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Cell Reports, Volume 2, Issue 2, 294-307, 02 August 2012
Transcriptome Analysis of the Planarian Eye Identifies ovo as a Specific Regulator of Eye Regeneration
Sylvain W. Lapan1, 2 and Peter W. Reddien1, 2, 3, ,
1 Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
Graphical Abstract
► The complete transcriptome of planarian eyes is determined ► The planarian eye expresses orthologs of eye disease genes ► The transcription factor Ovo is required for eye formation ► Similarities in eye progenitors for regeneration, tissue turnover, and embryogenesis
- Among the millions of invertebrate species with visual systems, the genetic basis of eye development and function is well understood only in Drosophila melanogaster. We describe an eye transcriptome for the planarian Schmidtea mediterranea. Planarian photoreceptors expressed orthologs of genes required for phototransduction and microvillus structure in Drosophila and vertebrates, and optic pigment cells expressed solute transporters and melanin synthesis enzymes similar to those active in the vertebrate retinal pigment epithelium. Orthologs of several planarian eye genes, such as bestrophin-1 and Usher syndrome genes, cause eye defects in mammals when perturbed and were not previously described to have roles in invertebrate eyes. Five previously undescribed planarian eye transcription factors were required for normal eye formation during head regeneration. In particular, a conserved, transcription-factor-encoding ovo gene was expressed from the earliest stages of eye regeneration and was required for regeneration of all cell types of the eye.
The vertebrate eye is a structure of striking complexity in form and function. Consequently, understanding the evolution and development of eyes has been a classic challenge. The human eye is also subject to numerous pathologies that are poorly understood, such as heritable retinopathies and age-related degeneration. Genetic studies in model invertebrates have the potential to advance the understanding of eye evolution and development, and of the functions of conserved genes associated with eye disorders.
The relevance of invertebrate models to vertebrate systems depends in part on whether homology exists between most bilaterian eyes, a difficult point to establish based on morphological studies alone. Establishing some degree of common ancestry between vertebrate and invertebrate (primarily Drosophila) visual systems has been a success of comparative molecular genetics. Two findings figure prominently in understanding the relationship between diverse animal eyes. First, two photoreceptor neuron categories, rhabdomeric (microvillar) and ciliary, exist throughout the Bilateria and rely on conserved R-opsin and C-opsin signal transduction pathways, respectively (Arendt, 2003). Second, embryonic development of multiple eye types involves Pax-6, Sine oculis, Eyes absent, and Otx gene family members. These observations have led to the suggestion that photoreceptor cell types were already present prior to the existence of the Bilateria, and that the common ancestor of the Bilateria utilized transcription factors for eye development still commonly used in extant eyes (Nilsson, 2009).
To explore many conserved features of eye biology, additional invertebrate model eyes must be established. The morphology, cell type composition, and set of gene activities present in ancestral bilaterian eyes remain largely unknown, and can only be inferred using data from multiple extant eyes belonging to diverse animal phyla. Invertebrate models are not yet established for extensive genetic study of several common aspects of eye biology not characteristic of Drosophila eyes, such as the formation of optic cups. Furthermore, many genes associated with eye disease have not yet been identified and studied in invertebrate models. Certain invertebrate species allow for study of regenerative repair of eye damage, an increasingly important therapeutic approach.
The planarian Schmidtea mediterranea is an emerging invertebrate model system that is highly amenable to gene function studies with RNAi. Planarians are ideal for the study of eye evolution because they are members of the Lophotrochozoa, the sister grouping of phyla to the Ecdysozoa (which includes Drosophila and Caenorhabditis elegans), and therefore exist at an important and understudied position in the animal phylogeny (Tessmar-Raible and Arendt, 2003). Planarian eyes are true cerebral eyes, as they connect via axon tracts to the brain (Agata et al., 1998), and express orthologs of Otx (Umesono et al., 1999), Sine oculis (Pineda et al., 2000), and Eyes-absent (Mannini et al., 2004). Planarians have rhabdomeric photoreceptor neurons (PRNs), pigment cells (PCs), and a pigmented optic cup structure - features that are common among cerebral eyes (Figure 1A). Photoreceptive organelles in the planarian eye face the optic cup in an inverse orientation, similar to the orientation of ciliary vertebrate photoreceptors with respect to the retinal pigment epithelium (RPE) (Figure 1A). Finally, planarians possess unique abilities to regenerate entire eyes, and replenish eye tissue throughout adulthood. These abilities require a population of adult regenerative cells (neoblasts) that includes pluripotent stem cells (Wagner et al., 2011).
Here we use eye purification and RNA-seq to identify most genes active in planarian eyes. These data demonstrate conserved features of the R-opsin signaling cascade, candidate rhabdomeric microvillus-regulating genes, and unexpected similarities between rhabdomeric and ciliary cell types. In addition, a high degree of similarity exists between the types of solute transporters expressed in planarian optic PCs and the vertebrate RPE, suggesting that optic pigment cells have an ancient role as an accessory eye cell type distinct from photoreceptors. We describe several conserved transcription factors expressed in the planarian eye that are functionally required for eye regeneration, including a central role for the conserved gene ovo in formation of all eye progenitors.
Planarian Eye Purification and the Eye Transcriptome
To obtain pure eye tissue for gene expression analysis, we developed a dissociation protocol for isolating ∼200 morphologically intact planarian eyes in 1–2 hrs (Figure 1B). Greater than 96% of cells in the purified eye preparation were indeed eye cells, as determined using an opsin RNA probe for PRNs and melanin as a marker for PCs (Figure 1B). RNA from purified eyes was used to generate a cDNA library for quantitative RNA sequencing (RNA-seq). Animals were fed prior to eye harvesting to stimulate growth and incorporation of new cells in eyes. All genes previously described to be expressed in planarian eyes, and many phototransduction-related genes, displayed at least 4-fold enrichment in our eye reads compared to reads from control tissue (ventral, anterior tissue enriched for neurons), in terms of fragments per kilobase per million fragments mapped (FPKM) values (Figures 1C–1E) (See Experimental Procedures for details). pax6A, pax6B, rax, six3, and dachshund are orthologs of genes with roles in eye development in other organisms, but have been described to lack detectable expression and function in the planarian eye (Lapan and Reddien, 2011; Mannini et al., 2008; Pineda et al., 2002; Pineda and Saló, 2002); all of these genes displayed FPKM values of zero (Figure 1D), demonstrating specificity of the data and further suggesting that these genes have no role in planarian eyes. We conclude that transcript levels in the RNA-seq data set can be highly predictive of gene expression in vivo.
For an in situ expression screen we selected unpublished planarian genes with similarity to genes in Drosophila and vertebrates and > 4-fold expression enrichment in the eye. Of these approximately 600 genes (Table S1. Information for Genes with Enriched Expression in Planarian Eyes, Related to Figure 1), nearly 200 were tested by in situ hybridization (Figure 2 and Table S2. Summary of In Situ Hybridization Results for Screened Genes, Related to Figure 2) in regenerating heads 7 days after amputation. PRNs and PCs can be identified by position and whether the eye expression domain curves toward (PRNs) or away from (PCs) the midline (Figure 2A). 93% of genes screened by in situ had detectable expression in eyes. 60% were expressed in PRNs, 21% were expressed in the pigmented optic cup cells, and 12% were detected in both cell types (Table S2. Summary of In Situ Hybridization Results for Screened Genes, Related to Figure 2).
Planarian Phototransduction Genes
Rhodopsin signaling components displayed eye-enriched expression in the RNA-seq data set (Figures 1C and 1E) and in situ hybridizations revealed expression in photoreceptor neurons for two R-opsin orthologs (Sánchez Alvarado and Newmark, 1999), two β-arrestin orthologs (Agata et al., 1998), Gα-q (Smed-gna-q), PLC-β (Smed-plcb), INAD (Smed-mpdz), PKC, and two Trp channel-encoding genes (Smed-trpc-1, Smed-trpc-2) (Fain et al., 2010) (Figures 1C, 1E, and 2B). Planarian PRNs also had abundant expression of genes encoding enzymes of the phosphoinositide cycle (Figures 1C, 1E, and 2B), which replenishes PIP2 after its hydrolysis by PLC (Wang and Montell, 2007). These include retinal degeneration a (Smed-dagk), retinal degeneration b (Smed-pitp), dpis (Smed-pis), and cds (Smed-cds).
