Genetic Dissection of Photoreceptor Subtype Specification by the <i>Drosophila melanogaster</i> Zinc Finger Proteins Elbow and No ocelli

<div><p>The <i>elbow/no ocelli</i> (<i>elb</i>/<i>noc</i>) complex of <i>Drosophila melanogaster</i> encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. Here we show that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the ‘dorsal rim area’ (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. Our data suggest that Elb/Noc genetically interact with Hth, and we identify two conserved domains crucial for this function. Furthermore, we show that Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. We propose that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease.</p></div

Elb and Noc are crucial for Homothorax function in DRA specification.

<p><b>A.</b>–<b>C.</b> Homothorax fails to ectopically induce the DRA fate in absence of Elb and Noc. (Ommatidial schematic to the left summarizes the only ommatidial subtype found inside the dashed white boxes; pink dot: nuclear Sens expression in R8). <b>A.</b> Exclusion of Rh6 (green) from the DRA (labeled with Hth, purple), in wild type flies (dashed white box). <b>B.</b> Complete loss of Rh6 upon over-expression of Hth (purple), under LGMR-GAL4 control. <b>C.</b> Co-expression of Rh6 (green) and Hth (purple) in <i>elb,noc</i> double mutant flies ectopically expressing Hth (green) in all photoreceptors. <b>D.</b> Expression of <i>elb</i>-GAL4 (visualized using UAS-lacZ:NLS) is never expanded into R1-6 by over-expression of Hth (green). Instead, strong expression of βGal (red) is observed in all inner photoreceptors R7 and R8 (co-labeled with Spalt, green). <b>E</b>–<b>G.</b> Homothorax fails to repress Senseless in absence of <i>elb</i> and <i>noc</i>. Whole mounted pupal retina expressing Hth in all photoreceptors (LGMR-<i>hth</i>; red) with homozygous clones lacking both <i>elb</i> and <i>noc</i> marked by absence of <i>arm</i>-<i>lacZ</i> (blue, E). The vast majority of strong Sens expression (green) is observed in R8 cells inside homozygous clones (F), as well as in close vicinity to mutant clones (G).</p

VP16- and <i>en</i>[R]-fusions of Noc specifically affect R8 rhodopsin expression.

<p><b>A.</b> Schematic of VP16:<i>noc</i> fusion cDNA generated in UAS-constructs for over-expression (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>B</b>,<b>C.</b> Mild effect of VP16:<i>noc</i> over-expression on DRA development: Rh3 expression (red) in the DRA is weak (arrows), yet detectable (B), and Hth expression (green) is normal (C, arrows). <b>D,E.</b> VP16:<i>noc</i> has a strong activating effect on Rh5 expression (blue), resulting in a high ratio of pR8 cells with Rh5. Since Rh6 expression appears normal (green), many R8 cells now co-express the two R8 rhodopsins (white arrows). <b>F.</b> Schematic of Engrailed fusion cDNAs generated for Noc (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>G.</b> Ectopic over-expression of <i>en[R]:noc</i> has no effect on R7 rhodopsin expression (Rh3 in red; Rh4 in cyan) and DRA specification (arrows). <b>H.</b> Expression of Rh5 (blue) is lost upon over-expression of <i>en[R]:noc</i>, and Rh6 (green) is the only R8 rhodopsin remaining.</p

Expression of <i>elbow</i> (<i>elb</i>) and <i>No ocelli</i> (<i>noc</i>) in photoreceptors.

