Genetic interactions between Ubx and Dcr1 affect haltere development

Our analysis of Ubx-miRNA interactions suggest that the haltere expressed miRNAs are incorporated into the Ubx instructed transcriptome to help regulate these transcripts during development. If this was to be the case, we would expect that disruption to this miRNA activity would be detrimental to Ubx function and regulation during haltere development. To test this proposal, we used a genetic interaction assay during haltere development. Animals that are heterozygous for a Ubx null allele develop halteres with distinctive partial homeotic transformations – the appearance of large sensory bristles. By combining Ubx and Dcr1 null alleles, we can use the appearance of these homeotic transformations as a read-out of Ubx activity. If miRNAs are required for Ubx directed haltere development, decreasing Dcr1 function, and therefore miRNA biogenesis should enhance any Ubx homeotic transformations within the haltere. We monitored the appearance of ectopic bristles in four genotypes – WT, Ubx-/+_{, Dcr1}-/+ _{and Ubx}-/+ Dcr1-/+ _{(Fig.5.11A-D). Ectopic bristles were detected in only the Ubx}-/+ _{and Ubx}-/+ _Dcr1-/+

genotypes (Fig.5.11C’-D’). A numerical analysis of these ectopic bristles was performed (Fig.5.11E). We find that the number of ectopic bristles appearing within

Ubx-/+ Dcr1-/+ halteres is significantly increased (p<0.001). This data suggests that there is a requirement for correct miRNA functionality within the developing haltere and that the primary role for this miRNA activity is in helping Ubx regulate the development of this appendage (Fig.5.11F).

In the previous chapter we described the miRNA profiles of wing and haltere imaginal discs at a specific developmental time point. Here we assessed the functionality of these alternative miRNA profiles and investigated to what extent, these haltere miRNAs are required for the correct development of the haltere appendage.

We were interested in understanding the potential significance of differential miRNA expression, in particular, was there a shared functionality between miRNAs which exhibit similar expression patterns when comparing wing and haltere tissue.

We had previously grouped miRNAs present in the wing and haltere dependant on their relative expression levels between the two tissues. Interestingly, we find that miRNAs which display similar, differential expression profiles tend to have similar sets of predicted target genes. We further show that miRNAs either enriched within the haltere or only expressed within the haltere (Halt Up & Halt Only expression groups) share many predicted target genes. This data suggests that the differential expression patterns of miRNAs may have functional consequences to the biology of the imaginal discs.

To explore this possibility further, we used gene ontology analysis to examine how similar or dissimilar each miRNA expression group is in terms of function. Through this analysis, we uncover significant differences when comparing gene ontologies of specific target genes from each miRNA expression group. These results lend weight to the notion that there may be a functional reason for the differential expression patterns seen between the wing and haltere.

We assessed to what extent Ubx regulation and function was integrated with the miRNA content found within the haltere. First, we re-analysed the potential for miRNA targeting of Ubx transcripts within the haltere using our understanding of the miRNA content within this tissue. Interestingly, we find no evidence that miRNAs enriched within the haltere, or miRNAs expressed at high levels within the haltere are less likely to target Ubx. In fact, the most statistically significant finding was that miRNAs down- regulated within the haltere (Halt Down expression group) are less likely to target Ubx. It is hard to discern if miRNAs not enriched within the haltere lack Ubx seed sites because they have decreased expression, or rather, because they do not target Ubx, there is reduced regulatory pressure to maintain their presence within the haltere. It is important to note that the miRNAs that are enriched in the wing do not necessarily have low levels of expression within the haltere. Overall, these results suggest that Ubx

is under a large degree of potential regulatory pressure by miRNAs within the haltere and that these regulatory interactions have evolved to maintain the strict regulation of

Ubx expression within this tissue.

