Investigating the targets of Abd-B using mRNA-seq
Because of the modest amount of information on secondary cells in the literature, and in order to further understand the secondary cell phenotype in Abd-B mutants, we performed an mRNA-seq analysis comparing the transcriptomes of wild-type accessory glands and iab-6cocu accessory glands. For our wild-type line, we used the iab-5,6rescue line. This line was made using the same PhiC31 system that we used to make the iab-6cocu flies, but differs in that a wild-type piece of DNA was integrated into the platform instead of the mutant piece. In this way, we hoped to obtain a genetic background as similar as possible between the two lines.
Total RNA was isolated from 100 accessory glands from each genotype and was sent for deep sequencing on an Illumina sequencer at FASTERIS SA (Geneva, Switzerland).
Bioinformatic analysis was also also performed at Fasteris. The HiSeq run yielded 66,943,897 reads, 36,740,061 for iab-5,6rescue and 30,203,836 for iab-6cocu. For iab-5,6rescue 71.0% of reads were mapped to the reference genome while for for iab-6cocu 70.6% were mapped. Counts were normalized as reads per million (RPM) by dividing by the total number of reads and multiplying by 1 million, leaving us 8,764 genes to compare. By simply dividing the number of reads per gene of iab-5,6rescue with iab-6cocu, we were able to determine the most down regulated genes in the iab-6cocu male accessory glands, as compared to iab-5,6rescue. The reverse operation was also performed in order to determine the most up regulated genes in iab-6cocu. Using an arbitrary cut-off value of 5x, we found that 73 genes were down-regulated in iab-6cocu flies by a factor of five or more relative to iab-5,6rescue flies. By contrast, 115 genes were found to be up-regulated by a
63 factor of 5. In a first step, we decided to focus our attention on the down-regulated genes, believing (for no real reason) that the loss of a product might account for the Abd-B phenotype more than an up regulated product.
Table 1. Top ten most down regulated genes from the mRNA-seq. This table is just a piece from the much larger table displayed in the appendix of the thesis (Table 1) with all 73 genes.
The predicted functions of the 10 most down regulated genes are summarized in Table 1 (see appendix Table 1 for list of the 73 candidate genes). According to the FlyBase predictions, the most common functions were listed as unknown (34.2% or 25 genes), serine-type endopeptidase activity (8.2% or 6 genes), transferase activity (8.2% or 6 genes), sodium:iodide symporter activity (6.8% or 5 genes) and transmembrane transporter function (5.5% or 4 genes).
As for the tissue in which these genes are expressed at a highest level, the FlyAtlas Anatomical Expression database indicates that 24 (32.8%) show highest expression in the malpighian tubules, 12 (16.4%) show highest expression in the testis, 9 (12.3%) show highest expression in the midgut and 2 (2.7%) show the highest level of expression in the accessory glands. The two showing highest expression in the accessory glands are CG11598 (Ravi Ram, Ji et al. 2005) and CG3349 (Ravi Ram and Wolfner 2007). These genes have previously been identified as genes
64 encoding Acps but have not been characterized. Since known Acps are secreted molecules it is interesting to note that 38 (52.02%) of the 73 genes have a signal sequence, though it is clear that the iab-5,6cocu phenotype is the result of a loss in the transcription of a specific Acp. Lastly, it is interesting to note that some of the genes seem to cluster to a specific genetic locus. CG12809, CG33783, CG33784, CG33631, CG5361 and CG33630 are all located next to each other within 13kb. The genomic co-localization of the genes, together with the RNA-seq results suggests that these genes are probably co-regulated as a gene neighborhood. A detailed account of the molecular characteristics and mRNA-seq results for the 73 genes can be found in the table 1 (appendix).
RNAi.
In order to further narrow down the list of candidate genes responsible for the cocu phenotypes, we decided to perform an RNAi screen of the candidate genes. Fly lines carrying UAS-RNAi constructs for most of our candidate genes were ordered from the Vienna Drosophila RNAi Center (VDRC). Males from the received lines (Table 6 in material and methods section) were crossed to the DI-Gal4 and DI-Gal4, UAS-GFP driver line females. The UAS-GFP marker, which is excluded from the secondary cell vacuoles, was used as an aid to visualize the cellular phenotype. Driver lines with and without a GFP marker were used separately in order to eliminate the effect, if any, of the UAS-GFP on the phenotype. Based on our examination under fluorescence and Nomarski microscopy, we did not observe any differences between the unmarked lines and the lines with the UAS-GFP marker. Below, I primarily show images derived from the GFP-marked secondary cells for visual simplicity.
