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Research on Phenotype D. Melanogaster Structure

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Establishing a Reference Phenotype

The testis of wild-type D. melanogaster is a thin and tubular structure, arranged spirally towards a seminal vesicle. At its very tip, eight germline stem cells (GSCs) are bound to a group of somatic hub cells. Each GSC is surrounded by to a pair of cyst stem cells (CySCs). Together, this rosette-shaped formation is known as the stem-cell niche, from which GSCs and CySCs divide to give rise to spermatogonia and spermatogonium-enveloping cyst cells, respectively. In Act5c>Oregon-R males, the well-maintained, temporally-monitored nature of spermatogenesis from this apical region means that growth occurs outwardly and down the trunk of testis, and therefore the organ’s width is more or less uniform from one end to another.

We have noted in the previous chapter how the two prime candidate miRNAs, both of which induced the elongation of cytoophidia within ovarian cells, seemed to instead produce bulged testes in males. Phenotypic manifestations vary; many displayed overgrowth in an apically localized manner. Oftentimes this phenotype is accompanied by a thinning of the trunk body of the testes itself. Conversely, some CytEl-miRNA-overexpressing testes displayed more obviously enlarged trunks than they do the apical tip. In any case, provided it demonstrated noticeable signs of bulging in comparison to Act5c>Oregon-R controls, the testes pair was therewith classified as a positive hit.

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Prevalence of Bulging is Doubled in Flies Overexpressing miR-1014 Compared to Flies Overexpressing Either miR-975 or CTP Synthase Isoform C

In ovaries, continuous overexpression of CTPsynIsoC produced greatly lengthened cytoophidia in both follicle and nurse cells (Azzam & Liu, 2013). We have seen evidence connecting the gene’s ubiquitous expression to more extensive cytoophidia formation in testes tissue as well. In males, however, this is accompanied by actual morphological changes in the form of increased incidences of apical tip overgrowth, observed in 23.45% of Act5c-GAL4>UAS-CTPsynIsoC individuals. We have already established in Chapter 4 that neither of these miRNAs appear to cause elongation of cytoophidia in testes despite doing so in ovaries; nevertheless, we wanted to ascertain whether their effects would manifest as testicular bulging instead. True enough, we saw much greater numbers of overgrown testes in flies overexpressing either miRNAs. The phenotype was recovered in 20.56% of Act5c-GAL4>UAS-miR975 (n=224; herewith Act5c>miR-975)) males, whereas in Act5c-GAL4>UAS-miR1014 flies, this number is nearly tripled (54.56%, n=408; herewith Act5c>miR-1014)). Student T-test show that whilst all of these overexpression lines significantly increased testicular overgrowth from controls, proportions between Act5c>CTPsynIsoC and Act5c>miR-975 are statistically only similar to one another (p>0.05). When compared to Act5c>miR-1014 numbers, however, differences are significant, indicating that miR-1014 overexpression is more likely to cause apical tip bulging than the overexpression events of either CTPsynIsoC or miR-975 flies.

Increased Spermatocyte Numbers is Causal to Apical Tip Bulging

Bulged testes of males overexpressing CTPsynIsoC, miR-975 and miR-1014 is obvious in the greater girth all three demonstrate compared to the control. Spermatogonial cysts – units of meiotically dividing cells formed immediately after GSC differentiation and detachment from the stem-cell niche – contain the germline-specific fusome (de Cuevas & Matunis, 2011). Anti-HTS elicits Adducin-like proteins within these organelles; as cells divide within cysts without cytokinesis, the fusome is therefore highlighted as ‘branches’ extending from one nucleus to the next. Both Act5c>CTPsynIsoC and Act5c>miR-975 testes clearly display increased numbers of fusomic branches, indicating that there are greater numbers of dividing cells. This phenotype was not as obviously discernible with Act5c>miR-1014 testes, in part due to the fact that bulging is often most severe within males of this genotype; the horizontal expansion in girth from the very tip meant that cellular division occurred outwardly, rather than towards the trunk of the testes.

Anti-Coilin staining displays this further; the three-way comparison between controls, Act5c>UAS-miR-975 and Act5c>miR-1014 the outward-spanning tendency of cells in overgrown miR-1014-overexpressing testes, over that of the other genotypes. Regardless, fusomic branching was greater in these testes compared to controls too, though to lesser degree than both Act5c>CTPsynIsoC and Act5c>miR-975, despite its size advantage.

The disarrayed pattern of branching in all three overexpression testes also conveyed that cell division is no longer occurring in a controlled manner. Anti-Armadillo further showed that this dysregulation may have occurred earlier in miRNA-overexpressing testes, in comparison to those overexpressing CTPsynIsoC.

Overgrowth is rarely bilateral in flies overexpressing miR-975 or miR-1014, and may only negligibly affect fertility.

