delivered subsequently [e.g., experiments 1–8, 22, and 23]; (Figure 2B). Second, continuous exposure to doxycycline begin- ning prior to or at E0 produces recombination in a dose- dependent manner, with some recombination seen even in the absence of 4HT [e.g., experiments 13, 18–21, 28, 32, and 33]; (Figure 2F). Third, initiating doxycycline exposure between E2 and E4 and delivering 4HT between E10 and E12 gives dose- dependent recombination that results in a relatively sparse collection of AP+ RGCs suitable for the analysis of single cell morphologies [e.g., experiments 2, 3, 11, 12, 24–27]; (Figure 2C,E). In particular, in experiments 2, 3, and 11 (with the Brn3aCKOAP/+ target), two experiments showed no labeled neurons and one experiment showed sparse labeling in more than half the retinas. Similarly, in experiments 24, 25, and 27 (with the Brn3bCKOAP/+ target), one third of the retinas have no labeled neurons and two thirds of the retinas have a sparse distribution of labeled neurons. Interestingly, several retinas with sparse labeling exhibited clusters of AP+ RGCs indicative of early clonal activation or repression (e.g., Figure 2C); an analogous clustering of AP+ neurons and their processes were observed in the brains of several of these mice, as seen in the coronal section through the superior colliculus in Figure 2D. Importantly, clustering of labeled RGCs was observed in retinas that received 4HT after E11, a time when there are already many hundreds of retinal progenitors and lateral migration of cells within a single retinal clone is severely restricted [10]. Thus, the data suggest that the clonal restriction reflects events that occurred earlier in development, such as epigenetic silencing of rtTA expression and/or rtTA-dependent activation of the CreER target in a subset of cells. It seems plausible to suppose that the spatial patterns of clonal restriction might reflect the same epigenetic process that silences CreER expression when doxycy- cline is not delivered in early gestation. The weak AP staining of all axon bundles in Figures 2B and C arises from a low-level background of AP expression in RGCs from the Brn3aCKOAP and Brn3bCKOAP alleles in the absence of Cre-mediated recombination. In sum, by using an appropriate regimen of doxycycline exposure, the R26rtTACreER locus can be used, even with highly recombinogenic target loci, to generate a sparse set of labeled cells at a developmental time point determined by 4HT injection. R26IAP: A Sensitive Cre-Dependent AP Reporter for a Broad Array of Tissues and Cell Types To create an AP reporter that is activated by Cre-mediated recombination with an efficiency higher than the relatively inefficient Z/AP locus and to eliminate background AP activity in unrecombined cells, a Rosa26 knock-in allele was constructed with an AP coding region in which the 39 half is inverted (R26IAP; Figure 1C). Upon Cre-mediated recombination, the 39 half of the coding region, which is flanked by loxP sites in head-to-head orientation, is flipped into the correct orientation in a subset of cells. On the assumption that a variable number of DNA inversion events occurs before dissociation of the Cre/loxP complex, we expect that the recombination process would produce a mixture of correct and inverted orientations in those cells in which recombination has occurred. Therefore, AP expression from the R26IAP allele should persist in a subset of recombined cells, with virtually no background AP activity in unrecombined cells. Cre- mediated inversion has been used previously in experiments in which extremely tight control of gene expression was essential, for example in the in vivo analysis of oncogenic fusion proteins [12]. As an initial test of the efficiency of Cre-mediated recombina- tion at the R26IAP locus, we examined AP expression by histochemical staining of adult tissues in R26CreER;R26IAP mice New Cre/lox Genetic Tools that had not been exposed to 4HT (Figure 3A,B). This analysis revealed 4HT-independent recombination at a frequency of hundreds of cells per retina and ,1,000 cells per 300 mm vibratome section of brain, roughly similar to the levels observed with R26CreER;Brn3aCKOAP/+ and R26CreER;Brn3bCKOAP/+ mice in the absence of 4HT. As noted in the Introduction, R26CreER; Z/AP mice average ,1 AP+ cells per retina or 300 mm brain section in the absence of 4HT [10]. In R26CreER;R26IAP mice, the number of AP+ cells increased by several orders of magnitude following a single IP injection of 200 mg 4HT at P3, resulting in AP-stained retina flat mounts that are completely opaque (not shown). Recombination in R26CreER;R26IAP mice is observed in a wide variety of tissues, as expected for expression of both CreER and AP from the Rosa26 locus. For example, Figure 3C and D shows scattered AP+ cells in the lens and cornea following intraocular delivery of 6 mg 4HT in adult mice. Using R26IAP to Assess the Efficiency of Different Methods for Cre Delivery The relatively high recombination efficiency of the R26IAP locus, together with the high sensitivity of AP histochemistry, suggested that the R26IAP locus might be generally useful for monitoring the efficiency of Cre-mediated recombination in a variety of experimental contexts. To test this idea, we have used R26IAP to compare Cre-mediated recombination following: (a) direct uptake of purified Cre protein fused to a poly-basic peptide (His-NLS-Cre; 13) injected into the peritoneal cavity or into the eye (Figure 3E–H), or (b) infection with a defective adenovirus vector expressing Cre (Ad-Cre) delivered by intravenous or intraocular injection (Figure 3I–O). Each of these methods of Cre administration induced recombination at the R26IAP locus, with the cell type, location, and efficiency of recombination reflecting the delivery route and vehicle. Intraperitoneal (IP) injection of His-NLS-Cre in adulthood led to recombination predominantly in epithelial cells lining the peritoneal cavity, as seen on the surface of the liver, stomach, and abdominal wall (Figure 3E–G); the only AP+ cells not lining the surface epithelium were occasional AP+ abdominal wall muscle fibers (Figure 3F). Similarly, intraocular (i.e. intravitreal) injection of His-NLS-Cre at P5 induced recombination principally in retinal cells in close proximity to the vitreal surface: RGCs, astrocytes, and superficial vascular cells (Figure 3H). Intraocular injection of Ad-Cre in adult mice resulted in transduction of Cre into retinal and corneal cells (Figure 3I and J), and tail vein injection of Ad- Cre in adult mice resulted in efficient recombination in the liver (Figure 3O) and scattered recombination in the kidney, with the highest efficiency in the renal pelvis (Figure 3K–N). Intraocular injections of His-NLS-Cre or Ad-Cre gave highly variable frequencies of recombination within the retina, ranging from few or no recombination events to nearly confluent AP staining across the retina, a variability that most likely reflects variation in needle placement, volume injected, and the fraction of the injected volume retained within the eye. In the absence of His-NLS-Cre protein or Ad-Cre virus or a genetically introduced Cre or CreER locus, no AP+ cells were observed in any tissues examined from R26IAP mice, including brain, liver, stomach, kidney, eye, and abdominal wall. The Utility of Brain Atlases Composed of Sparsely Labeled and Genetically Defined Neuronal Subtypes The role of cholinergic and catecholaminergic (dopaminergic, noradrenergic, and adrenergic) neurotransmission in brain function and disease has been an object of long-standing scientific PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7859