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Figure 1. RNAi experiment results for genes that showed a secondary cell phenotype. Names of the genes are embedded in the images. Their predicted function is noted below each image for each gene. The cross was DI-Gal4 X RNAi for the Nomarsky images (A-F) and DI-Gal4 UAS-GFP X RNAi for the fluorescence images (G-L). WT – wild type secondary cells (A) with nicely formed vacuoles, look like doughnuts with Nomarski, while in the rest of the pictures the vacuoles are gone or not the right size, they look like small bumps. Representative secondary cells from each panel on Nomarski images are marked with a circle with dashed line. In the GFP fluorescent images the vacuoles are clearly visible as black holes within the secondary cells with variable size, depending on the figure.
66 The male progeny from the crosses above were collected and subjected to two types of analysis. First, at least three accessory glands from virgin males were dissected to examine secondary cell morphology using Nomarski microscopy for the DI-Gal4 crosses (Figure 1 A-F) or using fluorescent microscopy for the DI-Gal4, UAS-GFP crosses (Figure 1 G-L). Second, an laying assay was performed to determine if any of the knocked-down genes exhibit an egg-laying phenotype similar to that seen in iab-6cocumutants (Gligorov, Sitnik et al. 2013).
Figure 2. Genes from the RNAi screen that showed a mild secondary cell morphological phenotype. Variable size of vacuoles can be detected in all images (A-J) but not an absence of vacuoles. Embedded in the image is the name of the gene whose knockdown causes the presented phenotype.
67 For few of the candidate genes, CG7882, CG9509, CG10514, CG14069 and CG31034 a detectable secondary cell phenotype of variable severity was detected (Figure 1 G-L). In general, these phenotypes ranged from displaying a lower number of smaller vacuoles to the loss of most vacuoles from the secondary cells. Many more showed a weaker and less penetrant phenotype, often consisting of more and smaller vacuoles than normal, in only one or two of the accessory glands dissected. The genes that exhibited this kind of phenotype are depicted in figure 2:
CG5106, CG31198, CG33630, CG9294, CG15406, CG6602, CG12809, CG13538, CG18088 and CG14681. The rest of the candidates screened didn’t display any secondary cell morphological phenotype.
Figure 3. Results the RNAi egg laying assay for genes CG7882 and CG9505. Both show the most drastic secondary cell morphological phenotype. Eggs were counted for 5 days after mating. The chart shows CG7882 (dark blue) and CG9509 (red) following the iab-6cocu(purple) egg laying phenotype. T test values for CG7882 p=0,009256 and CG9509 p=0,055006. (n=10)
The results of the egg-laying screen also varied from gene to gene (Table 2 of appendix – for the raw data coupled with secondary cell morphology data information). The top two candidate genes, CG7882 and CG9509 (Figure 3), showed a severe reduction of egg-laying.
Interestingly, these two candidates also displayed a severe secondary cell morphological phenotype. The next few genes with an egg-laying phenotype, CG15406, CG9036 and CG3285,
68 showed only mild or no secondary cell morphological phenotype (Figure 2 E; Figure 4 dark blue line). Interestingly, two of the genes that showed a severe secondary cell morphological phenotype, CG10514 and CG31034, showed no detectable egg-laying phenotype.
Figure 4. Results the RNAi egg laying assay for genes CG15406 (dark blue), CG9036 (purple) and CG3285 (light blue) that show an egg laying phenotype but no secondary cell morphological phenotype. Eggs were counted for five days after mating. The chart shows CG9306 and CG3285 following the iab-6cocu (red) egg laying phenotype with CG15406 following close as well. The T test results for these knockdowns are CG9306 p=0,126138 ; CG3285 p=0,011017; CG15406 p=0,009256. (n=10)
Based on the comparison between the two RNAi screens (the secondary cell morphology and the egg-laying screen), we decided to focus our future efforts on eight candidate genes, CG7882, CG9509, CG3285, CG14069, CG15406, CG3349, CG13793 and CG14292. All of them showed an egg-laying phenotype of variable degrees and three of them, CG7882, CG9509 and CG14069 showed a severe secondary cell morphological phenotype (Figure 1 J, K, H;
appendix Table 2). The other genes from the list were left aside because either they didn’t show an egg laying phenotype of sufficient severity.
Based on their amino acid sequence the predicted function for CG7882, CG3285 and CG15406 is in “general sugar transport”, while CG9509 is a predicted glucose-methanol-choline oxioreductase and CG13793 is a predicted neurotransmitter transporter. Finally, CG14069,
69 CG3349 and CG14292 have no predicted molecular function. We used TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) to predict their cellular localization based on sequence information. Five of them CG14069, CG15406, CG3349, CG13793 and CG14292 were predicted, with variable degrees of confidence, to be located somewhere in the secretory pathway. CG3285 was predicted with low confidence to be located in the mitochondria. As for CG7882 and CG9509 based on their sequence data this software cannot predict their location within the cell.
