Retinae develop an ectopic amacrine cell layer and supernumerary rod photoreceptors

<p><b>Copyright information:</b></p><p>Taken from "functions through to negatively regulate cell number in the developing retina"</p><p>http://www.neuraldevelopment.com/content/2/1/11</p><p>Neural Development 2007;2():11-11.</p><p>Published online 8 Jun 2007</p><p>PMCID:PMC1913510.</p><p></p> E18.5→8DIV retinal explants. DAPI-stained wild-type and explants. Rhodopsin expression in wild-type and + ECL retinae. Pax6 and syntaxin expression in amacrine cells in wild-type (e,e',g,g') and +ECL (f,f',h,h') retinae. Asterisks mark the ECL. The duplicated IPL is labeled by ipl' in (h'). Blue is DAPI counterstain. Average of the absolute number of DAPInuclei/layer in a standard counting field in wild-type (black bar; total DAPInuclei counted in 30 fields; ONL: 23,700; INL: 9,870; GCL: 1,776), without an ECL (grey bar; total DAPInuclei counted in 9 fields; ONL: 6,615; INL: 2,826; GCL: 498 nuclei) and +ECL (white bar; total DAPInuclei counted in 27 fields; ONL: 26,175; INL: 11,968; GCL: 1,674). Percentage of each retinal cell type based on total cell counts in wild-type (black bar; HC: 56 calbindin/7,183 DAPI; AC: 1,832 Pax6/11,696 DAPI; BP: 819 Chx10/9,302 DAPI; MG: 1,003 p27/18,465 DAPInuclei; 537 CRALBP/9,169 DAPI), without an ECL (grey bar; HC: 64 calbindin/12,960 DAPI; AC: 1,558 Pax6/10,304 DAPI; BP: 1,077 Chx10/10,171 DAPI; MG: 430 p27/9,966 DAPI; 332 CRALBP/6,773 DAPI) and +ECL retinae (white bar; HC: 11 calbindin/1,924 DAPI; AC: 2,068 Pax6/11,302 DAPI; BP: 646 Chx10/9,157 DAPI; MG: 395 p27/9,921 DAPI; 240 CRALBP/3,319 DAPI). AC, amacrine cell; BP, bipolar cell; HC, horizontal cell; MG, Müller glia

Loss of results in increased proliferation and reduced apoptosis at a late stage of retinogenesis

<p><b>Copyright information:</b></p><p>Taken from "functions through to negatively regulate cell number in the developing retina"</p><p>http://www.neuraldevelopment.com/content/2/1/11</p><p>Neural Development 2007;2():11-11.</p><p>Published online 8 Jun 2007</p><p>PMCID:PMC1913510.</p><p></p> BrdU labeling (red) of E18.5 wild-type and explants cultured 4DIV (a,b) or 6DIV (c,d). Arrowheads in (d) mark ectopic proliferating cells. Percentage of BrdUnuclei in wild-type (black bar; E13.5: 2,824 BrdU/8,235 DAPI; E16.5: 2,234 BrdU/10,663 DAPI; E18.5: 2,859 BrdU/27,380 DAPI; E18.5→2DIV: 4,371 BrdU/54,554 DAPI; E18.5→4DIV: 988 BrdU/55,300 DAPI; E18.5→6DIV: 0 in 9 fields) and retinae (grey bars; E13.5: 3,555 BrdU/10,413 DAPI; E16.5: 3,369 BrdU/15,707 DAPI; E18.5: 2,212 BrdU/17,642 DAPI; E18.5→2DIV: 3,298 BrdU/35,085 DAPI; E18.5→4DIV: 3,474 BrdU/97,499 DAPI; E18.5→6DIV: 54 BrdU/11,618 DAPI). BrdU-labeling indices of individual wild-type (squares) and (triangles) E18.5→4DIV retinal explants. E18.5→4DIV wild-type (g) and (h) retinal explants labeled with CcnD1 (red). Percentage of Ccdn1cells in wild-type (black bar; 2,480 CcnD1/21,329 DAPI) and without aberrant proliferation (grey bar; 3,156 CcnD1/26,328 DAPI) and with a proliferative phenotype (w/φ; white bar; 3,266 CcnD1/18,709 DAPI) at 4DIV. Ccnd1-labeling indices of individual wild-type (squares) and (triangles) E18.5→4DIV retinal explants. E18.5→4DIV wild-type (k) and (l) retinal explants labeled with pHH3 (red). Apical (Ap) to basal (Ba) ratio of pHH3cells in wild-type (black bar; 808 ap:791 ba pHH3) and without (grey bar; 971 ap:796 ba pHH3) and with (w/φ; white bar; 1,012 ap:480 ba pHH3) a proliferative phenotype at 4DIV. Ap:Ba ratios of pHH3cells in individual wild-type (squares) and (triangles) E18.5→4DIV retinal explants. Active caspase-3 (Ac-3) expression (red) in wild-type and E18.5→4DIV explants. Blue is DAPI counterstain. Percentage of apoptotic cells in the total population of wild-type (black bars; E18.5: 71 ac-3/18,341 DAPI; E18.5→2DIV: 532 ac-3/14,995 DAPI; E18.5→4DIV: 1,266 ac-3/27,321 DAPI; E18.5→8DIV: 294 ac-3/10,209 DAPI) and (white bars; E18.5: 67 ac-3/13,768 DAPI; E18.5→2DIV: 457 ac-3/13,195 DAPI; E18.5→4DIV: 488 ac-3/24,077 DAPI; E18.5→8DIV: 212 ac-3/14,377 DAPI) retinae. Distribution of individual wild-type (squares) and (triangles) ac-3-labeling indices at 4DIV

