Retinal expression of viral Cre dependent reporters after postnatal day 0.5 (P0) intraocular infection.

<p>A, Genetic backgrounds and viruses used. <i>Brn3b^Cre/+</i>, <i>Brn3b^Cre/Cre</i>, <i>Brn3a^Cre/+</i>, or <i>Brn3b^+/+</i> littermate pups were infected with a combination of Cre dependent AAV1.CAG.flex.tdTomato.WPRE.bGH virus and a constitutively expressed, Cre independent AAV1.CAG.GFP virus. B, Experimental time lines. Eyes of P0 pups were injected, and retinas were analyzed at either P3, P7 or after P21. Cells infected with the Cre independent virus should appear green, while Cre positive cells infected with the Cre dependent virus should appear red. Results from this experiment are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone-0091435-g005" target="_blank">figures 5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone-0091435-g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone-0091435-g007" target="_blank">7</a>. C, E, G, and I, flat mount preparations of adult retinas of indicated genotypes, demonstrating extensive expression of (Cre dependent) tdTomato red staining in <i>Brn3b^Cre/+</i> (n = 6), <i>Brn3b^Cre/Cre</i> (n = 4) or <i>Brn3a^Cre/+</i> (n = 12) retinas, and only very isolated expression in Cre negative, <i>WT</i> retinas (n = 3 for the Brnb litters and n = 4 for the Brn3a litters). Note that in some cases, red and green fluorescence are evenly distributed over the entire retina, while in others there is an apparent segregation of red and green fluorescence, most likely by subretinal distribution of the Cre independent, AAV1.CAG.GFP virus (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone-0091435-g006" target="_blank">figure 6</a>). White arrow in I points at one of the few red cells labeled in the <i>WT</i> retinas, shown enlarged in the inset in J. D, F, H, and J represent higher magnifications of retinas shown in C, E, G, and J respectively. Left panels are tdTomato, red fluorescence, middle panels are GFP, green fluorescence, and right panels are merge channels. In all panels, red arrowheads point at tdTomato positive, green arrowheads at GFP positive, and yellow arrowheads at double positive cells. White arrowhead in F labels wandering axons characteristic of Brn3b null (<i>Brn3b^Cre/Cre</i>) retinas. White arrowhead in J points at the tdTomato positive cell in I, most likely a Müller Glia. Scale bars in I = 1 mm and J = 50 µm.</p

Dre - Cre Sequential Recombination Provides New Tools for Retinal Ganglion Cell Labeling and Manipulation in Mice

<div><p>Background</p><p>Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations.</p><p>Methods and Findings</p><p>We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, <i>Brn3a^CKOCre</i> and <i>Brn3b^CKOCre</i>. When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by <i>Brn3a^CKOCre</i> or <i>Brn3b^CKOCre</i> is removed and replaced with a Cre recombinase, generating <i>Brn3a^Cre</i> and <i>Brn3b^Cre</i> knock-in alleles. Surprisingly both <i>Brn3a^Cre</i> and <i>Brn3b^Cre</i> knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the <i>Brn3a</i> and <i>Brn3b</i> genes. We use the <i>Brn3a^Cre</i> and <i>Brn3b^Cre</i> alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs.</p><p>Conclusions</p><p>Dre recombinase effectively recombines the <i>Brn3a^CKOCre</i> and <i>Brn3b^CKOCre</i> alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated <i>Brn3a^Cre</i> and <i>Brn3b^Cre</i> alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early postnatal development, opening up a large range of potential applications.</p></div

Sequential Dre to Cre recombination suggests ubiquitous Cre expression from the Brn3a and Brn3b loci.

<p>A–C, G–I, Adult retina flat mounts and D–F, J–L, hemispheres from coronal brain sections of mice with indicated genotypes. Note that, in CAG:Dre; <i>ROSA26^iAP</i> mice, germline Dre expression does not result in induction of AP positivity from the Cre dependent <i>ROSA26^iAP</i> locus (A, D - n = 4; G, J – n = 6). However, <i>Brn3^CKOCre; ROSA26^iAP</i> tissues show a reduced level of mosaic recombination in RGCs, and corresponding projection areas in the brain (B, E - n = 9; H, K – n = 13). In contrast, tissues from CAG:Dre; <i>Brn3^CKOCre; ROSA26^iAP</i> mice (C, F - n = 4; I, L – n = 13) show complete conversion to AP positivity, suggesting that the sequential Dre to Cre to AP recombination happened in the totality (or a vast majority) of the tissue. Scale bars in C, F, I and L are 1 mm.</p

Recombination strategy used to test sequential Dre and Cre recombination.

