In these studies, we describe the generation of Cited1-CreERT2 BAC transgenic mice which express tamoxifen-activated Cre recombinase in a discrete subpopulation of cells within the condensed MM known as the cap mesenchyme. These mice provide a unique reagent that induces efficient Cre-dependent recombination restricted to the cap mesenchyme following a single IP injection of tamoxifen, and incorporates a functional IRES-eGFP marker that could be used for enrichment of Cited1 positive cells in future studies. We have exploited this transgenic system to generate the first detailed in vivo fate map of cap mesenchyme cells over the course of nephrogenesis. By crossing Cited1-CreERT2 BAC transgenic mice with the R26RLacZ conditional reporter line, and activating Cre with tamoxifen, we were able to track the fate of cap mesenchyme derived cells at different stages of nephrogenesis. Using this approach our studies confirm what was predicted from the results of previous in vitro studies indicating that the cap mesenchyme gives rise to a wide variety of nephronic epithelial cell types. They also clarify conflicting in vitro data regarding the mixed ontogeny of the collecting duct epithelium by demonstrating definitively that the cap mesenchyme does not contribute to collecting duct epithelium within the adult kidney. We also demonstrate that the positioning of nephrons within the adult kidney depends on the stage at which they develop from the cap mesenchyme, and importantly, provide the first substantive evidence that these cells contain a population of self-renewing epithelial progenitor cells.
We chose to use Cited1
promoter elements to drive Cre expression given its unique and restricted expression domain in the cap mesenchyme. Previous studies from our laboratory have shown that Cited1 expression increases in the condensed MM from E12.5 and persists in the cap mesenchyme throughout nephrogenesis, but is absent in the adult kidney (Boyle et al., 2007
). Cited1 expression is distinct from that of Six2,
which is expressed strongly in the MM prior to UB invasion and in early condensations, and extends outside of the Cited1 expression domain to overlie UB branch points (Self et al., 2006
). These findings indicate that Cited1 is expressed in a subpopulation of Six2
positive cap mesenchyme, and will be important for future comparison with lineage tracing studies using other Cre lines expressed in the MM.
Initial characterization of this transgenic line demonstrated Cre-mediated recombination at the Rosa26RLacZ
locus in approximately 40–50% of cap mesenchyme cells 72 hours after a single injection of tamoxifen. In addition, there was widespread β-gal expression in nephronic epithelia throughout the adult kidney after a single IP injection at E13. As Cre-dependent recombination is a stochastic event (Nagy, 2000
), these findings suggest that with repeated injection of tamoxifen, this transgenic line will likely induce highly efficient recombination in the acute setting and will provide a powerful tool to study the effect gene deletions within the cap mesenchyme. Further characterization of this transgenic line indicated that Cre-dependent recombination reflects the expected pattern of Cited1 expression over the course of nephrogenesis (Boyle et al., 2007
). For example, we have previously shown that Cited1 expression at E11 is weak and restricted to a few cells in the MM. Correspondingly, when we injecteCited1-CreERT2
mice at E11 and examined cap mesenchyme lineage in the adult, we observed a low percentage of total nephrons labeled. This was not due to inefficient placental transfer of tamoxifen as we saw efficient Cre-dependent recombination within the developing myocardium at the same time point. In contrast, when we treated mice with tamoxifen at E13, by which time Cited1 expression in the cap mesenchyme is robust (Boyle et. al, 2007
), we saw recombination in a large proportion of adult nephronic epithelium. Furthermore, the declining percentage of labeled epithelium in the adult following injection at E15 and E18, respectively, is consistent with the fact that many primitive nephrons arise prior to these time points.
These fate mapping studies provide in vivo evidence that the cap mesenchyme gives rise to a wide variety of nephronic epithelial cell types populating the cortex and medulla of the adult kidney, confirming results predicted from classical tissue recombination experiments as well as in vitro lineage tracing studies. However, technical limitations of in vitro explant studies do not allow for the differential labeling and fate mapping specifically of cap mesenchyme vs. pre-tubular aggregates and stromal mesenchyme within the MM. Our studies, therefore, provide direct evidence that the cap mesenhcyme is the primary source of the epithelial progenitor cells that will comprise the mature nephron. In addition, these studies show, in vivo, that the cap mesenchyme does not contribute to collecting duct epithelium.
The transient nature of tamoxifen-induced activation of CreERT2
provides an opportunity to track the fate and spatial distribution of cells derived from the cap mesenchyme at different stages of nephrogenesis. This enabled us to address one of the key unanswered questions in this field: Is the cap mesenchyme repopulated over the course of nephrogenesis intrinsically or through the migration of extrinsic cells? Our studies show that a high proportion of cap mesenchyme cells still express LacZ 6.5 days after a single IP injection of tamoxifen. It is unlikely this is due to persistent tamoxifen-induced activation of CreERT2
as we demonstrated that tamoxifen-induced nuclear localization of CreERT2
was maximal 24 hours following injection with no detectable nuclear Cre after 96 hours. Furthermore, previous studies have characterized the kinetics of Cre activation in more detail, indicating that most of the Cre returns to the cytoplasm 48 hours after a single tamoxifen injection (Hayashi and McMahon, 2002
; Nakamura et al., 2006
). On this basis, if exogenous cells were being recruited to repopulate this niche, we would expect that a high percentage of cap mesenchyme cells present at the time of injection would not be labeled 6 days later, as the originally tagged cells would have been depleted by induction and differentiation and progressively replaced by unlabeled cells. These findings provide the first strong in vivo
evidence that an intrinsic, self-renewing population of progenitor cells reconstitutes the cap mesenchyme over the course of nephrogenesis.
By tracking the fate of nephronic epithelium labeled early vs. late in nephrogenesis, our studies also provide in vivo
evidence that deep nephrons extending into the renal papilla arise only during the early phase of nephrogenesis and that nephrons arising at later stages of development are restricted to the cortex and outer medulla. As deep and superficial nephrons contain all of the same cell types, this does not represent a shift in cell fate per se
, but provides in vivo
evidence that the positioning of nephrons within the adult kidney is temporally regulated. There are several possible explanations for this phenomenon. It is possible that nephrons arising later are physically ‘blocked’ from extending into the medulla by early nephrons that have already migrated into the papilla as corticomedullary patterning begins at ~E16 (Cebrian et al., 2004
). Given the fact that formation of the renal pelvis is poorly understood, a more intriguing explanation would be the differential expression of an unidentified chemotactic factor that directs migration of distal elements into the papilla. Perhaps this factor is down-regulated once an appropriate number of nephrons have arrived, leaving tubules arising later to occupy superficial positions. In any case, the temporally dependent organization of nephronic elements observed in our studies supports what has been predicted by classical anatomical studies of the adult kidney indicating that nephrons are organized into deep and superficial structures (Kriz and Koepsell, 1974
In summary, we describe the generation and characterization of Cited1-CreERT2 BAC transgenic mice, which express tamoxifen-regulated Cre recombinase exclusively in the cap mesenchyme. By crossing these mice with a Cre reporter line, we have used these mice to evaluate the fate of cap mesenchyme cells over the course nephrogenesis. These studies confirm the expected findings that cap mesenchyme gives rise to diverse nephronic epithelia in the adult kidney, and provide the first evidence that the cap mesenchyme contains a population of self-renewing epithelial progenitor cells.