In this study, we generated transgenic mice expressing Cre recombinase under the control of 203 kb of genomic DNA at the Ikaros locus. We used these animals to show quantitatively and in vivo that the Ikaros lineage is unbiased and contributes to both early- and late-born cell type production. In addition, we show that two alternative mouse lines generated here can be used as tools for in vivo clonal genetic modifications/lineage tracing in RPCs, or tracking of early-born neuronal cell types in the retina. We discuss below the significance and implications of these findings.
Cre Recombination in RPCs
Although the Ikaros protein is found in virtually all neuroepithelial progenitor cells forming the prospective rat retina (Elliott et al.,2008), the IKCre transgenic lines we generated do not achieve reporter expression in all mouse RPCs. Instead, some lines exhibit widespread, yet non-ubiquitous, Cre-reporter expression (IKCre-A and B), while others only label RPCs very sparsely (IKCre-F and I). Inter-line variability probably stems from the number of BAC copies integrated in the transgenic array and/or from the chromosomal surroundings at the integration site, which differ for each transgenic founder mouse.
A possible explanation to account for incomplete penetrance is suggested by the indirect detection method of Cre expression, combined with the low level of expression of the Ikaros mRNA in RPCs seen by in situ hybridization and PCR (Elliott et al.,2008). Although it cannot be ruled out that some regulatory modules are missing from the BAC clone, it is likely that Cre from the transgenic BAC is weakly transcribed, resulting in low protein amounts that only achieve recombination in a subset of Ikaros-positive RPCs. This conclusion is supported by our failure to detect the Venus protein from the transgene, whereas both Cre and Venus transcripts are detected by RT-PCR. As such, low transcription efficiency from the BAC would inconsistently produce enough Cre for recombination, but along with the typical decrease in translation efficiency associated to IRES sequences, would consistently fail to generate sufficient levels of Venus protein for detection. Once activated via permanent stop sequence excision, however, β-gal or YFP reporters used in this study are expressed from the potent R26 locus, which is unrelated to Ikaros regulation.
Ikaros appears to show very little cell specificity in embryonic stages, being expressed ubiquitously at low levels in most tissues while displaying higher expression in the liver, thymus, and striatum, for example (www.genepaint.org, and not shown). Consequently, it is difficult to evaluate and validate the specificity of expression of the Cre since we suspect a fully penetrant lineage labeling of all Ikaros-expressing cells would encompass most, if not all, adult cells. Moreover, all Ikaros antibodies tested so far only allow immunohistochemical detection in rat tissue, preventing the direct comparison of Cre and endogenous Ikaros expression patterns. It is notable, however, that albeit variably penetrant, our transgenic lines do show little embryonic tissue specificity, much like what we observe by in situ hybridization for Ikaros. Furthermore, subsequent post-mitotic retinal cell type restriction exhibited in the IKCre-G line is compatible with known Ikaros-specific expression in RGCs and amacrine cells. Importantly, unsolved specificity issues do not preclude the use of the IKCre-F line as a clonal Cre-driver in the early retina.
