The Abdominal-B locus spans approximately100kb and includes a number of different cis-regulatory elements (see general introduction). This size and complexity presents a problem for researchers in that the transgenic systems generally used to study cis-regulatory elements are size restricted. For example, P-elements generally do not exceed much more than 35kb, as insertion frequency drops dramatically with increasing transposon size. This makes the modeling of complex interactions between cis-regulatory elements difficult, as one cannot easily fit all one wants on such a small piece of DNA.
Another problem with the study of cis-regulatory elements on transgenes is that the location of integration is always subject to some sort of position effect. Depending upon the location of transgene insertion, different effects can often be observed (for a review see (Ryder and Russell 2003)). For this reason, multiple transgenic lines must be obtained and compared to determine a reliable readout. To make matters even worse, this all assumes random insertion into the genome. However, we know that P-elements do not insert randomly into the genome (Liao, Rehm et al. 2000). We also know that many cis-regulatory elements like insulators and PREs elements interact with each other in the nucleus and can bias transgene insertion site preference (Whiteley and Kassis 1997; Bender and Hudson 2000; Fujioka, Wu et al. 2009). Thus, a distinct bias in insertion sites can be expected that might skew the interpretation of one’s results.
It is for all these reasons that much of the work in the Karch lab is now performed by actually modifying the endogenous BX-C sequence. This is a time and labor intensive endeavor, but one that has proven quite successful (Cléard, Moshkin et al. 2006; Mihaly, Barges et al.
44 2006; Iampietro, Cléard et al. 2008; Iampietro, Gummalla et al. 2010). Still, outside of a few areas in the BX-C where our lab has made tools to speed up the process, systematic modification of the BX-C is still beyond our reach. During my graduate work, I have spent time trying to recreate the biological complexity of the BX-C within the experimental convenience of transgenic analysis. For this, I have turned to two tools to help bypass the problems associated with P-element transgenesis. The first tool was modified bacterial artificial chromosomes (BACs) to model BX-C complexity. The second tool was the PhiC31 integration system (Groth, Fish et al. 2004) to allow us to insert these BACs into the fly genome (Venken, He et al. 2006).
In 2001, a technique was developed to facilitate manipulations of large constructs, such as BACs, using in vivo homologous recombination in E. coli. This technology is now commonly called recombineering (Copeland, Jenkins et al. 2001). This technique uses homology regions to target specific modifications to precise sites in the bacterial genome or on BACs. Expression of three recombination genes (Red genes: Gam, Exo and Beta) from the λ bacteriophage is critical for this technology. Basically, the technique works as follows. First, a mutagenic fragment is designed containing DNA sequences identical to the regions flanking the area of DNA to be modified. Between these homology areas (of at least 50 bp and often 500 bp), any sequence modifications can be added. Next, E. coli deficient for many of the genes of its own recombination system are grown under conditions where the three phage red genes are induced.
These bacteria are made competent and transformed with the linear mutagenic DNA fragment.
Within the bacteria, the Gam protein starts by inhibiting the transformed linear DNA sequence from being degraded by the RecBCD nuclease. Then the second protein, Exo, a 5’-3’
exonuclease, generates a 3’ single stranded DNA on both ends of the linear fragment in the homology region. The Beta protein is then recruited to the 3’ ssDNA ends and helps target the
45 fragment to its complementary sequence in the genome or on the BAC. Recombineering is an efficient way to engineer large constructs without the need for the classical cloning tools like restriction endonucleases and ligases.
In 2006, BAC recombineering was successfully combined with the PhiC31 system, thus showing that large constructs can be efficiently integrated in the Drosophila genome (Venken, He et al. 2006). The PhiC31 integrase is an enzyme that mediates the site-specific recombination between two short DNA sequences: attB and attP. In its native setting, the PhiC31 integrase comes from the bacteriophage PhiC31, where it is used to integrate the phage genome into its bacterial host genome via and interaction between the phage attachment site (attP) and the bacterial target sequence (attB) (Thorpe, Wilson et al. 2000; Groth, Fish et al. 2004). Unlike other phage recombination systems, the PhiC31 system recombines between two divergent sequences instead of two identical sequences. This means that upon recombination, the sequences become hybrid sites between attP and attB sites, called attL (attachment left) and attR (attachment right) sites. These hybrid sites are no longer substrates for the PhiC31 recombinase.