Genes with predicted roles in cGMP signaling and intracellular calcium regulation, which are involved in phototransduction in multiple organisms, were also expressed in planarian eyes. Specifically, PRNs expressed orthologs of IP3-receptors (Smed-ip3r) and STIM (Smed-stim) (Figures 2B and S1), which regulate intracellular calcium levels (Smyth et al., 2010). PRNs also expressed genes encoding the cGMP pathway components guanylate cyclase (Smed-gucy-1, Smed-gucy-2), cGMP-dependent phosphodiesterase (Smed-cgs-pde), RGS7/9 (Smed-rgs6/7/9), a cGMP-gated ion channel (Smed-cng), and a hyperpolarization-activated cyclic nucleotide-gated channel (Smed-hcng) (Figures 1E, 2B, and S1). Many of these components mediate signaling downstream of C-opsin in vertebrates (Arendt, 2003), but we were unable to identify an ortholog of C-opsin in planarians. GO analysis of the RNA-seq data indicated that Rhodopsin signaling, cGMP signaling, and intracellular calcium regulation were all enriched categories in the data set of eye-expressed genes (Table S3. GO Categories Enriched in Eye Transcriptome versus Whole Planarian, Related to Figures 1–7).
Expression of Microvillus-Related Genes in Photoreceptor Neurons
Unexpectedly, orthologs of genes involved in the function and development of auditory hair cells displayed enriched expression in planarian eyes and were prominently represented in GO analysis (Figures 2B and 2C; Table S3. GO Categories Enriched in Eye Transcriptome versus Whole Planarian, Related to Figures 1–7). Like planarian photoreceptors, hair cells are sensory neurons with abundant apical microvilli. Microvilli are actin-based structures, and we identified enriched PRN expression of genes encoding actin (Smed-actin-1), the actin-regulating proteins Ezrin/Radixin/Moesin (Smed-erm), Clic (Smed-clic), Maguk (Smed-maguk), Gelsolin (Smed-gls), and the actin-based motor Myosin VI (Smed-myoVI). These genes have all been shown to have roles in morphogenesis and function of vertebrate hair cells (Gagnon et al., 2006; Kitajiri et al., 2004; Mburu et al., 2006, Mburu et al., 2010; Self et al., 1999). Auditory hair cells of human and mouse also express Usher syndrome genes, which function in morphogenesis of microvillus bundles (Frolenkov et al., 2004). Planarian PRNs expressed orthologs of three Usher syndrome genes, Myosin VIIA/Ush1B (Smed-myoVIIA), Sans/Ush1G (Smed-sans), and Cadherin 23 (Smed-cdh23) (Figures 2B and 2C). These genes are also expressed in vertebrate photoreceptors, despite the fact that the latter are ciliary. The function of Usher syndrome proteins in vertebrate photoreceptors is not understood (Williams, 2008), but many localize to the periciliary region and Usher syndrome can result in aberrant photoreceptor cilia morphology (Hunter et al., 1986). Many of the hair cell-related genes described here, including Usher syndrome genes, have not been described to be expressed in Drosophila photoreceptors. Therefore, our analysis describes unexpected similarity between a rhabdomeric photoreceptor and auditory hair cells, identifying genes that are candidates to regulate microvillus-like apical membrane specialization.
Genes of the Pigmented Optic Cup
The vertebrate RPE supports photoreceptor function by providing organic molecules, maintaining extracellular ion concentrations, phagocytosing aging photoreceptor components, and absorbing light energy (Strauss, 2005). Many genes expressed in planarian PCs were orthologs of genes important for the support function of RPE cells. The metabolic rate of the vertebrate retina necessitates high levels of available glucose. Planarian PCs expressed orthologs of Glut3 (Smed-glut3) (Figures 2B and 2C) and MCT transporters (Smed-mct-1 and Smed-mct-2) (Figures 2B and 2C), which transport glucose to the retina (Ban and Rizzolo, 2000) and remove lactic acid waste (Bergersen et al., 1999), respectively. Lactic acid uptake is coupled to proton uptake (Lin et al., 1994; Lin and Miller, 1991) and two genes encoding pH-regulatory proteins, a Na+/H+ exchanger regulatory cofactor (Smed-nhe-rf) and a sodium bicarbonate cotransporter (Smed-nabct), were expressed specifically in the planarian PCs (Figures 2B and 2C). High metabolic activity in the retina results in excess water production, which is eliminated using chloride gradients across the RPE (Strauss, 2005). Orthologs of the RPE-expressed chloride channels Bestrophin-1 (Smed-best-a) and Cftr (Smed-cftr) were strongly expressed in the PCs of planarian eyes (Figures 2B and 2C).
Planarians are similar to vertebrates, but differ from most other invertebrates, in the use of melanin as the primary eye shading pigment (Hase et al., 2006; Strauss, 2005). Among the most abundantly expressed genes in planarian PCs were those encoding orthologs of enzymes required for melanin synthesis in vertebrates, including Tyrosinase (Lapan and Reddien, 2011) (Figure 1D), Aromatic amino acid hydroxylase (Nishimura et al., 2007) (see "tph,"Figure 1D); and Dopa decarboxylase (Smed-ddc) (Figures 2B and 2C). PCs also expressed orthologs of Quinoid dihydropteridine reductase (Smed-qdr), which produces an essential cofactor of tyrosine/phenylalanine hydroxylase (Schallreuter et al., 2008); and Glutathione-S-transferase (Smed-gst), which catalyzes addition of glutathione during pheomelanin polymerization (del Marmol et al., 1996) (Figures 2B and 2C).
Peroxide is generated by melanin-synthesizing enzymes and is required for melanin polymerization, but it can also cause cytotoxicity in melanocytic cells (Mastore et al., 2005). The GO category for "Response to hydrogen peroxide" was significantly enriched in the eye RNA-seq data (Table S3. GO Categories Enriched in Eye Transcriptome versus Whole Planarian, Related to Figures 1–7). Two orthologs of catalase, Smed-cat-1 and Smed-cat-2, were expressed in the planarian PCs. Similar to tyrosinase, Smed-cat-1 was one of the most strongly and specifically expressed genes in the planarian PCs (Figures 2B and 2C and Table S2. Summary of In Situ Hybridization Results for Screened Genes, Related to Figure 2). Glutathione-S-transferase might also function as an antioxidant enzyme in the eye (Giblin, 2000; Singhal et al., 1999). Therefore, planarian and vertebrate optic PCs express similar melanin-synthesizing enzymes and enzymes that protect from oxidative damage.
Heterogeneity of the PRN and PC Populations
Recently, planarian photoreceptor neuron heterogeneity has been described, with pro-hormones and smad6/7-2 expressed in distinct domains of the PRN population (Collins et al., 2010; González-Sastre et al., 2012). Smed-soxB, Smed-best-b, Smed-pcdhP-1, and Smed-pthr were expressed only in anterior photoreceptors (Figure S2A). Smed-fzdP-1 was expressed primarily in dorsal and anterior photoreceptors, as well as progenitors near the eye (Figure S2A). Smed-zfp-2, Smed-pctaire, and Smed_19866 were expressed only in posterior photoreceptors (Figure S2B). We also identified a gene, Smed-actin-2, expressed in a subset of pigment cup cells, indicating that heterogeneity also exists within this population (Figure S2C).
Identification of an Eye-Specific Transcription Factor-Encoding Gene, Smed-ovo
Molecular regulators remain to be identified for key steps in planarian eye formation, including progenitor migration, homotypic cell aggregation, mesenchymal-to-epithelial transition, and cupping morphogenesis. In situ screening identified enriched expression in the planarian eye for many genes that are good candidates to encode proteins regulating these processes, including 14 kinases, 5 protocadherins, and 18 signaling receptors (Figures 2B, 2C, and S1).
