Complementation analyses.

<p>Individually established stocks are indicated by # in parenthesis.</p

Germline clone derived mutant embryos for <i>luna</i>Δ<i>1</i> or <i>luna</i>Δ<i>2</i>.

<p>Note same phenotypes as mutant embryos derived from heterozygous mothers. Stainings and stages are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096933#pone-0096933-g002" target="_blank">Figure 2</a>. Lower panels show Hoechst stain. Genotypes of germline clones are indicated above each panel. Compare to control prophase and metaphase embryos in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096933#pone-0096933-g002" target="_blank">Figure 2A and 2D</a>. DNA segregation defects were seen most prominently during prophase like chromatin stages, with fully segregated centrosomes ready for metaphase (A, B), combined with “nuclear fall out” defects, where centrosomes remain in the periphery and DNA has disappeared (indicated by white arrowheads in B, C). Yellow arrowheads indicate DNA bridges (A′, B′). (C) Asynchronous nuclear division stages combined with nuclear fallout. Upper left corner shows metaphase stage nuclei next to anaphase to telophase stages in the remaining part of panel. Control anaphase stage embryo (D), genotype as controls in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096933#pone-0096933-g002" target="_blank">Figure 2</a>. Compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096933#pone-0096933-g004" target="_blank">Figure 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096933#pone-0096933-t003" target="_blank">Tables 3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096933#pone-0096933-t004" target="_blank">4</a> for quantitative analysis.</p

<i>luna</i> mutants manifest themselves as “zygotic lethal like”: determining the lethal stage of <i>luna</i> mutants.

<p>Individually established stock are indicated by # in parenthesis.</p

<i>luna</i> mutant embryos show severe DNA segregation defects.

<p>Summary of defects quantified from different experimental approaches (first column). “% DNA segregation defect/bridges” represents nuclear figures during division stages, where DNA bridges remain between adjacent nuclei. Note that in the mutant scenarios generally between 50–100% of nuclear figures show bridges, whereas in the control germline clones or RNAi experiment it is mostly at 0%. These phenotypes can also be observed in early division cycles.</p

<i>luna</i> mutant embryos show division cycle asynchrony and increased nuclear fall out defects.

<p>Summary of defects quantified from 3 different experimental approaches (first column). Division stage asynchrony lists the different stages (1–3) and the percentage of nuclear figures found for every stage. Nuclear fallout lists how many individual centrosomes were found without associated DNA and how many such areas occurred in one embryo.</p><p>*embryo with DNA segregation defects/bridges.</p><p>**note that asynchrony is very rare/almost non-existent in control germline clone or control RNAi cohort (96–100% synchronous).</p

Map of the <i>luna</i> locus and mutant alleles.

<p>Open arrow indicates the <i>luna</i> locus, boxes below show exon containing areas, with non coding or coding sequences, indicated by white or black boxes, respectively. Black arrowheads point to the site and orientation of the PBac insertions used to generate deletion mutants (indicated by black lines).</p

DNA segregation and division asynchrony are the most prominent <i>luna</i> phenotypes in RNAi knock-down experiments.

<p>Graph of embryonic phenotype evaluation of 2 independent maternal <i>luna</i> RNA interference experiments for 2 independent RNA sequences and control (<i>white</i> RNAi). n = number of embryos analyzed for each genotype/experiment. Nuclear fall out was not quantified in this assay.</p

AKAP200 promotes Notch signaling in a PKA independent manner.

<p>(A) <i>AKAP200</i> has 2 splice variants. AKAP200-L, which can interact with the regulatory subunits of PKA via a tethering site coded for by exon 5 (blue). This exon is spliced out in AKAP200-S, eliminating its ability to interact with PKA. (B-E) Both AKAP200 isoforms can rescue PR number defects in the eye and lethality. Note in schematic of eye phenotypes, loss is indicated by a solid black dot and loss specifically of R7 is indicated by a hollow dot (B) Quantification of genotypes shown in (C-E) ***<i>p</i><0.0001 by chi square test (against <i>AKAP200</i>^<i>M30</i>, n = 514–726, from 3 independent eyes). (C-E) Tangential adult eye sections of indicated genotypes (C) Homozygous <i>AKAP200</i>^<i>M30</i> escaper displays PR number defects. Expression of <i>AKAP200-L</i> (D) and <i>-S</i> (E) via <i>tubulin-Gal4</i> rescues the <i>AKAP200</i> phenotype, suggesting that this phenotype is PKA independent. (F-J) PKA-independent effects of AKAP200 on N signaling. (F) Quantification of genotypes shown in (G-J), ***p<0.0001, by chi square test (n = 320–573 from 3–4 independent eyes). (G-J) Tangential adult eye sections of indicated genotypes. PR number defects caused by <i>sev-N</i>^<i>ΔECD</i> (G) is not modified by <i>PKA</i>^<i>-/+</i> (H), but is suppressed by <i>AKAP200</i> mutant (I), or both together (J). There is no statistical difference in the effect on <i>sev-N</i>^<i>ΔECD</i> of removing either one copy of A<i>KAP200</i> alone or together with <i>PKA</i>, suggesting that PKA may not be required for AKAP200’s effect on Notch signaling.</p