The miR-310C sequence and expression analysis

The miR-313 miRNA is positioned within an intergenic region of the Drosophila genome flanked by 7 other miRNAs all within 100-200 nucleotides distance of each other (Fig.3.3A). These miRNAs are likely transcribed together as a single poly- cistronic transcript that subsequently undergoes further processing via the miRNA biogenesis pathway. Sequence conservation analysis comparing miR-310, miR-311,

miR-312 and miR-313 pre-microRNA sequences show that they share high sequence

identity with each other (Fig.3.3B). Specifically, the seed sequence (labelled red Fig.3.3B) is identical in all four miRNAs. These miRNAs are likely genomic duplications that consequently diverged in overall sequence structure, whilst maintaining the seed sequence. Since all four miRNAs share the same seed sequence, they are all

predicted to target the Ubx 3’UTR. However their individual targeting strengths, as predicted by the PITA algorithm, vary due to differences in the remaining mature miRNA sequence (labelled pink Fig.3.3B).

To be viable regulators of Ubx, we verified that these miRNAs were transcribed and expressed in the haltere imaginal discs. We tested for expression of the primary miR-

310-313 (miR-310C) transcript using RT-PCR with oligonucleotides flanking the miR- 313 gene. We detected expression of this transcript in both the wing and haltere

imaginal disc tissue (Fig.3.3C). Having shown that the miR-313 miRNA was expressed at the transcript level, we determined any specificity in the spatial expression patterns within the developing haltere tissue.

To examine the spatial pattern of miR-310C expression within the developing haltere disc, we made use of a miR-310CNP5941 _{P-element insertion upstream of the miR-310C}

miRNAs (see blue triangle, Fig.3A). This P-element contains the GAL4 transcriptional activator CDS and was designed to function as an enhancer trap. These insertions can be used to drive the expression of a suitable UAS-reporter constructs revealing the spatial and temporal transcriptional activity at the site of insertion. Using this miR-

310CNP5941 insertion crossed to lines containing a UAS-mCherryNLS fluorescent

reporter transgene (Fig.2.3), we documented the spatial patterns of miR-310C expression within the haltere.

Fig.3.3 The miR-310C sequence conservation and expression analysis

(A) Genomic map of miR-310-313 cluster. Two P-element insertions are shown – miR-

310CEP2587 carrying multiple UAS sequences and miR-310CNP5941 containing a GAL4 coding

sequence (B). Alignment of the miR-310C pre-microRNA sequences showing evolutionary conservation. Mature miRNA sequences are shaded blue, seed sequences are shaded light blue (C). RT-PCR expression analysis of the pri-miR-310C transcripts in wing and haltere imaginal discs, third lane shows a No RT control reaction (D) miR-310CNP5941 (miR310C::GAL4) was used to drive mCherry (UAS-mCherryNLS) expression in the haltere imaginal disc. (D’) Enhanced view of haltere pouch region, areas of high miR-310C expression are denoted by *. (E) Schematic of haltere imaginal disc showing regions of high and intermediate levels of miR-

310C expression. (F) Expression of miR-310C co-stained for Ubx expression. (F’) Enhanced

view of haltere pouch showing variable Ubx expression and miR-310C expression. (F’’) An enhanced view of the haltere pouch showing only Ubx expression. Areas showing high miR-

310C expression and low Ubx expression are denoted by *. Scale bar for panels D-F & D’-F’’ is

RFP RFP DAPI DAPI RFP UBX 311 Ch2R.16475k Ch2R.16469k 310 qsm ₃₁₂313 2498 991 992 _{Nnf1a bl} 500bp UAS GAL4 EP258 7 NP594 1

A

D

F

F’

D’

B

*

*

*

*

*

*

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Fig.3.3 The miR-310C sequence conservation and expression analysis

313 rp49 200bp 200bp

C

mir-310-313 pre-miRNA Sequences

AUAU “seed” Sequence AUAU “mature” Sequence

No RT

RFP

mCherry expression was detected using an α-RFP antibody. We see that the expression pattern is largely contained within the pouch region of the imaginal disc. This section of the haltere corresponds to presumptive haltere appendage as opposed to the thoracic body. The transgene is active throughout the pouch region (Fig.3.3D’) but is specifically strong in two areas (denoted with * Fig.3.3D’). A schematic of the

miR-310C haltere expression pattern is shown (Fig.3.3E). To understand how the

expression of the miR-310C related to Ubx, we co-stained samples for UBX protein expression (Fig.3.3F-F’’). We see that the regions with strong miR-310C signal (* in Fig.3.3F’-F’’) correspond to regions with low levels of Ubx expression.

Overall our data shows that miR-313 is situated within a miRNA cluster, containing three other miRNAs which share an identical seed sequence. Using RT-PCR, we see that all four of these miRNAs are transcribed together as a poly-cistronic transcript within the haltere imaginal disc. Through the use of GAL4 promoter trap insertion upstream of miR-313, we analysed the spatial expression of the miR-310C miRNAs. The miR-310C exhibits a defined spatial pattern of transcriptional expression centred within the pouch region of the haltere disc. There are two specific areas of strong expression, each area correlates with reduced levels of Ubx expression. This data suggests that a possible function of the miR-310C may be to reduce Ubx function within this region.

3.5 miR-310C gain-of-function results in phenotypic changes linked to

Ultrabithorax loss-of-function.