We next investigated the potential recruitment of the haltere miRNA content into the

Ubx regulated transcriptome. Combining our sequencing data with available

transcriptomic studies, we show that there is greater potential for haltere miRNAs to target genes directly up-regulated by Ubx transcriptional activity. This finding suggests that the regulatory input of miRNAs into the Ubx transcriptome is required to fine-tune and buffer active transcription within the haltere, not to behave as primary regulators of gene-expression assisting Ubx in turning over the haltere transcriptome during development. In this manner we believe a substantial proportion of miRNAs within the haltere are sub-ordinate to Ubx, recruited by this Hox factor to help regulate and maintain the developmental programmes of the appendage.

In the previous chapter we observed little evidence to indicate that transcriptional regulation accounts for differing miRNA expression profiles. If Ubx does incorporate miRNA activity into its genetic programmes during haltere development, how does it achieve this? To try answer this question, we analysed Ubx transcriptomic data as before, looking for evidence that RBPs – common regulators of miRNA biogenesis, are differentially expressed due to Ubx activity, potentially facilitating the generation of divergent miRNA profiles. Interestingly, we find that three core components of miRNA biogenesis were up-regulated within the haltere. Our hypothesis is that enhanced miRNA biogenesis leads to increased levels of miRNAs in the haltere. We know little regarding the exact dynamics of miRNA biogenesis and mature miRNA stability. It is possible that some miRNA species require a greater level of biogenesis factors for efficient processing from the pri-miRNA. Alternatively, some miRNAs require constant processing to maintain their required levels. The reason for increased levels of RBP expression induced by Ubx and their relationship to miRNA processing are areas for future research. In this context, the haltere provides an excellent developmental model tissue for this work.

To determine the regulatory impact of interactions between haltere miRNAs and Ubx function during haltere development, we used a genetic interaction assay to determine the effect reducing miRNA function had on haltere development. Genetically disrupting the expression of Dcr1, a miRNA biogenesis factor in a Ubx deficient genetic background led to significantly greater homeotic transformations in the haltere. This data suggests that the main requirement for miRNA function during haltere

Ubx expression itself.

Overall we believe our analysis will provide an important addition in the effort to understand how Hox genes regulate developmental programmes through co-ordinated global changes in the transcriptome, using Ubx regulation of haltere development as a paradigm for Hox. In particular, by integrating our miRNA expression profiles with available transcriptomic data, it will be possible to elucidate and test candidate gene regulatory network motifs formed within the developmental programmes which build the haltere (Fig.5.12A). Through this experimental approach, we may uncover the regulatory pathways used by Ubx in specifying particular tissue and cellular fates. This knowledge may provide insight into understanding the potential risks of disruptions to Hox regulatory networks and how they may lead to developmental abnormalities and disease.

What role may global miRNA activity have in the development of the haltere? The primary functions of miRNAs are often documented as either having an ‘expression tuning’ or ‘expression buffering’ functions. During the development of the haltere, both modes of action maybe relevant. The increased miRNA content of the haltere may have evolved to fine-tune and buffer the haltere transcriptome, re-enforcing the changing transcriptional programme installed by Ubx and ensuring the correct development of this appendage (Fig.5.12B-E). Additionally, the co-ordination and integration of miRNAs to fine-tune and buffer Ubx expression cannot be overlooked (Fig.5.12F-G). In this manner, we hypothesise that the main role for miRNAs within the haltere could be viewed as a robust regulatory force which helped the canalisation of the haltere developmental programme induced by Ubx during the evolution of haltere morphology.

Fig.5.12 Ubx-miRNA integrated gene regulatory networks

(A) Overview of how miRNAs are potentially integrated into the Ubx regulated haltere transcriptome. (B-C) Example coherent feed forward network motifs where a miRNA forms an ‘Expression Tuning’ role. (D-E) Example incoherent feed forward network motifs where a miRNA forms an ‘Expression Buffering’ role. (F-G) Example network motifs where miRNAs form ‘Expression Tuning’ (F) and ‘Expression Buffering’ (G) network motifs to regulate Ubx expression.

Fig.5.12 Ubx-miRNA integrated gene regulatory networks

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