Ординатура / Офтальмология / Английские материалы / Retinal Development_Sernagor, Eglen, Harris, Wong_2006

The competence model (see Figure 5.3a) put forward to explain the majority of the above results suggests that the intrinsic competence of progenitor cells to respond to cues changes with time such that at any given point they have a limited repertoire of cell fates available for their progeny (Watanabe and Raff, 1990; Livesey and Cepko, 2001; Rapaport et al., 2001). This concept could have many explanations at the molecular level. The receptor composition and downstream events conveying extracellular signals may change with time, as for example the GlyRα2 expression is upregulated in late progenitors enabling rod production (Lillien, 1995; Young and Cepko, 2004). Extracellular signals may regulate the competence to produce different fates by modulating the activity of intrinsic cell fate determinants, as in the case of Notch signalling. The secreted molecule Gdf11 inhibits the production of RGCs by limiting the temporal window of Math5 expression. Gdf11 mutant mice exhibit an increase in RGCs, a prolonged Math5 expression and a delay in the expression of NeuroD and Mash1, bHLH genes that favour later neuronal fates, without any changes in progenitor proliferation (Kim et al., 2005). Glycogen synthase kinase-3β (GSK 3β) phosphorylates and inactivates NeuroD in early progenitors; a GSK 3β-insensitive form of NeuroD transfected early on favours ganglion cell production, not amacrine (Moore et al., 2002). Activity of GSK 3β can be modulated by extrinsic cues such as Wnt signalling and tyrosine kinase receptor activation, thus providing a possible link between a changing environment and the role of intrinsic factors. Finally, the cross-regulatory effects of transcription factors mentioned above could alter their relative expression levels with time, changing the favoured cell fates. These different aspects of cellular competence appear to change in parallel with

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Figure 5.3 (cont.) (a) The competence model: progenitor cells are initially equivalent and competent to produce only early-born cells. The two progenitors produce different early-born neurons by virtue of their exposure to different environments. For example, the top progenitor might receive much less Notch signalling and hence give rise to two ganglion cells, whereas the bottom one gives rise to another progenitor and an amacrine cell. In late histogenesis, the remaining progenitor has different intrinsic characteristics and also receives signals from a changed environment, thus becoming competent to give only late-born cells. (b) The progenitor mosaic model: at the beginning of differentiation the progenitor cells have already diversified, and they step through a series of preprogrammed differentiated cell production. In this case, our two progenitors differ greatly in their intrinsic programmes, for example the top one but not the bottom strongly expresses at the beginning of differentiation transcription factors that favour a RGC fate. The environment regulates cell survival and the maturation of the differentiated phenotype but not the cell fate decisions. (c) Stochastic choice model: intrinsic factors bias each progenitor towards the generation of particular cell types, with different probabilities for each type. Random fluctuations in the activity of different fate-influencing molecules can be amplified so that initially similar progenitors can produce different clones and the overall cell numbers and clonal compositions are determined by the probability for generation of each type. In this case again the environment does not play a significant role in fate determination.

A combination of the above models could explain the available data. For example, a progenitor population with some heterogeneity may initially be dependent on extrinsic cues for the stochastic production of early cell types and diversification of the progenitor pool. With time the now diversified progenitor population becomes more and more dependent on intrinsic cues.

a dynamic environment, as illustrated above, but it is not clear what the relative importance of intrinsic fate determination programmes is compared to external cues.

5.6 Contribution of progenitor heterogeneity to cell fate determination

Recent work puts into doubt the significance of the extrinsic cues, and consequently of the competence model. Cayouette et al. (2003) found that dissociated progenitors of mouse E16/17 retinas go on to produce progeny in similar proportions and clonal compositions to the in vivo progenitors. They suggested that extrinsic cues are not critical for the cell fate determination, which is dictated solely by an intrinsic programme, different for each progenitor, that is already in place by at least as early as E16 (see Figure 5.3b). The multiple effects of the extrinsic factors described previously could simply be fine-tuning mechanisms, regulating proliferation and differentiation in space and time (e.g. by local, transient feedback inhibition) and influencing the late stages of differentiation of committed, immature precursors (e.g. leukaemia inhibitory factor release from M¨uller cells may regulate the timing of rhodopsin expression in immature but committed rod precursors (Neophytou et al., 1997)). However, the number of different cell types, their relative order of birth and the clonal composition would be determined autonomously for each progenitor by an internal motor, driving progenitors in successive rounds of division to produce specific cell types. It should be noted that it is unclear, nevertheless, how this hypothesis can be reconciled with findings of cell fate changes when extracellular signals are inhibited (e.g. Young and Cepko, 2004). One might argue that the extracellular signals are primarily needed as permissive rather than instructive factors for differentiation of certain cell types, and that what changes with time and therefore dictates the cell fate is the intrinsic, autonomous change in the reception of the signal. However, the fact that progenitors were examined only after E16 does nothing to exclude the possibility that extrinsic signals have a role in diversifying the progenitor population during early differentiation.

