Ubiquitous Expression of CUG or CAG Trinucleotide Repeat RNA Causes Common Morphological Defects in a Drosophila Model of RNA-Mediated Pathology

Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington’s disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their ‘molecular hallmarks’ that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine ‘hairpin’ repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model

Reducing Dcr-2 levels is not rate limiting for the tergite phenotype.

<p><b>A-D</b>, Proportion of progeny within each phenotype category when repeat expression is driven by <i>da-GAL4</i> alone (left column), or in the presence of the <i>Dcr-2^L811fsX</i> mutant allele such that Dicer-2 levels are reduced (right column). <b>A</b>, Wild-type control, <b>B</b>, <i>4xrCAA_∼100 [line 1]</i>, <b>C</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>D</b>, <i>4xrCAG_∼100 [line 2]</i>. <b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>F</b>, <i>4xrCAG_∼100 [line 2]</i>. None of the observed changes were statistically significant (Fisher’s exact test).</p

Ubiquitous CUG and CAG RNA repeat expression leads to distinct localisation in muscle nuclei.

<p>Images from cryosections hybridised with fluorescent probes to detect repeat RNA. Images are representative of observations from multiple animals and independent transgenic lines for each repeat. <b>A</b>, Schematic of the repeat expression construct, indicating the location of the repeat and probe. <b>B-E</b>, Repeat transcripts detected with a complementary repeat probe. <b>B</b>, Progeny carrying the <i>da-GAL4</i> driver alone show no signal in the nucleus. <b>C</b>, Expression of <i>4xrCUG_∼100 [line 2]</i> leads to multiple foci throughout the nucleus. <b>D, E</b>, Expression of <i>4xrCAG_∼100 [line 1]</i>, or <i>4xrCAA_∼100 [line 1]</i> leads to between one and four sites of RNA concentration (arrowheads). <b>F</b>, Schematic of the construct giving repeat expression within the context of the GFP transcript, in this case RNA is detected using a probe against the GFP sequence. <b>G-K</b>, Repeat RNA localisation when expressed within a GFP transcript. <b>G</b>, No signal is observed using the GFP complementary probe against control EV progeny. <b>H</b>, <i>4xrCUG_∼100-GFP</i> expression leads to a similar pattern of foci as in <b>C</b>. <b>I, J</b>, Expression of 4x<i>rCAG_∼100-GFP</i> or <i>4xrCAA_∼100-GFP</i> leads to a similar pattern as in <b>D</b> and <b>E</b>. <b>K</b>, Expression of a single copy of GFP, not containing a repeat, leads to the formation of a single similar site of RNA concentration.</p

Tergite phenotype caused by ubiquitous expression of CAG or CUG repeat RNA in <i>Drosophila</i>.

<p><b>A</b>, wild-type flies show a regular arrangement of tergite bands along the dorsal abdomen (arrows). <b>B</b>, An example of the disrupted phenotype, whereby tergites do not fuse at all (white arrowheads), or fuse only partially (grey arrowheads). <b>C</b>, Phenotype severity was scored on a scale of 1–4, images show typical examples from each category, where category 1 is like wildtype; category 2, tergites disrupted but not split; category 3, one tergite split; and category 4, two or more tergites split. <b>D-E</b>, Graphs showing the proportion of progeny within each scoring category. <b>D</b>, <i>da-GAL4</i> driven expression of the EV control line (n = 161) and, <b>E</b>, <i>da-GAL4</i> driven expression of <i>4xrCAA_∼100</i> [line 1] (n = 148) gives no phenotype with almost all progeny like wild-type. <b>F</b>, <i>da-GAL4</i> driven expression of <i>4xrCUG_∼100 [line 1]</i> (n = 271) or <b>G</b>, <i>4xrCAG_∼100 [line 2]</i> (n = 343) gives a tergite disruption phenotype. <b>H,</b> Schematic (not to scale) showing the location of histoblast cells (black). Histoblasts proliferate and migrate to form the tergite bands (arrows). <b>I</b>, Expression within the histoblasts using <i>T155-GAL4</i> gives wild-type tergites in EV control progeny (n = 56). <b>J</b>, <i>T155-GAL4</i> expression of <i>4xrCAG_∼100</i> [line 1] (n = 25) gives a mild tergite phenotype, ***p<0.001 comparing the proportion with a phenotype in <b>I</b> and <b>J</b>.</p

Reducing Mbl levels does not enhance the tergite phenotype.

<p><b>A-D</b>, Proportion of progeny within each phenotype category when repeat expression is driven by <i>da-GAL4</i> alone (left column), or in the presence of the <i>mbl^E27</i> mutant allele such that Mbl levels are reduced (right column). <b>A</b>, Wild-type control, <b>B</b>, <i>4xrCAA_∼100 [line 1]</i>, <b>C</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>D</b>, <i>4xrCAG_∼100 [line 2]</i>. <b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i> shows a reduction in the proportion with any phenotype, but no change in the proportion with a strong phenotype with reduced Mbl levels. <b>F</b>, No significant effect was observed with <i>4xrCAG_∼100 [line 2]</i>. Comparisons were made using Fisher’s exact test, with significant results indicated above the proportion, where *p<0.05.</p

Reduced Dicer-1 processing can have opposing effects on CUG or CAG RNA-mediated tergite phenotypes.

<p><b>A-D</b>, Proportion of progeny within each phenotype category for expression with <i>da-GAL4</i> alone (left column), or in the presence of the <i>Dcr-1^Q1147X</i> mutant allele such that Dcr-1 levels are reduced (right column). <b>A</b>, wildtype control, <b>B</b>, <i>4xrCAA_∼100 [line 1],. </i><b>C</b>, <i>4xrCUG_∼100 [line 1],. </i><b>D</b>, <i>4xrCAG_∼100 [line 2]. </i><b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i> shows a suppression for both measures. <b>F</b>, <i>4xrCAG_∼100</i> [line 2] shows an enhancement for both measures. Comparisons were made between the populations using Fisher’s exact test, with significant results indicated above the proportion, where *p<0.05, **p<0.01 and ***p<0.001.</p

The GM-CSF receptor utilizes β-catenin and Tcf4 to specify macrophage lineage differentiation

Granulocyte–macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.