Application of STAgR.

<p>(A) Colony PCR of a 6xSTAgR reaction using two different promoters as well as both, the canonical and the SAM loop gRNA scaffold. The gel shows a colony PCR of 22 bacterial colonies, of which seven showed the amplicon indicative of the full length STAgR reaction (2444bp). (B) Exemplary colony PCR of STAgR constructs with 0 to 8 gRNA expression cassettes. (C) A STAgR plasmid containing four gRNAs or a mixture of four single gRNA plasmids have been transfected into P19 Cells expressing dCas9-VPR. (D) After 7 days mRNA was extracted and transcript levels of target genes have been compared via qPCR. Error bars depict standard errors of the mean.</p

Functional validation of STAgR.

<p>(A) Colony PCR of a 4xSTAgR reaction (using a string sequence containing a hU6 promoter and a canonical gRNA scaffold). 24 bacterial colonies are shown, of which six present the amplicon size indicative of the full length reaction (1596 bp). Additionally marked are amplicon sizes indicative of two (823 bp) and single gRNAs (458 bp). (B) Quantification of cloning efficiencies from three different 4xSTAgR reactions (n = 130). (C) A schematic showing constructs used for functional validation of STAgR gRNAs. A gRNA targeting the GFP ORF was either delivered in a single gRNA expression vector or on each of four different positions in STAgR vectors. (D) Functional validation of STAgR vectors shown in Fig 2C. HeLa cells stably expressing d2GFP and Cas9 have been transfected with vectors depicted above. Flow cytometry indicates that STAgR constructs are similarly efficient in mutating the ORF of GFP compared to a single gRNA vector. (E) Colony PCR of a 4xSTAgR reaction using four different promoters and SAM loop scaffolds. 24 bacterial colonies are shown, of which seven colonies incorporated the amplicon size indicative of the full length reaction (2043 bp). Shorter amplicons are indicative of gRNA subsets, which vary in size, depending on the incorporated promoter.</p

The STAgR protocol.