Transcription factors that control eye development in both Drosophila and vertebrates have received close attention for their capacity to elucidate the evolution of eye development. Here, we have identified ten conserved genes predicted to encode transcription factors with enriched expression in planarian eyes (Figures 2B and 2C). The gene with the most prominently enriched expression was a member of the Ovo family of zinc finger transcription factor-encoding genes (Figure S3), which includes the Drosophila gene ovo (shavenbaby) and mouse ovol1-3 genes. Expression of Smed-ovo (ovo) was detected in both PRNs and PCs, as well as in mesenchymal cells posterior to the eye in day 7 head blastemas (Figures 2B and 3A). After head amputation, ovo+ cells first became apparent throughout the dorsal anterior region, near the wound, by 2 days post-amputation (Figure 3A). At later regeneration time points ovo+ eye aggregates formed in the anterior blastema, while dispersed cells posterior to the eye remained visible. This pattern was similar to a recently defined population of regenerative eye progenitors in planarians (Lapan and Reddien, 2011). Accordingly, nearly all ovo+ cells at day 2 and day 7 of regeneration also displayed expression of six-1/2 and eya, which marks eye progenitors (Figures 3B and 3C).
In regenerating or intact animals, no ovo expression was detected outside of eyes or eye progenitors (see below). Furthermore, in the control (ventral anterior tissue) sequencing data, the FPKM value for ovo was near zero (Table S2. Summary of In Situ Hybridization Results for Screened Genes, Related to Figure 2 and Figure S4A). Other previously known eye-expressed transcription factors, eya, six-1/2, sp6-9, and otxA also have expression in the brain and/or elsewhere in the body (Lapan and Reddien, 2011; Pineda et al., 2000; Umesono et al., 1999). Therefore, ovo is the only known planarian transcription factor with expression exclusively in the eye and eye progenitors. It is one of only three transcription factor-encoding genes, together with six-1/2 and eya, that is expressed in all cells of the eye.
ovo Expression Is Required for Eye Regeneration
RNAi of ovo followed by decapitation resulted in animals that failed to regenerate eyes (Figure 3D). These animals also lacked detectable tyrosinase mRNA and Arrestin protein (Figure 3E), and no eye structure could be detected by differential interference contrast microscopy (DIC) (Figure S4B). We studied ovo(RNAi) animals using six-1/2/eya co-expression to label all eye progenitors, and otxA/eya and sp6-9/eya co-expression to label PRN and PC progenitors, respectively (Lapan and Reddien, 2011). ovo RNAi resulted in failure to generate any eye aggregate (primordium), and loss of most eye progenitors (Figures 4A and 4B). We observed no defects that were unrelated to the eyes, consistent with the highly specific expression of this gene. For example, other cell types of the head, such as sp6-9+/eya- cells of the head rim appeared unaffected (Figure 4A). Therefore, eyes and eye progenitors are strongly and specifically affected in ovo(RNAi) animals.
Formation of ovo+ Progenitors Requires Expression of Other Eye Transcription Factors
In gene regulatory networks involved in Drosophila and vertebrate eye development, several transcription factors that regulate eye formation are required for the expression of each other (Silver and Rebay, 2005; Zuber et al., 2003). We tested whether ovo+ progenitor cells are sensitive to expression of other planarian eye transcription factors. RNAi of six-1/2 and eya resulted in animals with greatly reduced or absent ovo signal in the eyes and eye progenitor region (Figures 4C and 4D). RNAi of the cell type-specific transcription factors otxA and sp6-9, required for PRN and PC regeneration respectively, also impacted the presence of ovo+ progenitors (Figures 4E and 4F). RNAi of otxA led to a 34% reduction in ovo+ cells; this reduction was primarily a result of loss of ovo+ PRN progenitors (Figures 4E and 4F), because most remaining ovo+ cells were sp6-9+. Similarly, RNAi of sp6-9 led to a 60% reduction in ovo+ cells, primarily a result of loss of ovo+ PC progenitors, because most remaining ovo+ cells were otxA+ (Figures 4E and 4F). These large decreases indicate that ovo-expressing cells, even in early stages of eye progenitor formation, are sensitive to the expression of six-1/2, eya, otxA, and sp6-9 transcription factors.
ovo Is Expressed in a Population of Eye Progenitors during Homeostasis and Is Required for Maintenance of Intact Eyes
The specificity of ovo expression for eyes and eye progenitors allowed us to ask for the first time whether subsets of neoblasts in uninjured animals are specialized for particular differentiation paths. In intact animals undergoing normal adult homeostasis, ovo was expressed not only in the eye but also in sparse cells posterior to the eye and anterior to the pharynx (Figure 5A). Adult planarians undergo perpetual tissue turnover (Newmark and Sánchez Alvarado, 2000), but the identity and origin of progenitors that replenish eyes in uninjured animals is unknown. ovo+ cells posterior to the eye in intact animals expressed eya and six-1/2 (Figure 5B), and those nearer to the eye also expressed markers for differentiated photoreceptor neurons (trpc-1) or PCs (tyrosinase) (Figure S5A). Therefore, ovo+ cells that exist posterior to the eye in intact animals have a gene expression profile seen elsewhere only in cells of the eye.
The localization of the ovo+ cells posterior to the eye in intact animals is consistent with the location of eye progenitors during regeneration following head amputation (Lapan and Reddien, 2011). Strikingly, these cells were eliminated within 1 day of irradiation (Figure 5A), a treatment known to specifically eliminate the dividing cells of the adult animal (the neoblasts) (Reddien and Sánchez Alvarado, 2004). Furthermore, many ovo+ cells in this prepharyngeal population expressed the neoblast-specific markers histone h2b (Figure 5C) and smedwi-1 (Figure S5B) (Reddien et al., 2005). It is unknown whether these ovo+ neoblasts are self-renewing or constantly produced from a more naive cell. These results suggest that eye progenitors constitutively exist in the neoblast population.
ovo RNAi in intact animals resulted in gradual loss of eyes, with pigment cups undetectable following two months of RNAi (Figure 5D). These un-amputated ovo(RNAi) animals also lacked six-1/2+/eya+ cells in the region of putative homeostatic eye progenitors (Figures 5E and 5F). Reciprocally, homeostatic RNAi of six-1/2 and eya, in addition to causing loss of eye tissue, caused a loss of ovo+ cells in the prepharyngeal region (Figures 5G and 5H). Overall, these data suggest that ovo, six-1/2, and eya are expressed in a population of eye progenitors during homeostasis as well as regeneration and are required for maintenance of this population.
Smed-soxB, Smed-foxQ2, Smed-klf, and Smed-meis Regulate Eye Differentiation and Morphogenesis
RNAi of genes encoding four additional conserved transcription factors, Smed-soxB, Smed-foxQ2, Smed-klf, and Smed-meis (Figure S3) resulted in PRN aggregates that were smaller in cross-section than in the control (Figures 6A–6C). All of these genes were expressed in PRNs and eye progenitors during regeneration (Figure S6A). Photoreceptor aggregates in klf(RNAi) animals were also displaced posteriorly relative to the pigment cup and descended unusually far toward the interior of the animal along the D-V axis, embedding within the brain (Figures S7A and S7B).
Small eye phenotypes were not accompanied by an evident decrease in the number of ovo+ progenitors in these RNAi animals (Figures S6B, S6C, and 6F), suggesting that these genes function primarily following progenitor specification. Because soxB expression was restricted to anterior PRNs, we assessed the presence of photoreceptor neuron subsets in soxB(RNAi) animals. Anterior PRNs (eye53-1+ [Collins et al., 2010] and best-b+) were strongly reduced or eliminated in soxB(RNAi) animals (Figure 6D). In contrast, posterior PRNs (pctaire+) were not affected (Figure 6D). soxB RNAi did not significantly impact numbers of PC (sp6-9+/ovo+) or PRN (sp6-9-/ovo+) progenitors (Figures 6E and 6F), and soxB is not abundantly expressed in the ovo+ progenitor population (Figure S6A). However, soxB eye signal is eliminated by ovo RNAi (Figure S6D). These data indicate that whereas ovo (along with six1/2, eya, and otxA) acts in general PRN progenitor specification (Figures 4A and 4E), soxB acts subsequently to promote differentiation of an anterior subset of PRNs.
