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. 2022 Oct 16;23(20):12388.
doi: 10.3390/ijms232012388.

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis

Cindy L Olmos-Carreño  1   2 , María Figueres-Oñate  2 , Gabriel E Scicolone  1 , Laura López-Mascaraque  2
Affiliations

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis

Cindy L Olmos-Carreño et al. Int J Mol Sci. .

Abstract

Clonal cell analysis outlines the ontogenic potential of single progenitor cells, allowing the elucidation of the neural heterogeneity among different cell types and their lineages. In this work, we analyze the potency of retinal stem/progenitor cells through development using the chick embryo as a model. We implemented in ovo the clonal genetic tracing strategy UbC-StarTrack for tracking retinal cell lineages derived from individual progenitors of the ciliary margin at E3.5 (HH21-22). The clonal assignment of the derived-cell progeny was performed in the neural retina at E11.5-12 (HH38) through the identification of sibling cells as cells expressing the same combination of fluorophores. Moreover, cell types were assessed based on their cellular morphology and laminar location. Ciliary margin derived-cell progenies are organized in columnar associations distributed along the peripheral retina with a limited tangential dispersion. The analysis revealed that, at the early stages of development, this region harbors multipotent and committed progenitor cells.

Keywords: UbC-StarTrack; chick embryo; ciliary margin; clonal analysis; stem/progenitor retinal cells.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Expression of the UbC-StarTrack in vitro and in ovo. (A) General overview of neurospheres generation and co-transfection with an UbC-StarTrack integrable EGFP plasmid and a non-integrable vector expressing mCherry. (B) Epifluorescence and phase contrast merge image of co-transfected neurospheres. Positively labeled cells for both plasmids were evident in retinal neurospheres. Green (EGFP), red (mCherry) and yellow (merge). (C) Diagram showing procedure followed for the in ovo electroporation of an integrable UbC-StarTrack vector (expressing EGFP) along a non-integrable UbC-StarTrack vector (expressing mCherry). Labeled cells were checked either two or eight days after the IOE of the three constructs. (D) Epifluorescence and phase contrast merge image of a horizontal section passing throughout the ventral part of the central retina at E5.5-6 (HH28). Note the EGFP-labeled axons (arrows) heading toward the formation of the optic nerve (ON). Co-electroporation was performed in the peripheral retina and labeled cells are located next to the central retina. Scale bar 100 µm. (E) Labeled green (integrated), red (episomal) and yellow (both) cells are shown in short-term E5.5-6 (HH28). Inset is shown below both in green and red channels at higher magnification. (F) Epifluorescence and phase contrast merge image of a retinal section at E11.5-12 (HH38). The peripheral retina, which extends from the proximity of the CM (arrowhead) towards the central retina, is shown. Cell clusters are located in columns along the radial axis of the retina occupying a limited area in the peripheral retina (arrows). Nasal retina is upwards and temporal retina is downwards. Co-electroporation was performed in CM. Scale bar 500 µm. (G) In the long-term, E11.5-12 (HH38), just the integrated UbC-EGFP labeling persists. Merging green and red channels are successively shown.
Figure 2
Figure 2
In ovo electroporation of the UbC-StarTrack. (A) General overview of IOE of UbC-StarTrack into the CM of chick embryos at E3.5 (HH21-22) and their analysis at E11.5-12 (HH38). (B,C) Confocal merged images of retinal whole mount (B) and retinal horizontal section (C) from chick embryos at E11.5-12 (HH38) after IOE of UbC-StarTrack at E3.5 (HH21-22). (B) Cell clones are observed from the external surface of the retina and individual cells are visualized from a perpendicular or oblique incidence in whole mounts. (C) Labeled cells observed from a longitudinal incidence, form columnar associations between the internal (basal border) and external (apical border) limits of the neural retina in horizontal sections. UbC-StarTrack labels cells in both the cytoplasm and nucleus, allowing the cells somas and processes to be seen clearly. (D) Graph representing the relationship between the number of clones and the number of cells detected in each of the five analyzed chick embryos. (E) Confocal images showing the characteristic morphology of chicken retinal cells in horizontal sections at E11.5-12 (HH38) after E3.5 (HH21-22) IOE with UbC-StarTrack. A labeled amacrine cell (a), a horizontal cell (b), a radial glia (c), a photoreceptor (cone) (d) and a retinal ganglion cell (RGC) (e) are shown. (F) Percentages of labeled cells located in the ONL, INL and CGL in each of the five chicken embryos analyzed. (G) Confocal merge images of all XFPs showing examples of sibling cells arranged in columns in the embryo retina. (H) Confocal image merge of all XFPs is shown on the left side. The inset shows an apparent homogenous clone in a single column. However, when analyzing the fluorescent expression at each confocal channel separately it is shown that the column is formed by two different clones: (1) a trilaminar clone formed by cells expressing mCerulean and mT-Sapphire in the cytoplasm (middle) and (2) a monolaminar (GCL) clone formed by cells expressing mCerulean, mT-Sapphire and EGFP in the nucleus (right).
Figure 3
Figure 3
Retinal layer distribution and the clonal size of cell-derived progeny after targeting CM progenitors. (A) General overview of layer distribution of clonally related cells. Retinal cell types are represented on the left side, whereas the seven group classifications depending on the clone layer distribution are represented on the right side. (B) Merge the confocal images of a trilaminar clone (a) with cells located at different retinal layers (ONL, INL and GCL), a bilaminar clone (b) formed by a photoreceptor, three horizontal cells and six undefined cells and a layer-restricted clone (c) formed by three RGCs. Circles represent cells belonging to the same clone. Detailed expression of XPF defining the color code of sibling cells as well as the schematic representations are shown in (c). (C) Pie chart displaying the proportions of the different layer distribution of sibling cells. (D) Venn diagram representing the number of cells belonging to clones formed by different combinations of cell types, such as RGCs, photoreceptors and INL cells. (E) Size of the clones distributed along the different retinal layers. Clones containing cells at all three layers were the larger ones. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; RGC: Retinal ganglion cell; Photo: Photoreceptor (cone).
Figure 4
Figure 4
Progenitor potential and clonal heterogeneity deciphered using UbC-StarTrack. (A) Schematic representation of the proportions of clones according to their cell type composition: mixed clones included at least two differentiated cell types, uniform clones only one and undefined clones were composed by cells for which an identity could not be designated. (B) A mixed clone was formed through three differentiated cell types: two photoreceptors, a horizontal cell and three RGCs. (C) Examples of mixed clones formed by both photoreceptors and RGC. Sibling cells could appear in the same horizontal retinal slice (a) or in adjacent ones (b). (D,E) Uniform clones were those composed by only one differentiated cell type, as photoreceptors (D) or RGCs (E). Cells from each clone are circled at each image. For all cells, the details of the XFPs expression is shown. Clone color codes are represented by the schematics. (F) Proportion of the clones that included RGCs in the five analyzed replicates. (G) Schematics displaying the progenitor potential deciphered by the clone composition. Mixed clones were derived from bi- or multipotent progenitors, whereas uniform clones arose from committed progenitors at E3.5 (HH21-22). There was a proportion of clones for which progenitor potential was not assessed due to the presence of sibling cells within the clone for which cell identity could not be found at the time of the analysis at E11.5-12 (HH38). (H) Scatterplot showing the accumulation of clones, named counts, of a determined size. Blue line shows the predicted fitting by local polynomial regression, and the grey shadow represents the standard error of the predicted model when using a 0.95 confidence interval. (I) Density chart comparing the distribution of the clonal sizes at E11.5-12, depending on the nature of the E3.5 RPCs: bi-multipotent, committed or undefined.
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