A critical question only touched on by our previous work concerned the potential specificity of Ikaros-expressing RPCs. While late RPCs do not express Ikaros, they could derive from the Ikaros lineage, or alternatively, represent distinct lineages that amplify late from a scarce and elusive population of early Ikaros-negative RPCs. In the first case, the Ikaros RPCs are likely unbiased in their potential, and their progeny would encompass all retinal cell types. In the second case, they would exhibit a degree of bias in competence that should be reflected in the lineage composition. Previous evidence in favor of unbiased Ikaros lineages was obtained by infecting early rat retinal explants with a GFP retrovirus, and showing that, 5 days later, clones could be composed of both Ikaros-positive and Ikaros-negative RPCs (Elliott et al.,2008). All progenitors at the time of infection were assumed to be Ikaros-positive. This current work was started in part as a way to validate this conclusion in vivo, and extend its significance by adding a quantitative type of analysis. One intuitive way to proceed would be to look for Ikaros-negative RPCs at early stages of retinogenesis, the progeny of which would appear negative upon Cre-mediated lineage labeling. The inability of our IKCre transgenics to achieve consistent Cre recombination in all Ikaros-positive RPCs, however, precluded such an approach. If low Ikaros expression is at the root of incomplete penetrance issues, even an Ikaros-Cre knock-in mouse produced by ES cell recombination might generate escaper RPCs that express Ikaros but fail to undergo Cre-mediated recombination. Therefore, instead of relying on a potentially false negative result, we feel a strong conclusion is best reached backwards, from the careful analysis of Ikaros-derived cell types in the mature retina. In this work, our qualitative observations in the IKCre-A line and clonal analysis in the IKCre-F line similarly indicate that early Ikaros-expressing RPCs have the potential to generate all cell types of the retina in largely appropriate proportions. Although Müller cells appear slightly over-represented in our clonal analysis compared to previously published study (see Table 1), this variation most likely stems from differences in the methodology used to estimate the proportion of Müller cells and from the mouse genetic background used in the different studies. Overall, these results support a model in which transient Ikaros expression acts as a permissive temporal competence signal to bias early-born neuron production rather than defining a limited range of cell fates.
Lineage tracing in the mouse retina proved a powerful tool and established the existence of multipotent progenitors able to give rise to all retinal cell types in radially-arranged arrays (Price et al.,1987; Turner et al.,1990). These seminal studies relied on retroviral infection of RPCs in vivo from E12.5, and typically generated clones that widely varied in size. Technical difficulties associated with in utero surgical injections, however, might have limited its widespread use for lineage tracing from early RPCs. Subsequently, a couple of studies cleverly exploited chimerism or X inactivation in mouse to confirm such conclusions and refine some observations like tangential dispersion (Williams and Goldowitz,1992; Reese and Tan,1998). Rather than labeling sparse clones, however, these latter genetic approaches differentially labeled one half of RPCs versus the other. Consequently, it is unclear how frequently the arrays obtained represented true clones.
We propose here that due to its very low rate of recombination, the IKCre-F line can be used to genetically label and track single RPCs from E10.0, making it relatively easy to analyze a large number of RPC lineages in vivo. Although it is inherently difficult to unambiguously demonstrate that all cell arrays in the IKCre-F line are actual clones, the observation of isolated Cre-recombined RPCs at E10.5, the progressive increase in array size over time, and the finding that the largest arrays observed here do not contain more cells than those observed by retroviral lineage tracing strongly suggest that most, if not all, cell arrays observed in the IKCre-F line are actual clones that derived from a single RPC. Although the classical retroviral lineage studies in the retina generated groundbreaking discoveries and have been extensively cited, conclusions on the potential of early RPCs were based on the analysis of 93 clones generated from virus infection at E12.5 and 222 clones at E13.5 (Turner et al.,1990). Considering the significance of this data for the field, the analysis of a larger number of clones from an earlier time point would be desirable, and in particular mandatory to test various models of retinogenesis. For example, a large number of clones will be required to test in vivo a stochastic model of retinal cell fate specification that we recently proposed from the analysis of lineages in culture (Gomes et al.,2011). This project further asks for lineage data at several intermediate stages of retinogenesis, and these results will be best compared to adult clone composition in the context of a unique and consistent mouse line.