Several RNAi phenotypes affected the pigment cup shape. Loss of photoreceptor cells by RNAi of PRN-specific genes, such as otxA, results in pigment cups with diminished apertures (Lapan and Reddien, 2011). Accordingly, in klf and foxQ2 RNAi animals, optic cups were more closed than in the control, or even completely circularized (Figures 6A and 6B), possibly explained if rhabdomeres provide support for optic cup opening. In meis(RNAi) animals, however, optic cups were also elongated and often more than one cupped structure was visible in each eye - a defect not characteristic of photoreceptor cell loss (Figures 6B and S7C). Although meis expression was not detected in the pigment cup, meis transcripts are detectable in tyrosinase+ PC progenitors (Figure S7D). Meis1 loss in mouse also disrupts optic cup morphogenesis, with optic cup duplication observed (Hisa et al., 2004).
Developing Eyes of Planarian Embryos Express Transcription Factors Involved in Eye Regeneration
Most eye formation studies in other organisms are performed in developing embryos. Therefore, we investigated whether the transcription factors that govern planarian eye regeneration might also be expressed during planarian embryonic development (Figure 7A). Recent work has shown that beginning at stage 6, Schmidtea polychroa embryos have expression of eya, six-1/2, otxA in the eyes, and lack expression of pax6A, rax, and dachshund in the eye domain (Martín-Durán et al., 2012), similar to the case in regeneration. We found that from early stages of S. mediterranea embryonic eye formation, ovo was expressed in the eyes and in mesenchymal cells posterior to the eye that were reminiscent of regenerative progenitor eye trails (Figure 7B). Double FISH (fluorescent in situ hybridization) with ovo enabled us to determine whether regeneration-related eye genes were expressed in the embryonic eye primordium and/or presumptive earlier progenitors. Embryonic eyes expressed all known transcription factors that function during eye regeneration: eya, six-1/2, otxA, sp6-9, klf, meis, foxQ2, and soxB (Figure 7C). Regionalization of expression in the embryonic eye mirrored the case in regeneration (Lapan and Reddien, 2011); for instance, otxA was expressed in the lateral eye primordium whereas sp6-9 was expressed in the medial eye primordium, and both were expressed in only a subset of ovo+ trail cells (Figure 7C). Furthermore, neither the eye primordium (Martín-Durán et al., 2012) nor the ovo+ trail cells (Figure 7D) expressed pax6A, rax, dachshund, or another Pax6 ortholog, pax6B. Therefore, findings regarding genetic regulation of eye regeneration are relevant to planarian embryonic development.
Planarian Eye Formation in Regeneration, Homeostasis, and Embryogenesis
The cellular events bridging the pluripotent state and cellular differentiation during planarian regeneration and homeostasis are poorly understood, and the eye is emerging as a system to study adult lineage specification in this animal (Lapan and Reddien, 2011). ovo is the first identified gene that is expressed in all eye progenitors and is specifically expressed in eye cells, and therefore is a marker that facilitates study of progenitor dynamics during various modes of planarian eye formation. Our investigations of ovo expression lead to three conclusions concerning planarian eye formation. (1) Isolated eye progenitors are present early in regeneration (by 2 days after decapitation), prior to the appearance of an aggregated eye primordium. Therefore, specification at a distance from the aggregated eye occurs from the onset of regeneration rather than only at later stages of eye growth (Lapan and Reddien, 2011). (2) Eye progenitors exist in uninjured intact animals in a dorsal prepharyngeal domain, similar to regenerative progenitors but at lower density. These data indicate that some neoblasts are specialized for specific lineages during normal tissue turnover conditions of adult life. (3) During embryogenesis, ovo+ cells are present in the eye and in a trail-like formation posterior to the eye.
Further experiments are required to formally determine whether, like regenerative progenitors (Lapan and Reddien, 2011), homeostatic and embryonic eye progenitors move toward the eye primordium. However, our ovo expression analyses indicate that migration of differentiating mesenchymal cells, rather than patterning of an epithelium, might be a feature of all modes of eye formation in planarians. Our findings advance the use of the planarian eye as a system for exploring the extent of similarity between embryonic, regenerative, and homeostatic modes of organ formation within a species and between species.
Eye Purification and Transcriptome Analysis
Canonical phototransduction cascades have been described for ciliary and rhabdomeric phototransduction based on functional investigations in vertebrates and Drosophila, respectively (Arendt, 2003). However, a diversity of phototransduction strategies likely exists among animals. For instance, horseshoe crab (Limulus) and Drosophila light sensation both begin with an R-opsin/PLC cascade, but Drosophila phototransduction culminates in Trp channel opening (Katz and Minke, 2009). By contrast, most evidence from Limulus indicates that opening of cyclic nucleotide-gated (CNG) ion channels (preceded by IP3-dependent intracellular calcium release and changes in cGMP levels) is responsible for photoreceptor depolarization (Garger et al., 2004). We observed robust expression for orthologs of nearly all central Drosophila phototransduction components in planarian photoreceptors, including two Trp channels. However, we also detected enrichment for intracellular calcium regulation and cGMP pathway-like components, including CNG channels. Based on the high levels of Trp channel gene expression, we hypothesize that these are the primary mediators of membrane depolarization following light detection. The roles of CNG channels in planarian eyes are unknown, but electrophysiological recordings combined with RNAi could be used to test possible functions, such as in the regulation of light response or light/dark adaptation.
The planarian pigmented optic cup has numerous similarities to the RPE of vertebrates. Melanin is not commonly found in protostome eyes; however, it is the primary pigment of both planarian PCs and the RPE. We expand upon this similarity by demonstrating that the enzymes used in melanin production and for protection from oxidative damage (a consequence of melanin production) are similar between these cell types. Because of the wide divergence in identity of ocular shading pigment among invertebrates (Vopalensky and Kozmik, 2009), the common synthesis of melanin in planarians and vertebrates need not indicate common ancestry of optic PCs. However, we identified additional similarities between planarian PCs and the vertebrate RPE with respect to expression of numerous metabolite and solute transporters, consistent with a common function of these cell types as components of a transport epithelium. This similarity supports a model in which pigmented cells already existed as an accessory cell type separate from photoreceptor neurons in the ancestral bilaterian eye.
The RPE is commonly affected in eye diseases, including age-related macular degeneration (AMD), edema, and inherited retinal degeneration syndromes. A molecular explanation for pathogenesis is lacking for most of these diseases. Planarian PCs express orthologs of two RPE chloride channels underlying eye diseases. Best-1 is mutated in Best vitelliform macular dystrophy, and Cftr mutations underlie vision defects in cystic fibrosis patients (Marmorstein et al., 2009; Xiao et al., 2010). Furthermore, RPE degeneration in AMD correlates with peroxide accumulation and declining Catalase function, which is also responsible for degeneration of other melanocytic cell types (Venza et al., 2012; Wood et al., 2009). catalase-1 is very abundantly and specifically expressed in the planarian pigment cup. Molecular similarities between Drosophila ocular PCs and the vertebrate RPE have been difficult to identify (Charlton-Perkins and Cook, 2010). The planarian eye should therefore prove useful as an invertebrate model system for investigating orthologs of mammalian RPE genes involved in pathogenesis, as well as for inferring ancestral functions of ocular PCs.
Transcription Factor Control of Planarian Eye Formation
Comprehensive sequencing of RNA from intact eyes identified three genes that encode transcription factors and that are expressed in all cells of the eye and their progenitors—six-1/2, eya, and ovo. The requirement for six-1/2 and eya in planarian eyes and eye progenitors is previously described (Lapan and Reddien, 2011; Mannini et al., 2004; Pineda et al., 2000) and both are homologs of well-known eye development genes (Figure 7E). We propose a model for eye regeneration in which six-1/2, eya, and ovo expression specifies all eye progenitor formation, with additional expression of otxA specifying PRNs and additional expression of sp6-9 and dlx specifying PCs (Figure 7F).
How do available planarian eye data impact models about transcriptional regulators governing eye formation in the common ancestor of the Bilateria? Planarian data support the inclusion of Otx and Eya as ancestral bilaterian eye genes, and of Six-1/2 as an ancestral eye gene for the protostomes (Figure 7E). Our data also support the possibility that Meis and SoxB family genes encode transcription factors with ancestral roles in eye biology. Meis genes regulate eye progenitor proliferation in Drosophila and vertebrates as well as optic cup morphology in mouse (Figure 7E) (Bessa et al., 2002; Heine et al., 2008; Hisa et al., 2004). The SoxB family sox-neuro and fish-hook genes are expressed in the Drosophila eye disc (Mukherjee et al., 2000), and Sox2 is required for neural progenitor maintenance and differentiation of retinal ganglion cells in vertebrates (Matsushima et al., 2011).