Figure 5. Preliminary in situ hybridization results for genes CG7882, CG9509, CG15406, CG10514, CG31034 and CG14069. A signal at the tip of the accessory gland can be seen coming from the secondary cells. There is background staining in the lumen of the glands, making definitive clear results lacking. Names of the genes are embedded in the images (A-F). Four of them are genes selected for the final 8, A, B, C and F.
Preliminary in situ hybridization results.
For some of the 73 candidate genes (CG7882, CG9509, CG15406, CG10514, CG31034 and CG14069) in situ hybridization was performed in order to establish the location of their expression within the accessory glands (Figure 5). For this, we used DNA probes made from exonic sequences from the candidate genes. The preliminary results seem to indicate that all of them are specifically expressed in the secondary cells. Convincingly strong secondary cell
70 staining could be obtained from CG10514, CG31034, CG15406 and CG14069. The results for CG7882 and CG9509 were less convincing, but the weak staining observed seemed to be secondary cell specific. In situ hybridization for the rest of the 73 candidates has been attempted, but has been difficult to achieve as transcripts seem to be expressed at a low level and developing a reliable protocol for in situ hybridization on accessory glands has been difficult.
Abd-Bm ISH.
In order to prove our assertion that the Abd-Bm form is only expressed in the secondary cells and the Abd-Br form is expressed in the ejaculatory duct of the accessory glands we designed in situ hybridization probes that could distinguish between the two isoforms. Indeed when in situ hybridization using these probes was performed on iab-5,6rescue male accessory glands, we could clearly see staining in the secondary cells with the Abd-Bm probe and not with the Abd-Br probe while the result for ejaculatory duct staining was reversed (Figure 6).
Figure 6. Differential expression of the two Abd-B isoforms. The images are from in situ hybridization experiments where probes specific for the Abd-B m and the r isoforms were used to distinguish between the two.
Panel A is staining with Abd-Bm specific probe, while the m isoform appears in the secondary cells of the accessory glands (A), isoform r is detected in the ejaculatory duct only (B). Surprisingly, the Abd-Bm isoform is still detectablt in iab-6cocu accessory glands. The black arrows in the images point to stained areas.
We also performed the same staining on cocu males. The Abd-Br probe gave the expected staining; the ejaculatory duct was clearly stained. However, when we stained with the
71 Abd-Bm probe we got a clear staining of the secondary cells in the accessory glands of the iab-6cocu males (Figure 6 C). This result was unexpected as antibody staining for Abd-B shows that Abd-B protein is completely lost in secondary cells of cocu males (Figure 9F, results part II). As I mentioned above, however, in situ hybridization on accessory glands has been extremely difficult to accomplish and Abd-B is not that highly expressed. This finding still must be confirmed, though it leads to some intriguing possibilities regarding Abd-B regulation in the secondary cells.
Antibody development.
In order to further explore the importance of the eight selected candidate genes, we have started the process of producing antibodies against all eight proteins. To do this, we designed
~50 amino acid long sequences from each target genes and expressed them as 6-his tagged fusion proteins (using pQE31) in the SG13009 bacterial strain. Thus far, we have been able to express and purify three out of the eight antigens and sent two (CG9509 and CG15406) to be injected into rabbits at the Pocono Rabbit Farm & Laboratory (PRF&L, PA 18325, USA) using their “49 Day Mighty Quick Protocol” (PRF&L – proprietary protocol).
Vacuolar markers.
Our work has shown that a loss of Abd-B in the secondary cells causes a loss of their characteristic vacuoles. Yet, although we used this phenotype as an indication of dysfunction in the secondary cells, we still have no clue as to their function in vivo. We have assumed that these vacuoles might store molecules for secretion into the lumen of the accessory gland to be included into the male seminal fluid. However, we have never observed a change in the number or size of
72 the vacuoles in dissected accessory glands from virgin or mated males. Even males dissected just after mating seem to display normal secondary cell vacuoles. Thus, we wondered if these vacuoles were secretory or something else. In order to begin to unravel this mystery, we decided to look vesicular trafficking markers.
