per section is shown, with wild-type normalized to one. The percentage of increase of M phase cells increases with postnatal age. C, Cell cycle analysis by flow cytometry reveals ptc+/- animals have an increased percentage of cells in S phase at each postnatal age studied. D, The total retinal thickness of P3 ptc+/- animals is the same as wild-type littermates, although the outer nuclear layer thickness (stained with rhodopsin) and the number of ganglion cells (stained with Brn3.2) at P3 are both significantly increased. *p < 0.05; **p < 0.005.
Figure 3.
Figure 3.
ptc+/- mice have extended proliferation at the retinal margin. A, P16 retina from ptc+/- mice has PH3-positive mitotically active cells (arrow). No PH3 labeling was observed in wild-type littermates. Sections of the peripheral margin of ptc+/- (B) and wild-type (C) mice at age P20, labeled for BrdU. The ptc+/- mice show proliferating cells at the retinal margin (arrow), whereas the wild-type mice have labeled cells nearly exclusively in the ciliary body. More frequent BrdU injections were made to analyze the number of dividing cells at the retinal margin. Animals received injections every 2 hr for 48 hr during P14-15 and were killed on P20. ptc+/- mice had more BrdU+ cells in the retina and in the nonpigmented epithelium of the ciliary body than wild-type littermates (D). Inset, Schematic diagram of the location of the BrdU+ cells that were counted. GC, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. *p < 0.05; **p < 0.005.
Figure 4.
Figure 4.
Proliferating cells at the retinal margin of ptc+/- mice are retinal progenitors.A, ptc is a retinal progenitor marker expressed in nearly all progenitors in the neuroblast layer of P0 retina. Proliferating cells at the retinal margin (B, D, arrows) of P20 ptc+/- mice express the progenitor markers ptc (B) and Chx10 (D), whereas wild-type littermates do not (C). Wild-type mice have very few BrdU-incorporating cells at the margin of the retina. Sections of the peripheral margin of wild-type (E) and ptc+/- (F) mice at age P20, labeled for BrdU (green), show a population of dividing cells at the ptc+/- retinal margin (F). The same sections stained with antibodies against the neural progenitor marker nestin (red) show a distinct population of labeled cells at the ptc+/- (F) retinal margin, but not in wild-type (E) littermates. These nestin-positive cells in ptc+/- mice are coincident with BrdU incorporation, identifying the proliferating cells at the ptc+/- retinal margin as undifferentiated progenitors (F, arrow). ptc+/- mice continued to incorporate (G) BrdU (green) and (H) Ki67 (red) at the retinal margin even at P90 after daily injections of BrdU on P85-89. These BrdU-positive cells also express nestin (arrow in G), and other nestin (red)-expressing progenitors are present at the retinal margin of these mice. No BrdU, nestin, or Ki67 was detected in the retinas of wild-type P90 littermates (data not shown). The asterisk indicates staining of an artifactual space between the outer nuclear layer and retinal pigment epithelium as a result of sectioning. GC, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 5.
Figure 5.
Progenitors from the retinal margin of ptc+/- mice proliferate in response to growth factors and differentiate into neurons. Anterior neural retinas from P10 ptc+/- mice and wild-type littermates were grown in dissociated cultures for 5 d. A, Ninety-five percent of BrdU-positive (green) cells in these cultures express the progenitor marker nestin (red). B, Other BrdU-positive cells had neuronal morphology and express neuronal genes, such as Tuj-1 (red), as confirmed by confocal microscopy. C, Similar to CMZ-derived progenitors in other species, progenitors from P10 ptc+/- mice proliferated in response to growth factors such as EGF and basic FGF during the 5 d culture period. *p < 0.05; **p < 0.005.
Figure 6.
Figure 6.
pro23his mice undergo severe photoreceptor degeneration. pro23his mice transgenically overexpress a mutated rhodopsin gene that causes severe photoreceptor death (Olsson et al., 1992). A, Recoverin (red), a photoreceptor marker, labels the outer nuclear layer (ONL). pro23his animals generate an ONL, which degenerates to a monolayer by P20. B, P20 pro23his animals that received injections of BrdU daily from P13-15 did not have increased proliferation at the retinal margin compared with wild-type littermates. Arrow indicates BrdU (green)-positive cells in the ciliary body epithelium. GC, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 8.
Figure 8.
Cells in the ptc+/- retinal margin proliferate more in response to injury. The number of BrdU-labeled cells at the retinal margin was quantified in each of four mouse genotypes at age P20. Concomitant photoreceptor death stimulates progenitors at the margin of ptc+/- mice to divide. *p < 0.05; **p < 0.005.
Figure 7.
Figure 7.
ptc+/- progenitors proliferate more, upregulate progenitor markers, and regenerate neurons in response to injury. ptc+/- and pro23his mice were mated to produce animals with both genotypes. ptc+/-;pro23his mice (P20) continued to express the progenitor markers at the retinal margin. A, ptc expression is markedly increased (arrow) at the retinal margin. Mice received injections of BrdU on P13-15. At P20, tissue was analyzed for the presence of BrdU-labeled proliferating cells at the retinal margin and with additional immunohistochemical markers to identify newly generated photoreceptors. Tissue was stained with markers for BrdU (green) and recoverin (red) to label photoreceptors. Progenitors at the margin (arrow) of ptc+/-;pro23his mice (B) proliferate more than in ptc+/- alone. Examples (C) are shown of double-labeled, newly generated photoreceptors (arrows). Double-labeled cells (arrow) were confirmed by confocal microscopy (F, F′). BrdU (green)-positive progenitors are capable of differentiating into other retinal neurons in addition to photoreceptors, including Tuj-1 (red)-expressing cells (D, E, E′). GC, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
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