In contrast to Otx, Eya, Six-1/2, Meis, and SoxB, several transcription factors that have prominent roles in Drosophila and/or vertebrate eye formation—Pax6, Dachshund, Rax, and Six3—have no apparent role in planarian eye formation (Figure 7E) (Lapan and Reddien, 2011; Mannini et al., 2008; Martín-Durán et al., 2012; Pineda et al., 2002), a conclusion extended by our eye transcriptome and embryonic eye expression data. Therefore, planarian data does not support the argument that these genes are ancestral regulators of eyes. Loss of a role for any of these genes in eye biology during planarian evolution is clearly a reasonable possibility, especially in cases such as Pax6 where data from multiple other phyla support an eye role for the gene. In addition to the case of the planarian eye, however, the development of Drosophila larval ocellus, branchiostome eye cups, Limulus eyes, and Platynereis adult eyes also does not depend on Pax6 (Vopalensky and Kozmik, 2009). Furthermore, orthologs of the Drosophila eye regulator Dachshund do not have major functions in mouse eye development (Davis et al., 2008), and an ortholog of the vertebrate eye regulator Rax is not known to be involved in Drosophila eye development (Arendt et al., 2004). Six3 family transcription factors predominate in vertebrate eye development, whereas most invertebrates depend on Six-1/2 (Vopalensky and Kozmik, 2009). Analysis of additional animal eyes will therefore be an important direction for further testing evolutionary hypotheses related to these genes and eye evolution.
We describe a number of transcription factors conserved among the Bilateria that regulate planarian eye formation, but for which the functions in other animal eyes has not been extensively investigated (Figure 7E). These include ovo, klf, and foxQ2. Our prior work also indicated prominent roles for dlx and sp6-9 in optic cup formation (Lapan and Reddien, 2011). One gene encoding an Ovo-like transcription factor, shavenbaby, is known to exist in Drosophila. This gene is well-studied in epidermal morphogenesis, but roles for this gene in the eye have not been reported. The chromosomal locus responsible for the blind-sterile phenotype in mouse contains the ovol2 gene (Li et al., 2002). These mice exhibit small eye size as well as early onset cataracts; however, other genes are contained in this locus and no causal role for ovol2 in this phenotype is known. Mouse ovol2 is expressed in the surface ectoderm of the ventral forebrain at E8.75-9.5 (Mackay et al., 2006), the site of optical eminence formation. Interestingly, ovol2 knockout mice die at E10.5 without displaying any sign of optic vesicle formation typical of this stage, despite the fact that D-V expression domains examined in the neural tube are maintained and overall embryonic development is not delayed (Mackay et al., 2006; Unezaki et al., 2007).
We demonstrate an unbiased technical approach to systematically survey the molecular basis of eye formation and function. This approach identified novel and conserved genetic features of eyes in planarians, further indicating the power of planarians as a system for study of animal eyes. Few transcription factors in animals display eye-specific expression and are required for formation of all eye cells, and ovo is a newly discovered one.
The Klf family of zinc finger transcription factors includes many members with diverse roles in development, but prominent functions in retinal development have not been described. In Drosophila, luna causes disrupted differentiation in the adult eye (De Graeve et al., 2003) and krüppel causes defects in organization and axon pathfinding of photoreceptors in the larval ocellus (Schmucker et al., 1992). We were unable to assign orthology of the planarian gene to a specific subfamily of Klf genes.
FoxQ2 is a subfamily of the Forkhead transcription factor family that has been lost from mammals. FoxQ2 has been studied in basal deuterostomes, where it is involved in early embryonic polarity (Yaguchi et al., 2008; Yu et al., 2003).
Little is known about the representatives of this subfamily in C. elegans (fkh10) or Drosophila (fd102c).
This planarian gene encodes a protein with three C2H2 zinc finger domains, which could not be definitively assigned to a particular zinc finger protein family in other species. It is unclear whether this gene encodes a transcription factor.
smad6 and smad7 orthologs encode inhibitory Smad proteins. Members of this family are induced by TGF-beta signaling and cause negative feedback regulation of pathway activity (Moustakas and Heldin, 2009). TGF-beta proteins are widely used in eye development in both vertebrates and Drosophila, but specific roles for inhibitory Smads are not described. A role for Smed-smad6/7 in planarian eye regeneration has recently been reported (González-Sastre et al., 2012).
Smed-Crooked Legs
This encodes a C2H2 zinc finger-containing transcription factor that, in Drosophila, is induced by ecdysone and mediates metamorphic processes (D'Avino and Thummel, 1998). A role in the Drosophila eye is not described. A deuterostome ortholog has not been described.
Nfi transcription factors have multiple described roles in neurogenesis and axon growth in mammals (Piper et al., 2010), but no roles in retinal development are described. There is no described function for the Drosophila ortholog, nuclear factor I.
Vertebrate Nkx1 homeobox genes are broadly expressed during development (Bae et al., 2004) but there are no described roles for these genes in the eye. The Drosophila ortholog of this homeobox gene family is slouch, which is implicated in muscle patterning but has not been described to function in eye development.
Experimental Procedures
Tissue Isolation
Large asexual Schmidtea mediterranea were used for eye preparation. Animals were fed twice prior to eye harvest. Head tips were amputated posterior to the eyes and collected into CMFB + 1 mg/mL collagenase (Sigma) + 0.05% NAC and agitated. Freed eyes were rinsed and transferred to Trizol for RNA collection. For control (ventral anterior) tissue, heads were amputated above the pharynx and then coronal amputation was made to remove dorsal tissue, including eyes.
cDNA Sequencing and Analysis
Approximately 1 ug of RNA was used to construct Illumina libraries for 36bp single-end sequencing on the GAII platform. Libraries were constructed using Illumina reagents and according to commercial protocol. FPKM were determined using the Cufflinks program Cuffdiff. See Extended Experimental Procedures for further detail.
Gene Cloning
For cDNA library generation, RNA from heads was isolated using Trizol purification, and used as template for first-strand reverse transcription with Superscript III (Invitrogen). Cloning was otherwise permformed as described (Lapan and Reddien, 2011).
RNA Synthesis and RNAi
For probes used in RNAi experiments, riboprobe template was produced with the same primers as were used for cloning, except that gateway adapters were not present on primers, and T7 promoter sequence was added to the 3′ primer. Probe synthesis and RNAi were otherwise performed as described (Lapan and Reddien, 2011). In homeostasis RNAi experiments, animals were fed dsRNA food every 4 days for 6 weeks.
Histology and Imaging
Whole-mount FISH and antibody staining was performed as described (Lapan and Reddien, 2011; Pearson et al., 2009). Embryos were killed in 2.7% HCl following manual decapsulation and were otherwise treated the same as adult worms for histologic analysis. Images were acquired with a Zeiss confocal microscope (LSM 700).
Full Eye Purification Protocol
Asexual Schmidtea mediterranea 1–1.5cm in length were used for eye preparation. Animals were fed with liver every 2 days for three feedings prior to eye harvest, and eyes were collected 1 and 2 days after the final feeding. Whole animals were transferred from planaria water to Ca/Mg-free medium (CMF) for two minutes, and then placed onto a CMF-soaked filter paper covering a round petri dish lid of equal size. Head tips were amputated ∼1 mm posterior to the eyes and collected into CMFB (CMF + 1% BSA). Head tips were then transferred to a CMFB soaked filter paper, bisected, and transferred to a 15 ml conical tube containing 10mL of CMFB + 1 mg/mL collagenase (Sigma) + 0.05% NAC. The tube was placed on a nutator for 5 min, and then contents were decanted into a medium-sized petri dish. Head pieces were aspirated and ejected repeatedly from a 232 (Samco) transfer pipette, taking caution not to create bubbles. Pipetting was interrupted periodically to check for the presence of freed eyes, which were transferred to a new dish of CMFB as they appeared. Eyes were then immediately transferred to a dish of iso-osmotic (Peter and Schürman, 2001) medium on ice while additional eyes were collected. When no more eyes appeared in the dissociation medium, the eyes in iso-osmotic medium were transferred to Trizol for RNA collection.