<p>A, Genetic loci and recombination events. The CAG:Dre transgene (a), expresses ubiquitously Dre recombinase that targets the roxP sites of the <i>Brn3^CKOCre</i> locus (b), removing the endogenous exons of the Brn3 gene and replacing them with the Cre open reading frame. After Dre recombination, primer pairs Pr4 (Brn3a) or Pr6 (Brn3b) are rendered nonproductive by the loss of the reverse strand primer and novel primer pairs Pr5 (Brn3a) and Pr7 (Brn3b) are generated, between the forward strand primer remaining 5′ of the roxP site in the 5′UTR and a reverse strand primer placed in the Cre gene. The Brn3 locus starts expressing the Cre gene, which targets the inverted loxP sites of the <i>ROSA26^iAP</i> locus (c), reversing the inverted exon, resulting in productive transcription of the AP protein. B, C, Crosses generating the triple transgenic mice and the appropriate controls, for <i>Brn3a^CKOCre</i> and <i>Brn3b^CKOCre</i> alleles. Note that, to control for the germline expression of the <i>Brn3^CKOCre</i> loci, the crosses were performed in both male to female combinations. D, PCR genotyping demonstrating the presence of the AP (primer pair Pr1), Dre (primer pair Pr2) and Cre (primer pair Pr3) genes, in the various genetic knock-in combinations. E, PCR reactions demonstrating the insertion of the roxP site in the 5′ UTR of the <i>Brn3a^CKOCre</i> (iii, Pr4) and <i>Brn3b^CKOCre</i> (v, Pr6) loci. Note that all samples have one wild type chromosome, showing a band of 220 bp for Brn3a (Pr4) and 200 bp for Brn3b (Pr6), but only the Dre – negative <i>Brn3a^CKOCre</i> (iii) or <i>Brn3b^CKOCre</i> (v) genetic combinations show a roxP insertion shifted by ∼ 30 bp up (top band). In contrast, in the triple transgenic combinations (ii and iv) in which Dre recombination has occurred, the roxP insertion band is removed (top band, Pr4 and Pr6), and the Brn3-Cre reactions (Pr5 and Pr7) become positive.</p

Whole tissue sequential Dre to Cre recombination in CAG:Dre; <i>Brn3^CKOCre; ROSA26^iAP</i> mice happens before E9.5.

<p>A, wholemount staining of E12.5 <i>ROSA26^rtTACreERt</i>; <i>Brn3b^CKOAP</i> embryo demonstrating <i>Brn3b^AP</i> labeling of the germinal ridge. Inset shows 8x magnification of the gonad. B, C, wholemount E9.5 embryos showing complete AP recombination in CAG:Dre; <i>Brn3a^CKOCre; ROSA26^iAP</i> mice (C, n = 6) and sparse mosaic recombination in <i>Brn3a^CKOCre; ROSA26^iAP</i> (B, n = 9) controls. Whereas no AP positive cells are visible in E9 CAG:Dre; <i>ROSA26^iAP</i> embryos (D, n = 4), sparse recombination can be seen in <i>Brn3b^CKOCre; ROSA26^iAP</i> (E, n = 6) and full recombination in CAG:Dre; <i>Brn3b^CKOCre; ROSA26^iAP</i> (F, n = 7) littermates. Scale bars in A, C, F are 0.5 mm.</p

tdTomato positive cells in AAV1-FLEX-tdTomato infected <i>Brn3a^Cre/+</i> and <i>Brn3b^Cre/+</i> retinas express the RGC markers Neurofilament Light Chain (NFL), Brn3a and Brn3b.

<p>Eyes from <i>Brn3a^Cre/+</i> or <i>Brn3b^Cre/+</i> were infected with the Cre dependent AAV1-FLEX-tdTomato virus at P0, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone-0091435-g005" target="_blank">Figure 5</a>. At p21, eye cups were prepared and briefly inspected under a fluorescence microscope for degree of infection. Highly infected eyes were then processed for sectioning and immunostaining. Sections from either <i>Brn3a^Cre/+</i> (A–C) or <i>Brn3b^Cre/+</i> (D–F) retinas were stained with antibodies against NFL (A, D), Brn3a (B, E) and Brn3b (C, F), shown in the left panels, in conjunction with endogenous tdTomato red fluorescence, shown in the middle pannels. Images were taken and quantitations performed on areas showing highest levels of infection, as judged by tdTomato fluorescence. Red, green and yellow arrowheads point at examples of red and green single positive or double positive cells. A’–F’ Box-whisker plots representing quantitations of experiments shown in A–F. For each experiment, the numbers of marker, tdTomato, or double positive cells were normalized to the total number of DAPI positive cells in the GCL. Significance levels shown were calculated with the Kolmogorov – Smirnov test for comparisons of two unknown distributions: ns = not significant, *p<0.05, **p<0.01 For number of quantitated images, cells counted, averages, standard deviations and significance levels by Kolmogorov-Smirnov and Student T tests, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone.0091435.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone.0091435.s002" target="_blank">S2</a>. Scale bar in F = 25 µm.</p

AAV1-FLEX-tdTomato infection at P0 results in RGC specific tdTomato expression, which is already detectable at P3.5 and P7.5.

<p>Retina samples are from P3.5 (A), P7.5 (B) pups or adults (C–E). Panels in A, B are tdTomato (top), GFP (middle), and merged (bottom) images. Panels A-D are from <i>Brn3b^Cre/+</i>, and E from from <i>Brn3a^Cre/+</i> mice, infected at P0 after the protocol described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091435#pone-0091435-g005" target="_blank">figure 5</a> A, B. Red, green and yellow arrowheads point at examples of red and green single positive or double positive cells. Note that the dendritic arbor of the double positive cell in B is clearly visible in both red and green channels. C, Projections along the z direction (left) and x direction (right) of a stack from a densely labeled <i>Brn3b^Cre/+</i> retina, showing an overwhelming majority of red fluorescent cell bodies stratified in the GCL (g) layer, whereas abundant numbers of green fluorescent bodies are seen throughout the Inner and Outer Nuclear Layer (i and o). D, Projections along the z direction (top) and y direction (bottom) of a stack from a sparsely infected <i>Brn3b^Cre/+</i> retina showing a displaced RGC, with its axon (white arrowheads) and dendritic arbor, seen both from the flat mount and transversal perspective. E, z projection of a stack from a densely labeled <i>Brn3a^Cre/+</i> retina, showing single and double labeled cell bodies, and tdTomato labeled RGC axons (white arrowheads). Scale bars in B, C, D and E = 50 µm.</p