Transcription factor Olig2 defines subpopulations of retinal progenitor cells biased toward specific cell fates - PubMed (original) (raw)

Transcription factor Olig2 defines subpopulations of retinal progenitor cells biased toward specific cell fates

Brian P Hafler et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression-profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix-loop-helix transcription factor, Olig2. The embryonic Olig2(+) RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The later, postnatal Olig2(+) RPCs also made terminal divisions, which were biased toward production of rod photoreceptors and amacrine cell interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases toward producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2(+) cells behaving as ganglion mother cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Olig2 is expressed by a subset of RPCs. (A) Microarray analyses using Affymetrix were conducted on 90 single retinal cells. The values for Olig2 in each single cell are shown as a heat map, with a value of >2,000 as the maximum intensity color and all other values scaled linearly. The RPCs were taken from across development, beginning at E12 and ending with P5, and examples of other retinal cell types are shown for comparison. Values are given in

Dataset S1

. Values were taken from refs. , , , and . (B) In situ hybridization was carried out for Olig2 probe at the indicated ages. The neuroblastic layer (NBL) and outer neuroblastic layer (ONBL) are the location of RPCs, with the inner neuroblastic layer (INBL) the location of differentiating neurons.

Fig. 2.

Fig. 2.

Olig2 expression history in the retina was analyzed by crossing an Olig2 knock-in mouse strain (Olig2-Cre+/−) (34) to a conditional tdTomato reporter strain (Ai9) (35). (A–C and E–G) Expression patterns of the reporter, tdTomato (red), and indicated cell type-specific markers (green) were compared at P20 using immunohistochemistry. Projected image stacks and single confocal plane images of regions labeled with dotted lines (Insets) are shown. (D) Drawing indicating the locations of the different retinal cell types within the retinal laminae. (H) Distribution of cells with Olig2-Cre expression history across distinct retinal cell types was quantified on image stacks (40X, 212 × 212 μm) collected from three independent retinas (red bars). The average percentage and SD of tdTomato+ cells that became each of the retinal cell types are shown. The overall frequency of each cell type in the mature retina is shown for comparison (gray bars) (41). (I) The percentage of each cell type with Olig2-cre expression history, as assessed by immunohistochemistry with the indicated markers, was quantified on image stacks collected from three independent retinas. The frequency of tdTomato+ photoreceptors was calculated as the percentage of outer nuclear layer (ONL) nuclei. OS, outer segements. (Scale bars, 32 μm.)

Fig. 3.

Fig. 3.

Morphology and composition of clones following postnatal infection of littermates of Olig2-tva-ires-cre+/− retinas with a virus using the EnvA protein or a control virus using the VSV-G envelope protein. Infection was carried out in vivo at P0 or P3 and analysis was carried out >P21. (A–E) Micrographs of clones generated by infection of Olig2-tva-ires-cre+/− at P3 using GFP retrovirus with EnvA, with each panel depicting a clone type. (F) A drawing of the retina, with examples of different cell types. (G and H) Quantification of all postnatally generated clones was carried out for multiple retinas. The overall frequency of each cell type across all clones are shown for P0 (G) and P3 (H) infections and comparisons were made for each cell type's frequencies between EnvA and VSV-G infections using the unpaired Student t test. *P <0.05. (See also Table 1 and

Dataset S2

). GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; R, rod photoreceptor. (Magnification: 40× in A–E.)

Fig. 4.

Fig. 4.

Clones generated by infection of embryonic Olig2-tva-ires-cre+/− retinas with an LIA retrovirus using the EnvA protein. Infection was at E13.5 via ultrasound-guided injections and analysis was conducted >P21. Clones from EnvA (A–D) were almost entirely one or two cells; those from a control virus were significantly larger (E) and resembled those published previously (4). (F) The distribution of clone sizes following infection with EnvA or the control virus are shown. (G) The frequency of one-cell clone types for EnvA or control virus infection are shown. (See also Table 2 and

Table S1

.) (Magnification: 40× in A–E.)

Fig. 5.

Fig. 5.

Heat map showing expression of bHLH genes in individual cells that express Olig2. The Olig2 gene and other members of its bHLH Clade E (56) as well as members of bHLH Clade A are shown. All members of these clades with a value of 1,000 in at least one of these cells are shown. Bright red is a signal value of >2,000 and all other values are scaled from 0 to 2,000. Values were taken from the Olig2+ subset of cells listed in

Dataset S1

and shown in Fig. 1. The values for this figure are shown in

Table S2

.

Fig. 6.

Fig. 6.

A model of the progression of RPCs over time. RPCs are able to proliferate to produce very large clones of hundreds to thousands of cells when randomly marked very early in retinal development. _Olig2_-expressing RPCs divide only once to produce two neurons, even early in development. The type of neurons produced by the _Olig2_-expressing RPCs varies over time, as indicated. The terminal divisions and the production of varied types of daughters is a behavior similar to that of the GMCs in the Drosophila ventral nerve cord. The model also represents the behavior of the more proliferative RPCs (shown, Upper). These cells also vary over time in terms of gene expression and have variable division patterns. The cells may also have programs of gene expression that direct them to make larger clones of particular daughter cell types, as yet undiscovered. Although not shown here, there are _Olig_2− RPCs that also make terminal divisions (e.g., those that make clones of MG and rods in the neonatal period).

References

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