cDNA Sequencing and Analysis
Approximately 1 ug of RNA was used to construct Illumina libraries with Illumina reagents and following the commercial protocol. Each library was analyzed by 36 bp single-end sequencing on one lane of an Illumina Genome Analyzer II. Read quality analysis by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) showed good per base quality scores and good per sequence quality scores (37-38) for greater than 90% of reads. Reads were aligned to two sets of contigs: a reference transcriptome (Adamidi et al., 2011) and contigs assembled de novo from our Illumina reads using Trinity (Inchworm) RNA-seq assembler (Grabherr et al., 2011). Bowtie (Langmead et al., 2009) was used for alignment, with only the best single alignment reported. The five most five prime bases were excluded from analysis to avoid adaptor contamination. Of the eye tissue reads, 18,786,198 (67.07%) and 23,669,529 (84.51%) reads aligned to transcriptome and de novo (Trinity) contigs, respectively. For control tissue the numbers were 24,088,254 (78.68%) and 20,463,476 (66.84%), respectively. FPKMs were determined using the Cufflinks program Cuffdiff (Trapnell et al., 2010) with default parameters. Duplicate read removal (Samtools) lowered FPKMs overall, but did not significantly affect the list of top enriched genes. Robustness of data was addressed by randomly dividing read data for each condition into three subsets, re-mapping, and comparing enrichment across "replicates" using DESeq (Anders and Huber, 2010). This analysis resulted in only small differences from the Cuffdiff analysis (average difference < .3 in Log2 ratios).
Top Enriched List and GO Analysis
A minimally redundant list of enriched genes was created by blasting eye-enriched de novo contigs (obtained using Trinity) against eye-enriched planarian reference transcriptome contigs (Adamidi et al., 2011) and removing the overlapping de novo contigs. FPKMs of zero were assigned a default value of 1 in order to compute ratios. REFSEQ human protein (H. sapiens) and Flybase GN (D. melanogaster) BLAST annotations were generated by blasting translated sequences (blastx) of transcripts from the enriched gene list. GO analysis based on the biological process annotation was performed using the DAVID (Dennis et al., 2003) functional annotation clustering tool. A set of the best ortholog of each enriched eye gene (cut off of 4-fold enriched for eye/control) was compared a set of the best ortholog from each contigs in the reference whole-planarian transcriptome (Adamidi et al., 2011). Only orthologs identified by BLAST with an E value < 0.1 were used in the GO analysis.
Transcript Sequences for Principal Genes
For genes that showed eye signal in the in situ hybridization screen, transcript sequences used in BLAST analyses and reported in Table S1. Information for Genes with Enriched Expression in Planarian Eyes, Related to Figure 1 were curated manually by comparing reads mapped to the genome versus gene predictions mapped to the genome (e.g., see Figure S3). All published planarian gene predictions (Adamidi et al., 2011; Robb et al., 2008; Sánchez Alvarado et al., 2002; Scimone et al., 2010) were used, and the gene model with the best support (exon structure matching regions with mapped Illumina reads) was selected for further use. If ambiguity remained, gene sequences were determined by RT-PCR and Sanger sequencing.
Phylogenetic Analyses
S. mediterranea amino acid sequence predictions were aligned by Clustal W (Thompson et al., 1994) with sequences of putatively homologous proteins from other metazoans. Sequences were trimmed by Gblocks (Castresana, 2000) under the lowest stringency settings. Phylogenetic trees were constructed by Bayesian inference using MrBayes (Huelsenbeck and Ronquist, 2001) with > 2,000,000 generations, and > 1,500 burn-in trees discarded.
Thanks to Irving E. Wang for the illustration in Figure 1A. Thanks to Jessica Witchley for generating the Illumina library. Thanks to Josien van Wolfswinkel, Mansi Srivastava and Jared Owen for bioinformatic assistance. P.W.R. is an early career scientist of the Howard Hughes Medical Institute and an associate member of the Broad Institute of Harvard and MIT. We acknowledge support from the NIH (R01GM080639) and the Keck Foundation.
Accession Numbers
The GenBank accession numbers for Smed-ovo, Smed-klf, Smed-foxQ2, Smed-meis, and Smed-soxB are JX091074, JX091075, JX091076, JX091077, and JX091078, respectively.
Supplemental Information
- Table S1. Information for Genes with Enriched Expression in Planarian Eyes, Related to Figure 1 (XLS 1440 kb)
- The table shows contigs with greater than 4-fold enrichment in FPKM (eye versus control), FPKM > 15 in the eye sample, and a sequence length > 300 bp. Contigs with no clear ortholog (E value > 0.1) are in gray. Genes that are mentioned in the text or that appear in a main figure are named based on orthology, if possible. Genes that were screened but that are described only in supplemental material are given a numerical identifier. In future analyses, numerical names can be replaced by verbal names based on orthology or gene function. "Adamidi" sequences are from a reference planarian transcriptome (Adamidi et al., 2011), and sequences beginning in "comp" are derived from de novo read assemblies (See Extended Experimental Procedures). An asterisk indicates that a gene was previously described as eye-expressed in the literature.
- Table S2. Summary of In Situ Hybridization Results for Screened Genes, Related to Figure 2 (XLS 51 kb)
- In situ hybridization results are categorized by expression in PRN, PC, or both. "none" indicates that no signal was detected by in situ hybridization. "not clear" indicates that signal was present in the animal but was not clearly enriched in the eye. In situ hybridization signal strength above background was approximated visually with a number on a scale of 5 (highest) to 1 (lowest).
- Table S3. GO Categories Enriched in Eye Transcriptome versus Whole Planarian, Related to Figures 1–7 (XLS 63 kb)
- Worksheets display enriched or depleted GO biological process (BP) categories among orthologs of planarian eye-enriched genes (relative to the whole planarian transcriptome).
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Publication Information
Received: February 28, 2012
Revised: May 12, 2012
Accepted: June 22, 2012
Published online: August 2, 2012


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Home > Consumer Protection > Consumer News & Information > FDIC Consumer News - Spring 2003 |
FDIC Consumer News - Spring 2003
Special Report on Fraud |
3. Sorry, that is incorrect.
The correct answer is "True."
With credit cards, a thief might use your card or obtain a new card in your name, perhaps by stealing a pre-approved card application from your mail and having the card sent to a different address.
See full story...
Last Updated 06/18/2003 | firstname.lastname@example.com |

subset: commoncrawl


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"Don't spend money you don't have" and "distinguish between essentials and frills" are lessons just about everybody learns at some point in their lives, whether from a basic economics course or from one's parents explaining that a credit card is not magic.
Why, then, can't Congress and the White House figure it out?
At the Daily Beast, [...]
The Teenagers Have Stolen the Credit Card – and Gotten Themselves Elected
March 8, 2010 by Leave a Comment


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J K Rowling The works of J K Rowling, not least the Harry Potter series. |
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28th June 2008, 06:50 PM | #1 (permalink) |
www.arieltelsa.etsy.com Join Date: Jun 2008 Location: Pennsylvania
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| If you could ask JK Rowling anything I actually did meet JK Rowling.. I won that contest back in october to go to New York to see her read. When I got up to meet her I sort of blithered and told her I loved her. Yeahhh. But seriously, I've always had these two questions I really want the answers to. How did James and Lily (and Frank and Alice) "thrice-defy" Voldemort? And what were the two times Slughorn took Felix Felicis? What would you ask her? |
28th June 2008, 10:42 PM | #2 (permalink) |
Where matter vanishes... Join Date: Feb 2008 Location: Maryland
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| Re: If you could ask JK Rowling anything Which book part does she most regret seeing cut from its counterpart movie, and which movie scene was least faithful to her vision as depicted in the counterpart book. Welcome to The Chrons, Arieltelsa, and many happy returns! |
16th July 2008, 10:22 PM | #4 (permalink) |
Reetou Diplomatic Corp Join Date: May 2001 Location: North-west UK
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| Re: If you could ask JK Rowling anything Not sure if she's been asked any of these before: If, having reached later books, there's one (or more) things in earlier books she wishes she'd added, not included, or written differently in hindsight? I assume there's several, so which was the most awkward to work round? Are there any sub-plots she's cut during editing she wishes had survived to the final print? And vice-versa? At what point did she realise the effect she was having on children's reading habits? Does she regret the over-hype that her books have caused? Is she concerned about the environmental impact of her over-hyped launches (especially 5-7) and the hundreds of thousands of copies which will be read once or twice then binned/etc.? Which is her favourite personal in-joke? Which scene in the series most closely resembles one from her own life? If she'd been at Hogwarts, which of the named characters would she most have been like? |
17th July 2008, 04:01 AM | #8 (permalink) | |
Actum pro novus diem | Re: If you could ask JK Rowling anything Quote:
Now that made me laugh. What could I do with a thousand pounds...get it exchanged for Australian dollars to start with... | |
17th July 2008, 06:00 AM | #9 (permalink) |
Senior Member Join Date: Oct 2007 Location: PACIFIC:
Posts: 954
| Re: If you could ask JK Rowling anything Her books aren't that bad but there have been many great children's series over the years. How did she manage to get the movies made while the series was still being written and have them be decently done. Most series get movies made well after they were written and often the movies are horrible. For example, Lloyd Alexander's Chronicles of Prydain were widely read until Disney released The Black Cauldron animated in the style of their Gummy Bears TV show and killed all further interest. |
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Ranked among the nation's most integrated healthcare systems, Community Health Network is Central Indiana's leader in providing convenient access to exceptional healthcare services, where and when patients need them—in hospitals, health pavilions, workplaces, schools and homes.