There are currently other mouse lines available to achieve sparse Cre-mediated recombination in the retina (Badea et al.,2009b; Yun et al.,2009; Brzezinski et al.,2011). In these lines, a drug such as tamoxifen is used to activate Cre recombinase activity at a specific time, and limiting dilutions can be applied to restrict recombination to a subset of RPCs. Such pharmacological approaches, however, have notoriously variable efficiencies, and finding the right drug dosage to achieve clonal recombination is often not straightforward. Another recently developed strategy for clonal analysis in the retina combines the conditional expression of the tumor virus A (TVA) receptor in a specific population of cells and retroviruses expressing the avian leukosis virus EnvA protein to specifically infect subsets of the Cre-recombined cells, thus achieving clonal lineage labeling even from non-clonal Cre-driver lines (Beier et al.,2011). This method, however, involves the use of several mouse lines as well as concommitant retroviral injections, which are inherently difficult and similarly subject to experimental variations. The IKCre-F line described here offers an alternative genetic tool for lineage analysis in the retina without the need for pharmacological induction or in utero retroviral injections.
Notably, the use of Cre recombinase makes the IKCre-F line versatile, as recombination can turn on a reporter (as in this study), but can also inactivate or over-express a gene of interest in a lineage-specific manner, thus allowing a clonal type of conditional genetics in the mouse retina. Additionally, most conditional Cre-driver lines currently available in the retina achieve recombination either in specific neuronal populations or in a widespread manner in RPCs. Because the clonal Cre line described here is unbiased in the laminar (tangential) dimension, but instead active in radial arrays, it is bound to be a useful addition to the mouse retina genetic toolbox. We anticipate the IKCre-F line could prove useful in a number of situations. For example, it could be used to (1) assess the cell-autonomous function of a particular gene in neurogenesis, or (2) look at the morphology and behavior of mutant cells amid wild-type neighbors if a mutated gene disrupts retina lamination, thus obscuring its mechanism of action. Next, (3) this line opens up the possibility to address whether neurons born from a common lineage make preferential connections or form functional units in the retina, a recent area of interest in the developing cortex (Yu et al.,2009; Li et al.,2012), or (4) whether the large number of neuronal subtypes arise randomly in the retina, or if single lineages exhibit a bias towards certain subtypes, an as yet unanswered question.
Cre Recombination in Postmitotic Neurons
Five IKCre lines out of nine generated in this study expressed the reporter in isolated cells in the adult retina. Although superficially this pattern is very different from radial arrays, the fact that recombination appears limited to amacrine and RGC strongly suggests that Cre is expressed under the influence of a different set of Ikaros enhancer(s) in the IKCre-G line. Indeed, these neurons represent the most abundant cell types in which Ikaros protein is found post-mitotically (Elliott et al.,2008). Given that many other types, including the most numerous photoreceptors, express neither the Ikaros protein nor the Cre-reporter, two points can be made: (1) accurate post-mitotic Ikaros expression might rely on a devoted set of CIS regulatory sequences that are at least partly present on the BAC transgene; (2) in such a post-mitotic context, Cre expression from the BAC transgene clearly exhibits an Ikaros cell-specificity that could not be verified in RPCs-expressing lines due to ubiquitous Ikaros expression in the embryonic retina (see above). It remains unclear, however, why horizontal cells that were previously shown to express Ikaros failed to consistently undergo Cre-mediated recombination in the IKCre-G line. Of note, while amacrine cells and RGCs are born during embryogenesis, Cre-reporter in the IKCre-G line is rarely detectable before birth. This could represent the delay necessary for Cre-mediated deletion of the stop signal at the Rosa locus in differentiating cells, or the inherent nature of Ikaros regulation in mature postmitotic neurons.
Much like sparse recombination in IKCre-F allows to track and genetically modify isolated RPCs, sparse recombination in the IKCre-G line could be exploited to visualize the morphology of single amacrine and RGCs cells. While the cytoplasmic YFP reporter used in this study is not optimal to outline neuron processes, several better-suited reporters could be used, including a recent highly efficient alkaline-phosphatase at the R26 locus (Badea et al.,2009a). This could be used to address in vivo the function of a gene in neuronal polarization, neurite branching, or axon navigation towards the optic nerve and the brain, in the case of RGCs. Potentially, this line could also be interesting to selectively study the connections that amacrine cells and RGCs establish together in the inner plexiform layer.