The above results suggest the presence of a rich mosaic of heterogeneous retinal progenitors at least during later differentiation in the mouse. Several other pieces of evidence suggested a degree of heterogeneity exists from the very beginning of retinal differentiation. Fate-mapping studies have shown correlations between progenitor origin and expression of molecules in subsets of progenitors and the fate of their progeny. Moody and colleagues found that blastomere origin of Xenopus retinal neurons biases their cell fate choice. The large majority of neuropeptide Y- or dopamine-positive amacrines originate from specific blastomeres (Huang and Moody, 1995) and, critically, transplantations of individual blastomeres showed that some, but not all, are intrinsically biased even at this early cleavage stage to produce an amacrine subtype (Moody et al., 2000). Particular blastomeres have a bias in producing specific classes of 5-hydroxytryptamine amacrines despite the fact that these cells have no spatial bias in their distribution in the retinal antero posterior and dorsoventral quadrants (Huang and Moody, 1997).

Progenitors carrying certain markers were found to be biased in the cell fates they produce. Alexiades and Cepko (1997) labelled progenitor cells and their progeny permanently by

using fluorescent latex microspheres coated to antibodies against VC1.1 and syntaxin-1a, markers of differentiated amacrine and horizontal cells. By co-labelling with 3H-thymidine, they showed that embryonic progenitors expressing VC1.1 and syntaxin were biased towards giving more amacrine and horizontal cells, whereas VC1.1-negative progenitors gave more cones. Similarly, later progenitors labelled thus gave amacrine and rod cells. p27Kip1 and p57Kip2, two cyclin-dependent kinase (cdk) inhibitors, are expressed in complementary domains in the mouse retina and amacrine cells seem to originate from the p57-positive population (Dyer and Cepko, 2000b, 2001).

Further indication for heterogeneity comes from results that suggest some fate-influencing genes are expressed in subsets of progenitors. In a recent genomic study, Livesey et al. (2004) identified a small number of genes such as Otx2 and SFRP2, which are expressed in subsets of progenitors; however, the authors note this pattern may reflect cell cycle variation of expression as opposed to heterogeneity. The vast majority of progenitor-specific genes seem to have a ubiquitous expression pattern.

Apart from the predominant multipotent progenitor demonstrated in the cell lineage experiments (Turner and Cepko, 1987; Holt et al., 1988; Wetts and Fraser, 1988) could there be also unipotent progenitors? Studies in the mature teleost retina have shown the presence of a rod-only precursor (Mack and Fernald, 1995) and there has been a report of a mouse E14 progenitor producing a clone of 33 rods (Turner et al., 1990).

If there is heterogeneity among retinal progenitors that contributes to their production of particular daughter cell types, a question arises about whether this heterogeneity is deliberately programmed or comes about as a matter of chance and early environmental exposure (see Figure 5.3). There may, for example, be fluctuations in the levels of Ath5 and other intrinsic determinants that neighbouring progenitors have in their nucleus and these different levels may influence the probability that the progenitors will produce a ganglion cell. Similarly, progenitor cells may have been exposed to small variations in signalling through the Notch or other pathways also resulting in slight heterogeneities in their potential. In this view, retinal progenitors are not developmentally programmed to be different from each other, but differences arise as a consequence of unprogrammed variations in the way similar cells develop.

5.7 The mechanism of cell cycle progression and cell fate determination

A highly conserved molecular mechanism underlies progression through the cell cycle. Coordination between cell cycle and fate determination is essential, not least because of the birthdate effect on cell fate and the need to generate the appropriate numbers of earlyand late-born neurons. Moreover the observation that, in most cases, differentiated cells have permanently exited the cycle and cannot divide suggests that the process of differentiation is somehow linked to the cell cycle (for a possible exception in the retina see Dyer and Cepko, 2000a; Fischer and Reh, 2001).

Classical transplantation studies in the ferret cortex (McConnell and Kaznowski, 1991) suggested that an environmental signal acts before the terminal S-phase of the cell cycle to

impart the fate of the postmitotic daughter cell. So one important aspect of this issue is the timing of the cell fate decision during the cell cycle, indeed whether there is such a decision by which a cell irreversibly commits to a fate.

The second important aspect is how the coordination between cycling and differentiation is achieved. Is differentiation dictating cell cycle exit or does the cessation of the cycle allow differentiation towards particular fates to take place? Recent studies in many systems, including the retina, have illustrated a bidirectional relationship between components of the cell cycle machinery and cell fate determinants.