<p><b>A.</b> Schematic of the <i>elb</i>/<i>noc</i> locus with GAL4 enhancer traps shown as red triangles. <b>B.</b> Schematic summarizing Rhodopsin expression in the three main ommatididal subtypes of <i>Drosophila</i>. L  =  lamina; M  =  medulla. <b>C.</b> Domain structure of Elb/Noc proteins: like most members of the NET family of transcriptional repressors they contain a single C2H2 zinc finger domain at the C-terminus (light blue), as well as a conserved SPLALLA motif of unknown function at the N-terminus (purple). In between the two lies a Groucho-binding motif (yellow). <b>D.</b> GFP fluorescence of <i>elb</i> > UAS-eGFP observed under water immersion. Fluorescence localizes to the central photoreceptors of each ommatidium (R7 or R8), as well as non-neuronal cells (green blur). <b>E.</b> Cryostat cross section through an adult eye expressing βGalactosidase (<i>elb</i> > <i>lacZ</i>). Strong expression was observed in R7 and R8 of the Dorsal Rim Area (red dashed box). Additional expression exists in the brain, in many R8 cells, as well as few R7 cells throughout the retina. <b>F.</b> Elbow expression (<i>elb</i> > <i>lacZ</i>:NLS; red) co-localizes with DRA marker Homothorax (Anti-Hth; green; white arrows). <b>G.</b> Expression of <i>elb</i>-GAL4 driving UAS-<i>lacZ</i>:NLS in the adult retina (visualized on a Cryostat cross section): expression is restricted to R7 and R8 nuclei, as well as non-neuronal cone cell nuclei above the R1-6 level. <b>H.</b> Adult expression of <i>noc</i>-GAL4 is virtually indistinguishable from <i>elb</i>. Expression is strongest in R7 and R8 in the DRA (red dashed box), as well as subsets of R7 and R8 cells outside the DRA. <b>I. </b><i>No ocelli</i> expression (<i>noc</i> > lacZ:NLS; red) also co-localizes with DRA marker Homothorax (Anti-Hth; green; white arrows).</p

The DRA fate is lost in <i>elb</i>,<i>noc</i> double mutants.

<p><b>A.</b> Nuclear localization of Exd (cyan) in R7 and R8 of wild type DRA ommatidia, marked with Rh3 (red). <b>B.</b> Exclusion of R8 marker Senseless (green) from wild type R8_DRA cells (arrows), positively marked with Exd (purple). <b>C</b>–<b>F.</b> Exclusion between Extradenticle (Exd) and Senseless (Sens) is lost in R8_DRA cells, in <i>elb,noc</i> double mutants (phenocopying <i>homothorax</i> loss-of-function). <b>C.</b> Co-expression of Exd (indirectly labeled using <i>hth</i>-lacZ:NLZ), and Sens (green) in <i>homothorax</i> LOF (LGMR > <i>hth^HM</i>). <b>D.</b> Identical co-expression of Exd (purple) and Sens (green) is observed in <i>elb,noc</i> double mutants. <b>E.</b> Exclusion of Exd (red) in the DRA (dashed line) and Sens (green) in a whole mounted pupal retina from wild type flies (labeled with ElaV, blue). Weak co-staining is sometimes observed, at that developmental stage. <b>F.</b> Strong co-expression of Exd (red) and Sens (green) in R8 cells of all DRA ommatidia (dashed line), phenocopying <i>hth</i> loss-of-function. <b>G</b>. Morphological defects of <i>elb,noc</i> double mutant DRA ommatidia: typical enlarged rhabdomere diameter of central photoreceptors R7 and R8 (yellow arrows) is lost specifically (black arrows) in <i>elb,noc</i> double mutants clones touching the dorsal eye margin (marked by absence of photoreceptor pigment granules to the left of the green dotted line). The same phenotype was described for mutant clones of <i>hth^B2</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#pgen.1004210-Wernet1" target="_blank">[2]</a>. Note that due to the absence of enlarged inner photoreceptor rhabdomeres the extent of the DRA could not be marked inside <i>elb</i>,<i>noc</i> mutant tissue (hence the dotted red line).</p

Mutagenesis of conserved Elb domains interferes with different Otd functions.

