rst Transcriptional Activity Influences kirre mRNA Concentration in the Drosophila Pupal Retina during the Final Steps of Ommatidial Patterning

Background: Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. Methodology/Principal Findings: By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles we showed that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore we demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. Conclusions/Significance: These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level

RT-qPCR quantification of relative rst mRNA levels in <i>rst^CT</i> mutant and two <i>rst^D</i> wild type-like revertants together with corresponding eye phenotypes.

<p>Despite its severe rough eye phenotype, <i>rst^CT</i> (which expresses a truncated version of the Rst protein) shows an essentially wild type rst mRNA temporal profile. Both <i>rst^D</i> wild type-like revertants however, are defective in rst mRNA expression, which is completely absent in <i>RTW6</i> and displays a striking departure of wild type temporal profile in <i>RTW8</i>, showing that the requirement for <i>rst</i> wild type function can be bypassed in these mutants during pupal eye development.</p

Genetically lowering <i>kirre</i> dosage in the <i>RTW6</i> revertant causes defects in adult ommatidial organization.

<p>(<b>A</b>) Scanning electron micrographs. Compound eye phenotype from a <i>RTW6/Df(1)w^67k30</i> heterozygote female, compared to those from her parent genotypes, <i>RTW6/Y</i> and <i>Df((1)w^67k30)/+</i>. Leaving only the <i>kirre</i> copy present in the RTW6 chromosome when no functional copies of <i>rst</i> are present in the eye lead to a mild but clear disruption of ommatidial patterning showed in the inset: lack or mispositioning of mechanosensory bristles (arrowheads), bristle duplications (arrow) and fused ommatidia (asterisk). (<b>B</b>) Dissecting stereomicroscope images. Head from a <i>RTW6</i> male where <i>kirre</i> function has been almost completely knocked down by expressing one copy of the <i>UAS-kirre RNAi 3111</i> transgene under the control of the <i>GMR-Gal4</i> eye driver (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022536#s4" target="_blank">methods</a>). Eyes from a male carrying only the driver and a <i>RWT6</i>/Y are also shown. Note the clear increase in the severity of the phenotype, particularly the higher number of fused ommatidia and mispositioned bristles, compared to the <i>RTW6/Df(1)w^67k30</i>eye in (<b>A</b>).</p

<i>kirre</i> silencing does not appear to influence rst mRNA concentration in the retina.

<p>(<b>A</b>) Relative quantification of kirre mRNA levels at 24% APF in retinae expressing one copy of either <i>UAS-kirreRNAi 3111</i> or <i>UAS-kirreRNAi 27277</i> transgenes, driven by <i>GMR-Gal4</i>, compared to those expressing the driver alone or the transgenes alone. (<b>B</b>) Relative quantification of rst mRNA levels at 24% APF in the retinae of the flies with the same genotypes as in (<b>A</b>). (<b>C</b>) Relative quantification of kirre mRNA levels at 34% APF in retinae expressing one copy of <i>UAS-kirreRNAi 27277</i>, driven by <i>GMR-Gal4</i>, compared to those expressing the driver alone or the transgene alone. (<b>D</b>) Relative quantification of rst mRNA levels at 34% APF in retinae with the same genotypes as in (<b>C</b>). In neither time point significant differences in rst mRNA levels between <i>kirre</i>-silenced retinas and the controls could be detected.</p

Comparative temporal quantification of relative kirre mRNA levels in wild type and <i>rst^D.</i>

<p>The symbols representing each genotype are indicated in the graph. Although qualitatively similar to wild type, kirre mRNA levels in <i>rst^D</i> retinae reach significantly higher concentrations at their 34% APF expression peak when compared to their basal levels.</p

<i>rst</i> RNAi silencing leads to an increase of kirre mRNA levels at the time of cell sorting.

<p>(<b>A</b>) Relative quantification of rst mRNA levels at 24% APF in the retinae expressing one copy of either <i>UAS-rstRNAi 951</i> or <i>UAS-rstRNAi 27223</i> transgenes, driven by <i>GMR-Gal4</i>, compared to those expressing either the driver alone or the transgenes alone. (<b>B</b>) Relative quantification of kirre mRNA levels at 24% APF in the retina of the flies with the same genotypes as in (<b>A</b>). A clear increase in kirre mRNA levels can be seen in the <i>rst</i>-silenced retinae.</p

Comparative temporal quantification of relative kirre mRNA levels in wild type, <i>rst^CT</i> and the two <i>rst^D</i> wild type-like revertants.

<p>The symbols representing each genotype are indicated. All mutants show a “rst-like” temporal profile of kirre mRNA expression, although in the case of rst^CT, changes in concentration relative to wild type are very small.</p

Adult eye phenotypes and protein expression pattern plus mRNA level quantification of <i>rst</i> during the first 43% of pupal retina development.

<p>(<b>A</b>) wild type control (Canton S) and <i>rst^D</i> adult compound eyes. Note the glassy and brownish appearance of the mutant. (<b>B</b>) Comparative pattern of Rst protein localization in wild type control and <i>rst^D</i> mutant retinae, visualized by anti–rst Mab 24A.5. In the mutant IOC sorting does not start before 32% APF and the Rst continues to be detected at IOC/pc1 border by 43%APF (<b>C</b>) Quantification of relative mRNA levels of <i>rst</i> in wild type and <i>rst^D</i> mutant retinae during the first half of pupal development by RT-qPCR. In the mutant, starting around 20% APF,rst mRNA concentration rises sharper and falls significantly slower than in wild type, although both express similar basal levels at the beginning and end of the first half of pupal development. See text for details.</p

Ubiquitous overexpression of a transgene encoding the extracellular portion of the Drosophila Roughest-Irregular Chiasm C protein induces early embryonic lethality

The cell adhesion molecule Rst-irreC is a transmembrane glycoprotein of the immunoglobulin superfamily involved in several important developmental processes in Drosophila, including axonal pathfinding in the optic lobe and programmed cell death and pigment cell differentiation in the pupal retina. As an initial step towards the "in vivo'' functional analysis of this protein we have generated transgenic fly stocks carrying a truncated cDNA construct encoding only the extracellular domain of Rst-IrreC under the transcriptional control of the heat shock inducible promoter hsp70. We show that heat-shocking embryos bearing the transgene during the first 8hs of development lead to a 3-4 fold reduction in their viability compared to wild type controls. The embryonic lethality can already be produced by applying the heat pulse in the first 3hs of embryonic development, does not seem to be suppressed in the absence of wildtype product and is progressively reduced as the heat treatment is applied later in embryogenesis. These results are compatible with the hypothesis of the lethal phenotype being primarily due to heterophilic interactions between Rst-IrreC extracellular domain and an yet unknown ligand