5.7.1 Cell cycle components influence fate determination

Inhibition of cell cycle progression using the DNA synthesis inhibitors aphidicolin and hydroxyurea still resulted in the generation of diverse neuronal cell types in the Xenopus CNS, suggesting that cell cycle components are not essential for the generation of particular fates (Harris and Hartenstein, 1991). What roles then have been demonstrated for these components in determination? Ohnuma et al. (1999) found that p27Xic1, an inhibitor of the G1S-phase transition, promotes M¨uller cell fate over bipolars when overexpressed in the Xenopus retina and its inhibition reduces M¨uller cells. This effect is mediated by the cdk/cyclin-binding domain of p27Xic1, however it is worth noting that a mutant form of the mammalian homologue p21Cip1, which exhibits minimal cell cycle-inhibitory activity, has nevertheless the same M¨uller-promoting effect when overexpressed in Xenopus, suggesting the gliogenic effect of p27Xic1 is distinct from its action on the cell cycle. The authors hypothesized that the increasing p27Xic1 levels with development may push later cells to exit the cycle as M¨uller cells (Figure 5.4). On the other hand, cell cycle inhibition by p27Xic1 when Xath5 is overexpressed enhances the ganglion cell fate-promoting effects of Xath5, suggesting that early cell cycle exit promotes early cell-fates (Ohnuma et al., 2002). In mice, a mammalian homologue p27Kip1 was not found to affect cell fate, as mice lacking p27Kip1 did not have altered proportions of retinal cell types (Dyer and Cepko, 2001). Mice mutant in p57Kip2 however had increased calbindin-positive amacrine cells (Dyer and Cepko, 2000b), reflecting species-specific or cell-specific differences in the role of cell cycle inhibitors.

The tumour suppressor Retinoblastoma (Rb), which regulates cell cycle progression by repressing E2F-mediated-S-phase initiation, also has an effect in retinal differentiation. Mice with a conditional Rb mutation show deficits in rod differentiation, which indicates Rb has an effect on rod phenotype maturation subsequent to the cell fate decision; however, microarray analysis suggests Rb may be upstream of Nrl, a gene implicated in cell fate choice (Zhang et al., 2004). Overexpression of Prox1, the vertebrate homologue of prospero, forces progenitors to exit the cycle and promotes horizontal cell fate, whereas Prox1 mutant mice do not have horizontal cells (Dyer et al., 2003). Chx10, necessary for bipolar development, also regulates progenitor proliferation and its mutation causes the mouse ocular retardation phenotype (Burmeister et al., 1996); however, its effects on cellular proliferation and cell

5.7.2Several fate determination factors impact on the cell cycle

Many fate-influencing extrinsic factors are mitogens and they have a doseand receptor- composition-dependent effect on the coordination of proliferation and differentiation (TGF- α: Lillien and Cepko, 1992; Lillien, 1995; Lillien and Wancio, 1998; TGF-β: Anchan and Reh, 1995; Shh: Jensen and Wallace, 1997; Zhang and Yang, 2001). Notch signalling in the presence of proneural activity induces early cell cycle exit (Ohnuma et al., 2002). Retinal neurons in Ath5 zebrafish mutants exhibit delayed cell cycle exit (Kay et al., 2001) and there is some evidence from other systems that bHLH genes may activate expression of cell cycle inhibitors (Farah et al., 2000).

Apart from the transcriptional regulation of the two pathways, less studied mechanisms might involve post-translational events. For example, determination factors may be targets of Cdk-phosphorylation or protein degradation in other systems (e.g. Reynaud et al., 1999).

5.7.3 When is cell fate determined?

Studies on the timing of cell fate commitment have given inconsistent results. Adler and Hatlee (1989) found a postmitotic effect of the chick retinal environment biasing cells away from a photoreceptor fate. Ciliary neurotrophic factor was found to be able to divert rat postmitotic cells from a rod to a bipolar fate up until the time of opsin expression by the rod cell (Ezzeddine et al., 1997), although Neophytou et al. (1997) reported that this effect was a reversible rod differentiation arrest and not a respecification to the bipolar fate.

On the other hand, cells destined to become RGCs were found to express an RGC-specific marker, RA4, 15 minutes after terminal mitosis, suggesting ganglion cell specification occurred before or during M-phase (Waid and McLoon, 1995). Moreover, the negative feedback mechanism for amacrine production appears to act before the progenitor terminal M-phase (Belliveau and Cepko, 1999). These incongruities may reflect cell-type-specific differences or, at least for some cases, may point to a treatment-related plasticity of the cell fate even after the cell is on its way to a particular fate.

