In the CNS, fluorescence was first observed at E10.0 within the telencephalon (), and became more robust by E12.5–E14.5 (), then diminished by P0 (). We investigated the colocalization of GFP and endogenous Eomes protein () and determined that GFP was expressed in a larger population of cells. High magnification views of sectioned brains at E14.5 revealed that GFP was detectable in a subpopulation of cells expressing and colocalized with Eomes protein predominantly within the subventricular zone (SVZ), but GFP expressing cells were also present in the intermediate zone (IZ) to marginal zone (MZ) (). This absence of GFP expression in Eomes +ve cells of the SVZ is likely to be analogous to the delay in onset of detectable fluorescence observed in blastocyst stage embryos (discussed previously). We also noted that GFP fluorescence extended to more differentiated regions of the neocortex where Eomes protein was not present, likely due to perdurance of the fluorescent protein.
FIG. 3 GFP expression within the telencephalon of Tg(Eomes::GFP) embryos at midgestation and early postnatal stages. (a) Onset of GFP fluorescence in the central nervous system (CNS) at E10.0. (b,c) Wholemount brightfield and GFP overlays depicting GFP fluorescence (more ...)
We next determined colocalization of the GFP reporter with molecular markers representing the different cell types present in the telecephalon, including both distinct classes of neural progenitors and postmitotic cells. Pax6 marks a population of radial glial progenitor cells (Gotz et al., 1998
) fated for glutamatergic differentiation (Schuurmans et al., 2004
). Pax6 antibody staining revealed no overlap between Pax6 expression in the VZ and GFP expression in the SVZ and MZ, suggesting that the transgene was not prematurely activated (). However, staining with T-brain1 (Tbr1) antibody revealed a subpopulation of cells co-expressing GFP and Tbr1 (). Since Eomes (Tbr2) and Tbr1 are expressed in mutually exclusive populations of cells (Englund et al., 2005
), this result suggests that GFP fluorescence extended into regions containing postmitotic neurons and that this could result from perdurance of the fluorescent protein or downregulation of the reporter. Furthermore, staining with the M-phase marker phosphohistone-H3 revealed colocalization with a subpopulation of GFP expressing cells representing a non-surface dividing intermediate progenitor cell population ().
To establish if the expanded domain of GFP fluorescence observed in Tg(Eomes::GFP) brains was due to perdurance of GFP protein, we processed sequential sections to determine and compare the localization of GFP protein, GFP transcripts, and Eomes transcripts. GFP transcripts were more restricted than GFP protein (), and were localized to an identical domain as Eomes transcripts, residing predominantly within the subventricular and intermediate zones at E14.5 (), and the hippocampus at P0 (). Thus perdurance of the fluorescent protein results in the expanded domain of GFP fluorescence, such that the GFP reporter acts as a lineage tracer for postmitotic neurons that have recently extinguished Eomes having migrated into the cortical plate (cp).
FIG. 4 The expanded domain of GFP fluorescence within the telencephalon of Tg(Eomes::GFP) embryos is due to perdurance of GFP protein. (a) Brightfield and GFP overlay of vibratome sections of E14.5 brains illustrating GFP localization. (b,c) GFP expression by (more ...)
Within the developing limb, a focus of GFP fluorescence was observed at the base of digit IV (, ). Sections through the forelimb at E12.5 revealed that GFP was localized in a ring of cells around the condensations associated with the fourth digit (). In addition, Eomes transcripts within the forelimb at this stage were also restricted to the same ring of cells at the base of the fourth digit, and thus closely correlated with transgene expression ().