The miR-310C miRNAs seemed excellent candidates to test for possible regulatory interactions with Ubx due to their high scores through bio-informatic analysis as well as their apparent active expression within the developing haltere. To test if the miR-310C could regulate Ubx expression we used the GAL-UAS expression system to over- express the miR-310C within the developing haltere and look for changes in phenotype and Ubx expression.

To assess the gain-of-function effects of the miR-310C miRNAs, we made use of the

miR-310CEP2587 insertion upstream of miR-313. This P-element based insertion

contains UAS sites that be used to ectopically express downstream transcripts (in this instance, the miR-310C). Animals carrying this insertion were crossed to a NubbinGAL4 containing stock (Nubbin::GAL4), a driver which expresses specifically in the “pouch” region of both the wing and haltere imaginal discs. (Fig.2.4).

310C, we first looked for phenotypic changes appearing in the adult haltere

appendage.

miR-310C gain-of-function (GOF) led to noticeable phenotypic changes within the

haltere appendage. Specifically, the appearance of ectopic sensory bristles within the haltere structure (arrowheads Fig.3.4B & Fig.3.4B’). The wild type haltere appendage clearly lacks these large sensory cells (Fig.3.2A & Fig.3.2A’). The ectopic sensory cells seen in the miR-310C GOF animals would normally be found along the margin of the wing appendage and may be considered a “homeotic transformation”. Indeed when analysing halteres from animals that are heterozygous for a Ubx null allele, these ectopic sensory cells can be clearly seen (Fig.3.4C-C’). This similarity of phenotype resulting from a Ubx loss-of-function (LOF) allele and miR-310C GOF expression suggests that the miR-310C phenotype could be due to reductions in Ubx expression levels.

To test this further, we over-expressed the miR-310C in a genetic background that was heterozygous for a Ubx null allele. Any increase in phenotypic severity in these animals would suggest that the miR-310C was negatively regulating Ubx expression. We saw that this was indeed the case (Fig.3.4D-D’), there is a clear increase in phenotypic severity of this genotype. These data suggests that miR-310C GOF negatively regulates Ubx expression, resulting in marked phenotypic changes within the haltere appendage.

Fig.3.4 miR-310C gain-of-function leads to homeotic transformations.

(A-D) The ectopic expression of miR-310C miRNAs using the miR-310CEP2587 insertion leads to homeotic transformations. (A-A’) A WT haltere shows no large sensory bristles. (B-B’) miRNA overexpression using Nub::GAL4 leads to the appearance of large ectopic sensory bristles denoted by arrowhead. (C-C’) A haltere taken from a Ubx null heterozygote (w ;; abx1 bx3 pbx1/+) showing the appearance of ectopic bristles. (D-D’) miR-310C GOF in a Ubx null genetic

background leads to a severe haltere transformation. (E) Induction of clonal cells in haltere imaginal disc over-expressing miR-310C, marked by RFP. (F) The same clonal cells co-stained for Ubx protein expression. (H-J) Images show magnified area of the haltere imaginal disc. (H) Clonal cells marked by RFP. (I) Haltere disc showing Ubx expression. Decreased expression of

Ubx is seen in clonal cells marked with white dashed circles. (J) DAPI staining of haltere

imaginal disc, nuclei are still visible in clonal cells. Scale bars for panels A-D are 40µm, panels A’-D’ are 25µm. The scale bars for E-F represent 20µm and for panels H-J 10µm.

H

RFP

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UBX

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_RFPUBX

Fig.3.4 miR-310C gain-of-function leads to homeotic transformations

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WT

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B’

miR-310C GOF

C

C’

Ubx Null

D

D’

affected Ubx protein levels within the haltere. Using clonal analysis, we induced ectopic

miR-310C expression in clonal cell populations by crossing the miR-310CEP2587 _(UAS-

miR310C) insertion to Actin-GAL4 ‘FLP-OUT’ stock which also carried a hs-FLP recombinase and a UAS::myrRFP reporter construct (Fig.2.6). Progeny of this cross were exposed to 37oC heat shock treatment during first larval instar growth phases. This heat shock treatment induced the expression of the FLP recombinase which excises the FRT-Stop cassette which separates the Actin promoter sequence from the GAL4 driver. FRT excision can only occur when cells are dividing therefore the result of the heat shock treatment is a stochastic activation of the Actin GAL4 driver, which induces the expression of the target UAS-miR310C insertion and the UAS::myrRFP reporter. Haltere discs were dissected from white-pre pupae animals and stained for Ubx expression. This technique has the advantage of inducing ectopic miRNA expression in small subsets of cells marked with an independent RFP reporter which can then be compared with the remaining haltere tissue.

We used immuno-histochemistry to monitor changes in Ubx protein expression in miR-

310C over-expressing cells. Small groups of clonal cells, marked by myrRFP (see

dashed box Fig.3.4E) were co-stained with the nuclear stain DAPI and Ubx antibody (Fig.3.4F). Close inspection of these cells (Fig.3.4H) shows that they appear to have little detectable levels of Ubx protein (Fig.3.4I). This loss in Ubx expression is not attributable to the cell death within the clonal cell populations as there is clear staining of DNA within nuclei still present in these cells (Fig.3.4J).

Overall, through genetic analysis we see that miR-310C GOF leads to phenotypic changes during the development of the haltere appendage. Furthermore, we also see that this phenotype can be affected by changes in endogenous Ubx levels. Through clonal analysis in the developing haltere imaginal disc, we see that miR-310C GOF leads to a visible reduction in Ubx protein expression. Together these results show that

miR-310C is physiologically capable of negatively regulating Ubx expression levels.