<p><b>A.</b> Schematic illustrating the UAS-construct for over-expression of an Elb protein with an altered Groucho-binding motif (FKPY → IEGS, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>B.</b> Over-expression of mutated <i>elb</i>[Gro*] in all photoreceptors using LGMR-GAL4 leads to a specific loss of Rh3 (red), while Rh4 expression (cyan) is normal. <b>C.</b> In R8 cells, Rh5 (blue) is specifically lost, while Rh6 expression (green) is normal. <b>D.</b> Table summarizing gain-of-function phenotypes observed using different point-mutated forms of Elb. Every mutant produces a specific phenotype that can be broken down into four aspects: DRA specification, <i>rh3</i> expression, r<i>h5</i> expression, and <i>rh6</i> expression. Abbreviations: DN, dominant-negative; wt  =  expression like wild type; ‘-’  =  loss of expression; ‘+’  =  de-repression of expression into outer photoreceptors R1-6; ‘N/A’  =  DRA specification could not be assessed due to ectopic loss of Rh3 expression.</p

Model summarizing the roles of Elb/Noc action in photoreceptor cell fate specification.

<p>Model summarizing the role of Elb and Noc proteins in terminal specification of photoreceptor cell fates. <b>A.</b> Role of Elb and Noc in DRA ommatidia. Loss of function data suggests Elb & Noc function with Hth in specifying the DRA cell fates, both morphologically (rhabdomere diameter) and molecularly: repression of Sens in R8_DRA by Hth/Exd and Elb/Noc is crucial for these cells to express the DRA rhodopsin marker Rh3. <b>B.</b> Dissection of Otd functions through antagonism with Elb/Noc protein domains. The similarity of <i>elb</i>/<i>noc</i> gain-of-function phenotypes to the previously published <i>ocelliless</i> (otd) loss-of-function suggests Otd/Elb/Noc proteins interact genetically. The different transcriptional effects of Otd on <i>rh3</i> and <i>rh5</i> expression (activation in ‘pale’ ommatidia), as well as <i>rh6</i> (repression via Dve in outer photoreceptors) can be neutralized independently, using different mutated forms of Elb/Noc. The specific role of three conserved Elb/Noc domains (Sp/SPLALA, Gro-binding, Zn finger) in genetically antagonizing Otd function are indicated by inhibition arrows.</p

Mutagenesis of Sp/SPLALLA motif or zinc finger transform Elb into a dominant-negative.

<p><b>A.</b> Schematic illustrating the UAS-construct for over-expression of an Elb protein with an altered Sp/SPLALLA motif (mutated to TGIVIIV, using site-directed mutagenesis; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>B.</b> Ectopic expression of mutated <i>elb</i>[SPLALLA*] using LGMR-GAL4 leads to a loss of typical Rh3 expression in R8_DRA cells (white dashed box), while R7 expression appears normal in the rest of the retina. <b>C.</b> As a result, Rh6 (green) and Hth (purple) are co-expressed (white arrow) at the dorsal rim of the retina. <b>D.</b> Weak co-expression of Sens (green) and Exd (purple) was sometimes observed (white arrow), although not with 100% penetrance. <b>E.</b> Expression of Rh6 (green) is strongly expanded into outer photoreceptors R1-6, in LGMR > <i>elb</i>[SPLALLA*] flies. <b>F.</b> Expression of Rh5 (blue) appears normal in LGMR > <i>elb</i>[SPLALLA*] flies. <b>G.</b> Schematic illustrating the UAS-construct for over-expression of an Elb protein with a mutated zinc finger (both His were altered to Ala, using site-directed mutagenesis; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>H.</b> Over-expression of mutated <i>elb</i>[ZnF*] in all photoreceptors leads to a loss of DRA-specific Rh3 (red) expression in R8_DRA cells (white dashed box). <b>I.</b> In the R8 cell layer, expression of Rh5 (blue) is specifically lost, and Rh6 (green) is the only remaining R8 rhodopsin, including the DRA (white dashed box).</p