Community Health Network is Central Indiana's largest provider of behavioral health, with an extensive continuum of treatment options. More than 4,700 inpatient stays and 19,000 outpatient total client visits a year are provided to patients ranging from age four to senior adults. Clients may be seen in individual, family or group therapy, utilizing psychiatric assessment and evaluation in the development of personal treatment plans by physicians, psychologists, advance practice nurses, therapists and counselors.
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The Behavioral Health Pavilion, a full-service, 122-bed inpatient behavioral health hospital, is located on the Community Hospital North campus. Crisis services are available 24 hours a day, seven days a week for immediate evaluations in person.
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Search results for : | mige hufflepuff |
We know that four founders had created Hogwarts. Rowena Ravenclaw, Salazar Slytherin, Hegla Hufflepuff and Godric Gryffindor. What we never knew was their story. Harry Potter roleplay set in the times of the four founders.
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Hetalia Potter
Okay~! Well, I felt the need to put this up here, so here it is~! Hetalia Potter! A hetalia/HarryPotter crossover! Your Charries are students at Hogwarts~! You get to choose what house they're in, but just make sure the house fits their personality. Th
O U T O F C H A R A C T E R Name: nikki Gender: female Age: 15 How you found Potter's Army: andrian..ages ago Any other characters on Potter's Army: nikki granger, vanessa white and replacing timothy c. ashes with this character Have your read the
1st Year Hufflepuff & Slytherin Transfiguration
"Magic, magic, magic.." A young boy named Christopher sang, playing around with his wand, twirling it in the air. He was an early riser, and he was the first one in the classroom. "Fun, fun fun fun.."
[head of house] App, House in charge of is Hufflepuff
Your Full Name: Travis Andrew Blair/ Jorge Xavier Rameriez (RL writer) House you want to be responsible for: Hufflepuff Why do you want to become Head of House?: I feel like I can lead the site to success. With a background of full of Admin
House Information
If you can't decide on which house you wan't to be in, this thread will surely help you. In HP and PA there are 4 houses in Hogwarts. You are sorted into these houses as soon as you enter and CAN NOT change houses, so choose carefully. G R Y F F I
History Of Magic: 1st Year (Gryffindor and Hufflepuff)
After Anthony had followed Dara into the relatively empty History Of Magic classroom, he stopped right in his tracks. And no, it wasn't by a girl. It was a desk, in the most prime location imaginable. It was in the middle of a row of desks, off to the


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Answered by Ustadha Zaynab Ansari, SunniPath Academy Teacher
As-salaam Alaikum, my question is regarding Anal Sex. I know clearly that anal sex is forbidden in Islam. However my husband is not a well practicing Muslim. I try to practrice Islam as closely as possible and try to remove things in my life that may be sinful. My Husband is overall an understand person and does not create any obstacle in me trying to follow Islam. However, when it comes to Sex, he does not like it at all when I put restrictions on him. He likes to have full freedom in this aspect. He reallys wants to do anal sex and i stop him but then he gets very upset and mad at me. Due to this he is even fading away his understanding for me in other areas of life. He is frustrated, and some times even if I dont like it, he has still peformed anal sex with me. My question is, am I going to be punishable for this? In my heart, I know its wrong and I also try to stop him, but sometimes he just does not listen, will Allah hold me responsible for this? Am i sinful?
In the Name of Allah, the Gracious, the Merciful.
Dear Sister,
I pray this message reaches you in good health and spirits.
First, I would like to thank you for your question.
Many sisters believe that their husbands can demand anything and everything when it comes to the bedroom. However, this is not the case.
Sexual relations, even within a marriage, are subject to the rulings of Sacred Law. Islam is a complete way of life and there is an etiquette for every situation and every relationship.
For a healthy view of marital intimacy, we must consult the Sunna of our Beloved Prophet, Allah bless him and give him peace, who was the "best of you to his family."
Anal sex is prohibited in clear texts from the Prophet, Allah bless him and give him peace. A man who forces anal sex on his wife has sinned an enormity and is liable to being cursed by Allah, and we seek refuge in that!
This is not an area Muslim couples should play around with. Anal sex is repulsive and simply has no place in a Muslim's sex life. It runs counter to the spirit of Islamic marriage, which is premised on love and mercy. Not only is anal sex physically injurious, it also violates the rights of the wife, who is entitled to vaginal intercourse and the right to have progeny through this intercourse if she wishes.
Furthermore, anal sex is reminscent of the way homosexual males approach each other. So why would a Muslim man wish to engage in this action with his wife when there are so many other permissible ways of experiencing sexual pleasure?
Remember, each soul is accountable to Allah Ta'ala for what they have earned. Therefore, you are responsible before Allah Ta'ala according to the hadith of the Prophet, Allah bless him and give him peace, "No obedience to creation in disobedience to the Creator."
Help your husband to obey Allah by not consenting to this reprehensible action. Please suggest other alternatives to your husband. There are many options to explore that are conducive to a healthy, halal sex life.
If your husband still keeps forcing anal sex, then you need to seek counseling. If you say no, and your husband keeps on going, then this is considered marital rape, and no woman should tolerate this.
So please see what can be done about creating a healthy, happy sexual relationship wherein each spouse is respected and given the Islamic courtesy they deserve.
May Allah Ta'ala guide your husband to what is right.
And Allah Ta'ala knows best.
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The Messenger of Allah, peace and blessings be upon him, said, "Knowledge is only through study." While some knowledge can be gained from reading or casually listening to lectures, the best means to gain knowledge is through finding a qualified teacher and then setting up a systematic program of learning. Picking up a book or reading an article and trying to figure things out on our own is no substitute for learning from someone who has a direct link to our living tradition.
Through joining an online class at Qibla, you can benefit from convenient, online courses that will give you access to reliable scholars and our popular curriculum learning tracks. Knowledge gained in these courses will both build your iman and assist you in putting into practice what you learn. Don't give yourself less than you deserve, register today.


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It was the first snowfall 2011 in Pierce Idaho. She let me snap this photograph as she was coming back into the house. The snow is deep enough here that when I call her to come back inside, she has to hop and run, hop and run. Too funny to watch!
Miss Grizzy Bear LOVES The Snows!!
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The program is intended to deliver an all-around education in the theatre that will allow our students to either enter the profession, pursue graduate level studies, or another degree.
It is very much the philosophy of the program that our students develop an "educated imagination". To that end, our students participate in Studio lab courses, Masterclasses and (starting in second year) at least two mainstage productions a year. In addition, all of our students are required to take a healthy selection of lecture and seminar courses, with an emphasis on Dramatic Literature. It is, to say the least, a busy program.
We believe, though, that the rewards are evident: Our graduates have leading roles in Gemini-nominated television shows, perform at the Shaw and Stratford festivals and with professional Newfoundland theatre companies such as Rising Tide and Theatre Newfoundland Labrador. They also build props on Walt Disney cruise ships, work backstage in London's West End and pursue successful careers such as teachers, entrepreneurs and lawyers.
