White fur, red eyes – distinctive characteristics of an albino mouse, widely known to stem from a lack of melanin. Less well known is the phenotype of impaired vision, and there has been limited research exploring which molecular dysregulations in embryonic development lead to reduced visual function. In their recent publication, Slavi et al. from Columbia University examined embryogenic differences between pigmented and albino mice, to identify disruptions in retinal development. In albino animals – like in albino humans – the precise targeting of neurons from the retina to the brain is impaired during development. The study could pinpoint the deficit to a single culprit, CyclinD2, a known regulator of the cell cycle. In pigmented mice, the authors could replicate this targeting defect by disrupting the expression of CyclinD2, while in albino mice, the targeting defect could be prevented by enrichment with CyclinD2.
Impaired Equilibrium of Ipsilateral and Contralateral Retinal Ganglion Cells in Albino Mice
Retinal development is shaped by the precise temporal and spatial distribution of signaling proteins, ensuring proper embryogenesis. A center-to-periphery gradient dictates how development originates from the center and extends outward into the periphery by gradually incorporating additional cells. This is facilitated by the ciliary marginal zone (CMZ), the most peripheral region of the retina that contains retinal progenitor cells.
Seven retinal cell types arise from these progenitor cells, with their genetically driven specification following a precise spatio-temporal pattern. Retinal ganglion cells (RGCs) are part of the early born cells that specify first. They forward visual output from the retina to the brain, including onto the lateral geniculate nucleus (dLGN). One crucial part of development – underlying binocular spatial vision – is that some RGCs need to project ipsilaterally, i.e., to the brain hemisphere located on the same side as the eye, while others project contralaterally, i.e., to the other brain hemisphere. This balance is disturbed in albinism. Albino mice have a smaller population of ipsilateral RGCs in their retina, resulting in a disproportionally small ipsilateral input in the dLGN. This led Slavi et al. to investigate the molecular drivers causing the imbalance and to explore differences in the molecular program between pigmented and albino mice.
Molecular Markers Behind the Disrupted Balance
Through fatemapping of retinal progenitor cells, the authors found that the temporal-ventral CMZ possesses strong neurogenic potential, particularly for RGCs. In normal embryonic development, the CMZ initially generates Zic2-positive RGCs but transitions to Brn3a-expressing RCGs after 2 days. Zic2-positive cells project ipsilaterally, Brn3a-positive cells project contralaterally. In albino mice, two notable disruptions occur: Slavi et al found that the neurogenic output from the CMZ is reduced, with the number of RGCs being about 35% lower. Additionally, fewer cells expressed Zic2, resulting in decreased ipsilateral RGC projections.
Dysregulation of Cell Cycle CyclinD2 Prompts Contralateral Cell Fate
During development, cells need to grow and then divide into daughter cells, in a process known as cell cycle. The cell cycle consists of multiple phases called G1 (growth), S (DNA synthesis), G2 (further growth and preparation for mitosis), and M (mitosis, or cell division). The current phase of a cell can be identified by specific markers, i.e., proteins that are present in the cell during these phases. In the CMZ of albino mice, the researchers noticed a 35% reduction of cells in S and G2/M phases, even though the overall cell number was not affected. Instead, more cells were stuck in the G1-phase. This finding indicates that the G1- to S-phase transition is delayed and in turn, cells have longer differentiation periods.
The Columbia research group identified the culprit as the ccnd2 gene, the expression of which is reduced in albinos compared to pigmented mice. This gene encodes for CyclinD2, a cell cycle regulator that promotes G1- to S-phase transition. CyclinD2 downregulation in albino mice comes at a price: a delay in cell development, due to diminished concentration of CyclinD2, significantly impacts the cell fate. Fewer RGCs are born during a time where the developmental environment would initiate the expression of Zic2. When cells miss this time frame, the specification to ipsilateral RGCs is no longer possible, resulting in contralateral cell fate.
