Instrumentos de recogida de datos

We have discussed many intrinsic and extrinsic factors and the roles they play in retinal cell fate decisions, but there are other aspects to the story. For

example, the position and activity of a progenitor cell during its progression through the cell cycle may confer information about its final cell fate beyond its internal composition and the nearby environmental factors. It is well documented that the nuclei of cycling progenitor cells follow a predictable progression (Dyer and Cepko, 2001a). They migrate to the apical, or outer, surface of the retina to undergo mitosis. As they move forward through the first gap (G1) phase, these progenitor cells shift basally, toward the vitreal surface of the retina. At their basal nadir, progenitor cells pass through G1 and start the S phase of the cell cycle. They then move apically again in G2 phase before entering mitosis again at their apical peak. By studying the kinetic trends of progenitor cells, it was found that cells that travel much farther

basally than their neighbors tend to generate two neurons instead of another proliferating progenitor cell (Baye and Link, 2007). However, whether this phenomenon is linked to specific retinal fates or just neurogenesis in general remains to be determined.

In Drosophila and C. elegans, asymmetric distribution of cell fate

determinants prior to cell division is widely known to bias the cell fate decision- making process (Roegiers and Jan, 2004). Uneven distribution of mRNAs or proteins within a dividing parent cell can produce different fates between its two daughter cells. One identified factor that is asymmetrically localized at mitosis is Numb, a negative regulator of Notch. When Numb is misexpressed in a progenitor cell, it has a higher chance of producing rod photoreceptors at the expense of

amacrine cells, bipolar cells and Muller glia (Cayouette and Raff, 2003). Since Numb inhibits Notch signaling, these results are consistent with those where Notch itself was perturbed. Loss of Notch1 late in retinal development led to an increase in rod photoreceptors much like overexpression of Numb. Removal of Numb from the retina leads to a corresponding decrease in asymmetric cell divisions that produce a photoreceptor cell. However, symmetric divisions producing rod photoreceptors are increased, indicating that Numb is not generally required for this particular cell fate (Kechad et al., 2012). What are the determinants that drive rod photoreceptor fate in symmetric divisions versus asymmetric ones? Why does there need to be a difference in how these cells acquire their fates? The answers to these questions await further experimentation.

5.2 The cell cycle’s influence on cell fate

Underlying all of the stimuli (extrinsic and intrinsic) that influence retinal cell fate is the fact that this process must be coordinated with cell proliferation so that the retina produces the correct amount of total cells. Many extrinsic and intrinsic factors that we have discussed influence the cell cycle in addition to cell fate. Cell cycle regulators also aid in the cell fate decision-making process and perhaps, in some cases, help drive the specification of particular fates. One well- studied factor that is critically involved in the regulation of cell cycle exit is the retinoblastoma tumor suppressor protein (Rb) (Weinberg, 1995). Conditional knockout mice where Rb is deleted from the retina do in fact show a defect in the ability of progenitor cells to exit the cell cycle (Zhang et al., 2004). Interestingly, loss of Rb also leads to the production of fewer rod photoreceptors specifically (Zhang et al., 2004). This cell fate phenotype occurs only in rod photoreceptors and is

separate from the effect of Rb loss on the cell cycle. Instead, transcription factors that are important for rod development, such as Nrl, are absent in Rb deficient retinas (Zhang et al., 2004). These results demonstrate that factors can play

different roles in determining different cell fates and that, for some factors, their cell cycle roles are separate from other roles in cell fate determination.

Another family of cell cycle regulators, the cyclin kinase inhibitors (CKIs), can block the cell cycle at a number of places and, in some contexts, promote

differentiation. Overexpression of a CKI from Xenopus, p27Xic1, inhibits retinal progenitor cell proliferation, but also shows a specific cell fate effect. Muller glia

cells are increased, while bipolar cells are decreased (Ohnuma et al., 1999).

Interestingly, the domain that is responsible for kinase inhibition was not required to facilitate the cell fate switch, suggesting a potential novel mechanism (Ohnuma et al., 1999). Both gain and loss of the mouse homolog of this CKI, p27Kip1, indicate cell cycle phenotypes in a similar fashion as p27Xic1. However, in the case of the mouse, neither gain nor loss of function showed significant alterations in any retinal cell fates (Dyer and Cepko, 2001b). Perturbation of another related mouse CKI, p57Kip2, displays aspects of both a cell cycle and cell fate phenotype, much as in the case of Xenopus. When p57Kip2 was removed from embryonic retinal progenitor cells, these cells could not properly exit the cell cycle and subsequently died (Dyer and Cepko, 2000). On the other hand, when p57Kip2 was removed from postnatal retinal progenitor cells, there was an increase in amacrine cells (Dyer and Cepko, 2000). All of these experiments point to the precise regulation of intrinsic factors (transcription factors and otherwise) in response to extrinsic cues leading to a coordinated exit from the cell cycle and the acquisition of specific retinal cell fates. It is important to consider how all of these processes are co-regulated when

examining any experimental results dealing with cell fate determination.