Different endosomal pathways exist within the cells and each type of vesicle displays different markers depending upon the pathway in which they participate. The most commonly used markers are the Rab family of small GTPases. After consulting with our colleague Marcos Gonzales-Gaitan here in Geneva, we obtained a battery of fly lines containing UAS driven GFP-tagged vesicular trafficking markers to test. Rab4-GFP was used as a marker for the early and fast recycling endosomes while Rab5-GFP as a marker for the early endosomes. Rab7-GFP was used to mark the late endosomes and Rab11-GFP for the slow recycling endosomes (for review on Rabs see (Fukuda 2008; Hutagalung and Novick 2011)). Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-GFP was used to label sorting endosomes and multivesicular bodies while a “tandem of FYVE domains” (originating from the hrs gene) was used as a marker for sorting endosomes. The Smad anchor for receptor activation (Sara)-GFP marks sorting and early endosomes (for review see (Gonzalez-Gaitan 2003). Finally, two constructs from the Uninflatable gene (Uif) were used to mark the general secretory pathway, Uif-ecd-GFP is the extra cellular domain of Uif and Uif-C-term-GFP is the C terminal region of Uif including the transmembrane domain to mark the membrane of secretory vesicles (unpublished). We used our DI-Gal4 driver to drive expression of each construct and examined their localization within the secondary cells (Figure 7). To limit the overexpression effects, a Gal-80 expressing transgene was also included in the background. The results showed that Rab7, Rab11, Hrs, uif-C-term, FYVE and Sara are all located on the membrane of the large vacuoles. Meanwhile, Uif-ecd was
73 located within the vacuoles. The Rab4 and Rab5 proteins were not associated with the vacuoles at all. These results at first seem contradictory because the vacuoles harbor characteristics of different classes of endosomes such as, late endosomes (Rab7), recycling endosomes (Rab11 and Hrs), secretory endosomes (uif-exo, uif-c-term) and early endosomes (SARA). The result with SARA is a bit odd since SARA is usually seen coupled with Rab4 and Rab5 staining. These results, obtained by overexpression should be interpreted cautiously, because they seem to indicate that the secondary cell vacuoles may represent a new kind of intracellular vesicle.
Figure 7. Endosome markers. To determine the localization of markers for different endosome pathways with in the secondary cells the DI-Gal4 driver was used to drive of UAS-XXXX-GFP constructs in the background of gal80ts. Glands were dissected and imaged from flies after 1-2 days incubation at 25°C, they were initially kept at 18°C. Names of the genes/constructs tested are written on the images. Uif C-term, Hrs, Rab-11, Sara, FYVE and Rab-7 have vacuoles positive for these markers. Uif-ecd locates to the inside of the vacuoles. Rab-4 and Rab-5 don’t show any localization to the vacuoles.
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DISCUSSION
Here, I have described results showing that Abd-B is expressed in the secondary cells of the Drosophila male AG. Using mutations that specifically remove Abd-B from these cells, we have been able to uncover roles for this previously unstudied but important reproductive cell type. Furthermore, we show that Abd-B expression in these mesodermally-derived cells does not fit the classical domain model paradigm developed for the segment-identity function of the Hox genes in ectodermal tissues. And finally, with the aid of our collaborators whose work we will not present here, we demonstrate that the secondary cells of the male AG synthesize products necessary for maintenance of the seminal fluid’s effects on the female PMR (Gligorov, Sitnik et al. 2013).
New insights into Abd-B gene regulation
Due to the large size and complexity of the Abd-B regulatory region, we created a BAC- reporter construct to monitor Abd-B expression in the adult fly. When combined with fluorescent markers, this method allowed us to bypass the technical issues of antibody penetration and the laborious dissections needed for in situ hybridization or immunohistochemistry to identify a novel area of Abd-B expression in the adult. Overall, the BAC reporter is able to accurately reproduce the known, complex Abd-B expression pattern; in fact, it should be noted that our BAC construct reproduces the Abd-Bm expression pattern better than even an enhancer trap line inserted in the Abd-B promoter (Abd-B-Gal4LDN) (de Navas, Foronda et al. 2006). Furthermore,
75 by combining our BAC reporter with pre-existing deletion mutations, we were able to discover the function of a vital gene in an adult tissue without the need to create mitotic clones.
From the standpoint of Hox gene regulation, our discovery of the secondary cell enhancer is quite interesting because, unlike other cell-type specific enhancers from the BX-C, the secondary cell enhancer does not seem to be regulated by a domain initiator (Simon, Peifer et al. 1990;
Mihaly, Barges et al. 2006; Iampietro, Gummalla et al. 2010). Most cell-type specific enhancers from the BX-C are not intrinsically restricted along the A-P axis. They are restricted only to a specific cell-type and gain A-P restriction through clustering in a BX-C regulatory domain. For example, in a transgene assay, an enhancer from the iab-7 domain (called 11X) drives expression in the tracheal placodes in all segments. However, in the BX-C, this enhancer seems to be active
Mihaly, Barges et al. 2006; Iampietro, Gummalla et al. 2010). Most cell-type specific enhancers from the BX-C are not intrinsically restricted along the A-P axis. They are restricted only to a specific cell-type and gain A-P restriction through clustering in a BX-C regulatory domain. For example, in a transgene assay, an enhancer from the iab-7 domain (called 11X) drives expression in the tracheal placodes in all segments. However, in the BX-C, this enhancer seems to be active