To corroborate this finding, the authors examined a strain of pigmented CyclinD2 Knockout (KO) mice. These mice showed similarly dysregulated RGC populations. In addition, the region of the dLGN receiving ipsilateral input was 20% smaller in comparison to conspecifics with proper CyclinD2 expression. These findings suggest that CyclinD2 is a crucial component of a CMZ-specific molecular machinery. Proper cell-cycle progression ensures ipsilateral RGC neurogenesis, and this is necessary for proper retinogeniculate targeting.
The Price of Disturbed Balance: Implications for Depth Perception
In patients suffering from albinism, stereoscopic vision is compromised, a phenomenon that previous research attributes to improper targeting of the dLGN, similar to the targeting deficits in CyclinD2 knockout and albino mice. To test whether stereoscopic vision is also impacted in these animals, the authors exposed the mice to a visual cliff paradigm – an experiment in which the animals walk on a transparent surface. Half of the surface lies directly on grid with black and white tiles, while the grid under the other half is located far underneath the surface, which forms the illusion of a cliff drop. Normally, mice avoid the deep pat of the board. The researchers observed difficulties in both albino and CyclinD2-KO mice in differentiating between the shallow and deep parts of the board, indicating that the mice suffer from impaired depth perception.
To prove that this is not caused by other binocular abnormalities, the scientists conducted additional testing with Striatech’s OptoDrum. Visual acuity and contrast sensitivity were comparable between all studied groups, suggesting that the failure to distinguish between shallow and deep parts of the board is caused by impaired depth perception.
Ca2+-Administration as a Game-Changer
All RGC targeting dysfunctions that albinism exhibits have one thing in common – they are linked to downregulated CyclinD2. Slavi et al. postulate that its upregulation should restore RGC balance and proper depth perception. The group’s innovative approach was to upregulate the transcriptional regulators of CyclinD proteins, fos and jun. When intracellular Ca2+ levels increase, both these regulators exhibit higher expressions.
The critical task at hand was now to evaluate whether the elevated levels of Ca2+ in the cells, leading to the upregulation of fos and jun, would indeed result in an increased expression of CyclinD2. Consequently, increased Ca2+ indirectly contributes to maintaining a healthy phenotype by stabilizing CyclinD2 levels.
To verify this hypothesis, pregnant mice from the albino, pigmented, and CyclinD2 KO strains were injected with BayK-8644, a substance that increases intracellular calcium through L-type Ca2+-channel activation. As the authors expected, the offspring from the albino strain showed upregulated CyclinD2 in the ventral CMZ, resulting in properly regulated cell cycle progression since the proportions of cells in different phases were more balanced. Further to this, the scientists discovered a 50% increase in Zic2+ ipsilateral RGCs and a 40% expansion in the size of the dLGN’s ipsilateral RGC input region.
Most importantly, the mice from the albino strain performed well in the visual cliff paradigm, indicating restored depth perception. CyclinD2-KO mice displayed none of these improvements, which underlines the significance of cyclinD2 presence during CMZ neurogenesis.
Potential for Future Studies
This extensive and thorough inspection of CMZ neurogenesis by Slavi et al. highlights the key role CyclinD2 plays for proper retinal development. It is particularly important in the critical window of Zic2-expression for acquiring ipsilateral cell fate. They also showed that properly formed CMZ-derived RGCs are essential for depth perception.
An interesting outlook for further research is the fact that albino mice have greater defects in all tested factors when compared to CyclinD2-KO mice. This suggests that there is an additional dysregulated route at play, additionally to CyclinD2 deficiency, that triggers depth perception anomalies. Moreover, there is only limited understanding regarding the precise mechanisms through which the absence of melanin induces CyclinD2 deficiencies and disrupts the normal regulation of the developmental pathway. Hopefully, future research will unveil further components of the affected networks and how they are connected to the lack of melanin in albino specimens.
Blog author:
Emilia Kawecka, University of Tübingen, Student Assistant at Striatech GmbH
Original paper:
Slavi N, Balasubramanian R, Lee MA, Liapin M, Oaks-Leaf R, Peregrin J, Potenski A, Troy CM, Ross ME, Herrera E, Kosmidis S, John SWM, Mason CA. CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism. Neuron. 2023 Jan 4;111(1):49-64.e5. doi: 10.1016/j.neuron.2022.10.025