Where the affected tissue comes in a pair, cancers can manifest unilaterally or bilaterally. Here, we wanted to determine the frequency of such occurrences in our overgrowth phenotype, and whether fertility is duly affected by the ‘tumorous’ state of the tissue. We rarely observed bi-laterality; for all overexpression lines, its incidence was recorded in less than 4% of males. This presented us with a challenge in the subsequent fertility assay. As it was impossible to ascertain laterality without dissection, crosses to virgin wild-type females were done ‘blind’; after three days, males were retrieved and their testicular phenotype was verified. Exactly 100 single-pair crosses each were made with males of either Act5c>miR-975 or Act5c>miR-1014 genotype. Bilaterally-overgrown testes were subsequently recovered in 2% and 5% proportions, respectively.

Fertility was nonetheless uncompromised. Barring one of Act5c>miR-1014 (female in cross might have died before egg-laying, as she was dead prior to retrieval), all bilateral-male vials bore offspring within eleven days, although crosses with Act5c>miR-975 produced lower numbers than Act5c>miR-1014. Unilateral testes bulging appeared to have no effects, showing comparable fecundity to controls (single-pair crosses of Act5c>Oregon-R to wild-typed females. This lack of effect overgrowth has on fertility was supported outwardly by the projected ability of bulged testes to produce sperm. Despite how severely overgrowth may have distorted normal cellular division within a testis, it was always accompanied by a seemingly full seminal vesicle.

Whereas overexpression of miR-975 affects CTPsyn-related genes independent of sex, genetic consequences of miR-1014 overexpression display sex-specific signatures.

Tissue overgrowth is a characteristic hallmark of tumours. It is thus natural to speculate that the bulging testes phenotype observed with the overexpression of these miRNAs is attributable to known oncogenic factors. We narrowed a candidate pool down to four genes, all of which have either shown a connection to testicular or prostate cancer in past literature, and/or a demonstrated relationship to CTPsyn (Aughey et al., 2016; Fellenberg et al., 2010; Mahajan et al., 2007). The expression levels of these genes in Act5c>miR-975 testes tissue were compared to that within ovarian tissue. We did the same for Act-GAL4>UAS-1014 flies, as well as female-to-female and male-to-male comparisons between the two genotypes. Rp49 and GAPDH, both commonly utilized reference genes for D. melanogaster, were used for normalization. Fold-change (FC) values were derived after discounting expressional fluctuations observed in Act5c>Oregon-R controls, where FC is assumed to be zero for each gene of interest.

In Act5c>miR-975 flies, a congruency in expressional trends was apparent. Overexpression of the miRNA did not differentially effect a majority of the associated genes. Only Ack was downregulated in testes tissue where it was upregulated in ovarian tissue. Fc values were, however, very modest for both cases (-0.35 vs 1.53). Upregulation was unanimously observed with all other genes, independent of sex. The most drastic escalation in expression levels was shown to be in AllCtps within Act5c>miR-975 ovaries. Although this was also the case for testes, its numbers in this tissue were only doubled (1.91 vs 5.06; p<0.05). This was the only instance whereby the upregulation of associated genes were significantly different between sexes in Act5c>miR-975 flies. One outcome of note was that recorded CTPsynIsoC FC in males was almost identical to changes in AllCtps; this suggests that in testes, miR-975 overexpression may have most affected the expression of Isoform C, if not exclusively. Conversely, a bulk of CTPsyn upregulation in ovaries seemed to be attributable to the other two isoforms. However, as we are unable to distinguish isoforms from one another here, the ratios in which each would have contributed to the fold-change would remain unclear.

Due to their shared cytoophidia-elongating effects, we had expected an upregulation of CTPsynIsoC in Act5c>miR-1014 flies as well. As a matter of fact, more positive FC values were anticipated, especially in ovaries where miR-1014-driven lengthening of cytoophidia was often more extreme than miR-975-induced phenotypes.

It was therefore a surprise to see that neither CTPsynIsoC nor AllCtps levels were altered in these tissues (Figure 5.6-b), and although they were indeed upregulated in testicular tissue, FC values remained decidedly underwhelming (1.44 and 1.35 for AllCtps and CTPsynIsoC, respectively). The rise in Brat and IMPDH were comparatively greater; in the case of the latter gene, its recorded upsurge in ovaries was also significantly higher than that of testes (1.79 vs 1.17; p<0.05). The most jarring difference was seen with Ack: whilst its expression was increased twofold in testes tissue, the gene was almost as severely downregulated in ovarian samples (2.01 vs -1.54).