<p>(A) An Overview over STAgR procedure. STAgR allows simple and fast generation of multiplexing vectors in one overnight reaction. STAgR is also highly customizable as diverse strings and vectors can be used to assemble expression cassettes with different promoters and gRNA scaffolds. (B) Sequences of overhang primers used for generation of STAgR vectors.</p

Additional file 4: Figure S4. of CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening

Functional analysis of CORALINA gRNAs. (A) List of most frequently sequenced alteration generated with gRNA P1-20. (B-E) Top: Schematic depicting CORALINA-derived gRNAs targeting regions in or near various human genes (HS3ST3B1 gRNA H1-46, 46 bp protospacer, PCDH8, gRNA P2- 40, 40 bp protospacer, ZNF790, gRNA Z1-35, 35 bp protospacer, PIK3AP1, gRNA P3-35, 35 bp protospacer). Control gRNAs have been shortened from the 5′ end to yield a 20 bp protospacer. Right: Bargraph depicting percentage of NGS reads displaying indels after targeting wild-type Cas9 using CORALINA-derived gRNAs in HEK293T cells. Below: List of the most frequently sequenced alterations generated by CORALINA and control gRNAs. (PDF 320 kb

Verification of microarray results by qRT-PCR and qChIP.

<p>(A) Relative expression levels of <i>Gaa</i>, <i>Has2</i>, <i>Isl1</i>, <i>Kif1b</i>, <i>Mtmr2</i>, <i>Pax6</i>, <i>Pcsk1n</i>, <i>and Snca</i> in wild type (WT, shown in black) and Pax6^+/− (shown in gray) lenses were determined using qRT-PCR as described in Methods. B2m, Hprt and Ccni transcripts were tested as internal references, and all were found unchanged between the WT and Pax6^+/− lenses. The data are expressed relative to the unchanged expression level of B2m transcripts. For statistical evaluation of the results, p-values were calculated from paired Student t-tests. (B) Validation of Pax6-binding regions in lens chromatin by qChIPs. A and B are distal regions with Pax6 binding identified in ChIP-Chip experiments and P regions are binding regions around the proximal promoters. At each of these gene loci, a non-specific region (negative signals in ChIP-Chip experiments and no candidate Pax6 binding sites predicted) was also included as a negative control. In addition, Cryaa promoter (Cryaa-P) and +6 kb region serve as positive and negative controls respectively. The specific enrichments of Pax6 binding were detected at Isl1-A, Mtmr2-A, Snca-A, Gaa-P, Kif1b-P, Pcsk1n-B and Pcsk1n-A regions. The calculation of the cutoff value (0.100 of 1% input) for background signals and specific binding signals is described in Materials and Methods.</p

Twenty-seven genes relevant to lens placode formation and lens morphogenesis show differential expression in Pax6^−/− E9.5 mutated lens placodes.

<p>(A) A list of 27 genes includes a combination of well-characterized genes in lens biology and selected differentially expressed genes in Pax6 null (<i>Sey</i>) cortex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Holm1" target="_blank">[45]</a>. The differentially expressed genes in Pax6^−/− E9.5 wild type and mutated lens placodes were identified using the Illumina Mouse6 bead microarrays as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Huang1" target="_blank">[96]</a>. Twenty-four of the 27 genes were differentially expressed in at least 50% of experiments. (B) Relative expression levels of <i>Fat4</i>, <i>Trpm3</i>, <i>Pax6</i>, <i>Has2</i>, <i>Efnb2</i>, and <i>Nav1</i> in wild type (WT, black bars) and Pax6^−/− (open bars) lens placode and mutated ectoderm were determined using qRT-PCR as described in Methods.</p

Pax6 regulates expression of Snca.

<p>(A) Identification of Pax6-binding region by ChIP-Chip in lens chromatin and corresponding luciferase reporter constructs for transfection assays. (B) Pax6 regulates <i>Snca</i> promoter/distal region in cultured cells. Transient transfections were performed in P19 embryonic carcinoma and in αTN4-1 lens cell as described in Methods. (C) Prediction of Pax6 binding sites with novel Pax6 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Xie1" target="_blank">[48]</a>. (D) EMSA validation of Pax6 binding to the probes identified by motif 1-1, 1-2 and 3-3. PD/HD, recombinant Pax6 protein containing both Pax6 paired domain (PD) and homeodomain (HD). P6CON, DNA-binding concensus for Pax6 paired domain.</p

Down-regulation of Kif1b and Snca in Sye/Sey forebrains.

<p>In situ hybridization analysis of E14 wild type (WT) and Pax6^Sey/Sey mouse embryos. Ganglionic eminence, GE.</p