5.8Comparison with other systems

The cell fate specification in the vertebrate retina presents similarities in the types of molecular mechanisms used in other systems, and seems to employ a variety of strategies each of which, but maybe not all together, are found elsewhere. In the Drosophila eye, for example, the Atonal–Notch lateral inhibition pathway specifies the first photoreceptor, R8 (similarly it contributes to RGC specification in the vertebrates); however, the other seven photoreceptors in a cluster are recruited in a stereotypical sequential fashion. The Notch and Receptor Tyrosine Kinase–Ras–Mitogen-Activated Protein Kinase pathways are instrumental in this process and cooperate with or may induce different intracellular factors such as rough and lozenge, which go on to specify the fate (reviewed in Voas and Rebay, 2004). The Drosophila Prox1 homologue Prospero and the Otx/Crx homologue Otd are also involved in

photoreceptor differentiation. In general, parallels pointing to common evolutionary origins have been drawn between first-born retinal neurons in several species. Ath, an atonal/Ath5 homologue, also specifies the first photoreceptors to differentiate in the larval Platynereis, a species of Polychaeta, with two simple eye structures each having one photoreceptor and one pigment cell and the vertebrate RGC is similar to the invertebrate rhabdomeric photoreceptor because of similarities in their axonal projections, atonal/Ath5 specification of their fate, Brn3 and BarH expression and expression of r-opsins (vertebrate melanopsin) (Arendt, 2003).

Development of the Drosophila CNS resembles the vertebrate retina in that multipotent progenitors go through a series of divisions giving cell types dependent on their birthdate. Doe and colleagues developed an elegant model involving a fixed, sequential, transient expression of heterochronic genes in each neuroblast dictating the fate of the progeny born at the time of expression. They hypothesized these genes may work via chromatin modifications to translate a temporary neuroblast code to a permanent progeny identity (Isshiki et al., 2001; Pearson and Doe, 2003). Such a mechanism has not yet been found in the vertebrate eye, and the study of epigenetic modifications in cycling progenitors may prove a fruitful area of investigation.

In the vertebrate spinal cord, cell fate is determined by gradients of dorsoventral and rostrocaudal signalling molecules. This mechanism is clearly different from the ones that operate in the retina, however other aspects are more similar, as illustrated for example by the differentiation of multipotent progenitors into both motor neurons and oligodendrocytes. The sequential generation is dictated by the change in expression of transcription factors whose combined action dictates neuronal versus glial fate (Novitch et al., 2001; Zhou et al., 2001). In general, the employment of a transcriptional combinatorial code of homeobox genes and perhaps bHLH proteins to define neuronal classes along the dorsoventral axis is the predominant theme of cell fate specification in the spinal cord and resembles the action of intrinsic factors in the retina. Moreover, there is an influence of the birthdate on cell subtype within the motor neuron domain brought about through intercellular interactions of the daughter cells (reviewed in Jessell, 2000).

5.9 Concluding remarks

Although several mechanisms have been found to operate in directing the multipotent retinal progenitors to produce the different cellular fates, integration of these aspects in a single framework has been lagging. One important area of investigation will therefore be the establishment of connections between the different mechanisms, particularly how signalling pathways downstream of extrinsic cues interact with intrinsic factors and what their relative contribution to the fate decision is. Does a precursor move from an early, largely uncommitted, extrinsic signal-dependent programme to a later autonomous intrinsic programme and if so, when does this happen? Are these programmes strictly dictating the clonal composition of each progenitor, or are we dealing with a more stochastic determination, with the different molecules imparting biases to the progenitors for one fate or the other?

Related to this, it will be important to clarify the extent to which the decision for the cell fate is linked to a gradual differentiation process, and whether the competence of a precursor is restricted in a step-wise fashion so that the determination process initially leads to a generic cell type, such as a ganglion or bipolar cell and subsequently specifies subtypes. For example, Harris and Messersmith (1992) suggested that two extrinsic inductive events in the retina specify first a photoreceptor fate and subsequently a rod or cone fate and, in the same vein, the transcription factor Nrl may be one factor dictating rod versus cone specification, as mice mutant for Nrl lose their rods and display an increase in S-cone-like cells (Mears et al., 2001). On the other hand, the production of certain amacrine subtypes is influenced by blastomere origin, suggesting that subtype specification does not strictly follow a generic type specification (Moody et al., 2000) and, similarly, manipulation of BarHl2 expression affects the production of glycinergic amacrines at the expense of M¨uller, bipolar or photoreceptor numbers but has no effect on γ-aminobutyric acid (GABA) ergic amacrines (Mo et al., 2004).

Ultimately, the answer to many of these questions must link the fate-determining factors described in this chapter to the mature cellular phenotype. Some progress has already been made, particularly in studies of rhodopsin expression regulation; however, important matters are still open. Are there ‘master’ regulatory genes integrating the different signalling pathways to regulate all aspects of cell differentiation or are multiple parallel pathways acting in concert to produce the mature phenotype? What is the connection between the expression of genes specific to one cell type and the repression of genes specific to another cell type or to pluripotency?

Powerful new tools, including reverse genetics in the mouse, forward genetics in zebrafish, genomic analysis and in vivo time-lapse imaging, promise exciting developments in the coming years.

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