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Denis Bučković (2013) Computer control with Sphinx-4 speech recognition software. EngD thesis.
Andraž Glavan (2012) Economic and performance aspects of data storage in the clouds. EngD thesis.
Matej Hlad (2013) Graph database neo4j and performance comparison with relational database. EngD thesis.
Matej Jerič (2012) Framework for automatic execution of testing activities. EngD thesis.
Elmedin Osmanagić (2014) Windows Store application developmentWindows Store application development. EngD thesis.
Tine Orel (2012) Storage units to ssd drives and beyond. EngD thesis.
Janez Puntar (2012) The functionalities and performance comparison of SharePoint search engines. EngD thesis.
David Petrunov (2011) Presentation of reporting services technology. EngD thesis.
Marko Sedlar (2013) Web applications for notebook editing. EngD thesis.
Janez Sorta (2011) Data integration in social networks. EngD thesis.
Janja Škedelj (2013) Using Kano model in the improvement of software solutions. EngD thesis.
Nejc Škerjanc (2012) Performance comparison between NoSQL and relational database. EngD thesis.
Robi Tomc (2013) Cloud application development and deployment. EngD thesis.


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Are there special study areas in Bracken Library? Yes, the fourth floor is reserved for quiet study. Anyone studying there is asked to turn cell phones to vibrate or off, and to use quiet voices.
Group study rooms are available, as well, seating between 6 and 18 people. Reservations may be made online.
There is also space reserved for Ball State athletes to study on fourth floor, east (toward the north). Up to 174 athletes can study at any one time, monitored by personnel from Athletics. Hours for study are Monday - Friday, from 9 a.m. to 2 p.m. and Sunday - Thursday, from 8 p.m. to 10 p.m.
Where do I go to read class materials my professor has left at the library? These items are located at the service desks of the Architecture and Science-Health Science libraries, or at the Circulation/Reserves counter on the first floor of Bracken Library and the Educational Resources Center service desk on the lower level of Bracken Library.
To find out what is on reserve for a particular class, log in to CardCat with your Ball State username and password and click on "Course Reserves" at the top of the screen. Enter the instructor's name, course number or course name. If there is an electronic copy of the article on reserve, you can get a copy by clicking on "Details" to view the full record and then by clicking on the URL link in the record.
Reserve items are not available for hold or recall and you may only check out one reserve item at a time.
Where can I listen to music assigned by my professor? The Music Listening Center, located on the lower level of Bracken Library, has many listening stations. Go to the Music Listening Center Counter for assistance or for a list of course-related music.
Do the Libraries have copies of textbooks for BSU classes? The Libraries do not systematically purchase textbooks used in BSU classes because textbooks often change from one semester to the next.
However, the Libraries may have some of the books required for classes. The Libraries will often have copies of classic pieces of literature for humanities and social science classes. A Ball State faculty member may have also donated a copy of the textbook used for a class. Check CardCat to see if the Libraries have the book you need.
Some instructors put textbooks and other reading material on reserve. To find out what is on reserve for a particular class, log in to CardCatwith your Ball State username and password and click on "Course Reserves" at the top of the screen. Enter the instructor's name, course number or course name. If there is an electronic copy of the article on reserve, you can get a copy by clicking on "Details" to view the full record and then by clicking on the URL link in the record.
How can I reserve a study room in Bracken Library? There are many rooms available in Bracken Library which can be reserved by Ball State faculty, staff and students for group studying and learning and small conferences. These large and small rooms are also available for staff and campus organizations for meetings, project discussions, and planning. Reservations can be made online.
Where can I find current course catalogs? Online versions of both the undergraduate catalog and graduate catalog are available.
How can I get a faculty or graduate student carrel? Scholar carrels (study rooms) are available for use by graduate students in Bracken Library. Students may apply for these through the University Libraries' Assistant Deans' Office (BL 014) 765-285-1305. Preference is given to full-time doctoral candidates working on their dissertations.
How can I find out about new materials at the library? The University Libraries' Newly Acquired Materials web page lists all of the new books, videos, sound recordings, maps, and other materials that the Libraries have purchased each month. The list is divided up by academic discipline, so it's easy to find all of the new materials we've purchased to support your major!
Also, a selection of new books available for checkout can be found on the northeast side of the first floor, near the Bestsellers.
Are there proctoring services available on campus? Proctored computer-based testing service is provided to Ball State students through Testing Programs.
Ball State students enrolled in distance education classes who need a proctor should contact the Online and Distance Education office .
The Office of Testing Programs will provide proctors for non-Ball State students.
Can I take tests such as the GRE or Praxis I at Ball State? The Office of Testing Programs provides a wide range of testing services to Ball State students as well as the community. Their web site provides further information about specifics tests which are offered.


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Filed under: Addiction
Alcoholism is a chronic and often progressive disease that includes problems controlling your drinking, being preoccupied with alcohol, continuing to use alcohol even when it causes problems, having to drink more to get the same effect (physical dependence), or having withdrawal symptoms when you rapidly decrease or stop drinking. If you have alcoholism, you can't consistently predict how much you'll drink, how long you'll drink, or what consequences will occur from your drinking.
It's possible to have a problem with alcohol, even when it has not progressed to the point of alcoholism. Problem drinking means you drink too much at times, causing repeated problems in your life, although you're not completely dependent on alcohol.
Binge drinking — a pattern of drinking where a male consumes five or more drinks in a row, or a female downs at least four drinks in a row — can lead to the same health risks and social problems associated with alcoholism. The more you drink, the greater the risks. Binge drinking, which often occurs with teenagers and young adults, may lead to faster development of alcoholism.
If you have alcoholism or you have a problem with alcohol, you may not be able to cut back or quit without help. Denying that you have a problem is usually part of alcoholism and other types of excessive drinking.
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Michael Porter studied Vocational Graphic Design at Nottingham College of Art between 1963 and 1966. From 1967 to 1972 he studied at Chelsea College of Art obtaining a 1st Class Dip AD in Painting followed by a Post-graduate degree. From 1972 to 1973 he became a Fellow in Fine Art at Gloucester College of Art in Cheltenham. He currently is a Research Fellow at Falmouth College of Art and teaches at Wimbledon School of Art. He has taught on several Post-graduate courses and has worked as a Visiting Lecturer in thirteen institutions.
He has been the recipient of numerous awards in recent years including an Arts Council of Great Britain Minor Award in 1976, The Lorne Award, London University in 1999 and an Honorary Doctorate in Professional Practice at the University of Derby.
Michael Porter has exhibited extensively since 1976 in both group and solo exhibitions. He has had several solo shows at the Purdy/Hicks Gallery in London and in 2001 showed at the Tate Gallery, St Ives. His work has been included in exhibitions in France, Belgium, Poland Spain, Germany, Norway and Ireland. Many notable collections contain examples of his work such as The Arts Council, The British Council, Deutsche Bank, Unilever, the Victoria & Albert Museum and the Whitworth Gallery, Manchester amongst numerous others.
The main influence on his work is 'landscape in its widest sense' whether it is from people who work on the land, or music or literature that uses the land as subject matter. Traditional and contemporary artists who engage with this subject are also influential on him.


text: Stags finish third in Region 8-AAA meet By Dan Brown The Independent Wednesday, May 1th, 2013 Text size: A A A Tweet Back To Story Dan Brown/Independent -- Berkeley's Ezequiel Jenkins took the Region Championship in the high jump and the 4x100 relay. Jenkins finished fifth in the long jump. --


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Darren Barker made his return to the ring tonight in London, and dominated Kerry Hope en route to a fourth round corner stoppage victory.
Pacquiao-Marquez IV is the weekend's marquee event, but plenty of notable names and stars are in action this week, including Mikkel Kessler, Yuriorkis Gamboa, Darren Barker, James DeGale, Koki Kameda, Giovani Segura, and more.
Matchroom Boxing and Sky Sports won't have near the card they hoped for on Saturday, as Lee Purdy has withdrawn from his bout with Carson Jones due to illness.
Kelly Pavlik and Arthur Abraham won on Saturday, but Jorge Linares faced another setback. Bad Left Hook has the boxing results from around the world.
A round-up of boxing results from Friday night. Featured: John Murray, Yordanis Despaigne, Danny Garcia and more.
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