Interestingly, Ack continues to be the outlier in male-to-male comparisons. Apart from this gene, both Act5c>miR-975 and Act5c>miR-1014 testes tissue displayed similar trends of upregulation across the board. Ack levels were differentially affected; whilst unperturbed by miR-975 overexpression, it was found immensely upregulated under endogenously excessive levels of miR-1014. Exactly the opposite was true in female-to-female comparisons. A moderate increment (FC=1.53) was observed in Act5c>miR-975 ovaries, against the just as moderate decrement (FC=-1.51) in Act5c>miR-1014 ovaries. However, ovarian tissues did as a whole demonstrate a more varied response to miRNA than testicular tissues. Trends in AllCtps, CTPsynIsoC and Myc expression patterns under miR-975 were seen to contradict trends seen under miR-1014. This was particularly obvious with AllCtps. MiR-975 may therefore be more directly involved in CTPsyn regulation, whereas the effects of miR-1014 is likely to be a consequence of its activity elsewhere.

A final noteworthy observation is that IMPDH and Myc – both well-known carcinogenesis drivers – were consistently upregulated in males of either genotype. This indicates that at the very least, excessive levels of either miRNA may be conducive towards the activation of cancer-related factors, thus inducing the tumorous phenotype within testes.

Discussion

Testicular apical tip bulging in males displaces cytoophidia-elongation phenotypes in females of like, miRNA-overexpression genotype, despite a universally observed upregulation of CTPsyn

At the outset of this study, we had hypothesized that should the overexpression of a miRNA alter the lengths of ovarian cytoophidia, it should be directly correlated to altered cellular levels of CTPsyn proteins, specifically that of Isoform C. Previous studies utilizing the same tissue have demonstrated the direct correlation between increased CTPsynIsoC levels to increased cytoophidia lengths (Azzam & Liu, 2013; Huang et al., 2017). Elongated cytoophidia is therefore considered to be the consequence of miRNA-caused upregulation of CTPsyn. Immunostaining and qPCR has shown that this is indeed the case for both Act5c>miR-975 and Act5c>miR-1014 ovaries. As CTPsynIsoC was also upregulated in testes of like-genotyped males, we had assumed that staining would starkly show longer cytoophidia within testicular tissue as well.

However, this was not the outcome observed. In fact, cytoophidia are arguably shorter, more compact, and less numerous in miRNA-overexpressing testes than they were in controls. Subsequently, more than 20% and 54% of miR-975 and miR-1014 overexpressing flies, respectively, presented with overgrown testes. Bulging is often restricted to the apical region. Our past investigations into similarly overgrown testes obtained from CTPsynIsoC-overexpressing flies have given us reason to believe that the primary cause of bulging may have been the rapid lateral division of GSCs into gonialblasts (GBs) (Woo et al., 2018, unpublished data). Whereas GBs would detach physically away from GSCs under normal growth conditions, this was not the case where CTPsynIsoC was expressed excessively. Subsequent transit amplification events within developing cysts therefore occur outwardly, rather than down the ‘neck of the testes as it should be. Hyper-branching of fusomes within Act5c>miR-975 and Act5c>miR-1014 testes further demonstrate a burgeoning of spermatocyte population growth. As these events occur through spermatogenesis and testes maturation, their compounding effects ultimately promotes excessive tissue growth, leading towards the expansion-of-girth phenotype we now categorically recognize as ‘bulging’.

Cytoophidia have been shown to be abundant in wild-typed testes (Liu, 2010). In these flies, spatial and temporal separation between occurrences of micro and macro-cytoophidia are commonly observed (Liu, 2011). Whereas micro-cytoophidia are found in mitotically active transit-amplification regions, macro-cytoophidia are seen in later stages instead. Cytoophidia formation is a known mechanism of adaptive metabolic regulation (Aughey et al., 2014; Aughey & Liu, 2015; Strochlic et al., 2014). Incorporation of CTPsyn proteins into such filaments negatively affects its enzymatic activity (Noree et al., 2014). Interchangeably altering cytoophidia lengths within this relatively minute stretch of tissue is therefore speculated to not only be crucial in maintaining cellular homeostasis, but doubly serves as a means towards regulating spermatocyte division by way of manipulating CTPsyn activity profiles.

It must thusly be noted that alongside the numerical and density reduction of cytoophidia seen in Act5c>miR-975 and Act5c>miR-1014 males, nuclear-CTPsyn became detectably prominent in earlier-staged spermatocytes than they were in Act5c>Oregon-R controls. As aforementioned, these are phenotypes observed as CTPsynIsoC levels were upregulated in both genotypes. We hereby theorize that overexpression of either miRNA may have disrupted the concertedly organized ability of CTPsyn to tether into filaments, culminating in the greater presence of free-flowing active proteins. As CTPsyn is a potent driver of cell proliferation (Martin et al., 2014), it is in its excess that a microenvironment conducive towards population size growth is created, eventually manifesting as the apical tip bulging of testicular tissue.

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