In practical terms, this makes the recombination reaction unidirectional, unlike the bidirectional reactions of the CRE/lox and FLP/FRT systems (Groth, Fish et al. 2004).
In terms of a transformation tool, there are numerous advantages to the PhiC31 system over standard, P-element-mediated transformation systems. First, the system is site-specific. This means that once an attP or B site is inserted into the genome, any DNA sequence containing the complementary att site can be integrated into the same genomic locus. Integration into the same genomic locus means that all transformants can be compared against each other without worrying about varied position effects from random transgene insertion. Next, integration sites can be pre-screened for detrimental position effects and integration frequencies. Given a good
46 integration site, transformation efficiencies can be as high as 60% (% surviving injected offspring yielding transformed progeny) (Bischof, Maeda et al. 2007). Another great advantage of the system is that unlike P-element-mediated transformation, the system is not limited by the size of the construct, allowing much larger constructs to be integrated. Indeed, the only limitation in size seems to be the determined by the fragile nature of large pieces of DNA and their increased in viscosity when in an aqueous environment. These limitations are more limitations of the injection procedure used to create transgenic flies than the PhiC31 system itself.
By the time I joined the lab, numerous attP landing platforms had already been created throughout the fly genome. Many of these platforms were created through collaboration between our group and the group of Prof. Konrad Basler at the University of Zurich (Bischof, Maeda et al. 2007). These landing sites were partially pre-screened for both position effects and integration frequencies. Also, a stable, germline source of the PhiC31 integrase was supplied in these lines under the control of the vasa promoter. This system allowed for easy integration of a desired construct by simply adding an attB site and selectable marker to the construct, and injecting it into embryos.
Using this system, I set out to find new tissues in which Abd-B is expressed in the fly and then to understand the mechanism by which Abd-B expression is controlled in these cells.
Because recombineering could be used to quickly modify regions of the BAC to help find novel cis-regulatory elements, we thought this method would prove to be quite efficient. As is explained below, however, my project took on a different twist, as we quickly discovered that the enhancer I was attempting to locate was in an area where we have tools for quick genetic manipulation.
47 Creation of BAC reporters/fusions for Abdominal B.
In order to discover new tissues in which the Abd-B gene functions, we undertook the creation of transgenic reporters that accurately reproduce the Abd-B expression pattern throughout development. Previous studies indicated that the Abd-B gene is expressed as two isoforms, the Abd-B m and r forms, and that the expression of these two isoforms requires separate elements located within a large cis-regulatory region spanning >90kb of DNA (Zavortink and Sakonju 1989). As the Abd-Br isoform is thought to be primarily involved in the formation of the external genitalia (Foronda, Estrada et al. 2006), we decided to concentrate our study on the Abd-Bm isoform, which is involved in determining segment identity. BACR24L18 is a BAC of ~172kb that contains the Abd-B, and much of the abd-A region of the Bithorax complex (BX-C).
Figure 1. Extend of DNA contained in the Abd-B BAC A) Molecular map of the abdominal region of the Bithorax complex numbered in kb according to (Martin, Mayeda et al. 1995)(Genbank U31961). The abd-A and Abd-B transcription units are drawn below the DNA line along with the extent of the segment-specific iab cis-regulatory domains iab-2 through iab-9. B) The rectangle depicts the extent of the BAC used in this study. Note that it lacks the B,C and γ promoters specific for the Abd-Br form. The Gal-4 coding sequence was inserted within the 5’UTR of the Abd-Bm form. C) The structure of the vector sequences used to propagate the BAC and to select the integration within the Drosophila genome. Note the presence of two gypsy insulator sequences flanking the mini-white sequences to prevent possible position effect on white expression (see material and methods for further details).
By recombineering, we reduced BAC24L18 to contain mostly the iab-5 to iab-8 domains required for Abd-Bm expression (removing many of the Abd-Br alternative promoters and its regulatory elements) and the Abd-Bm coding sequence (Figure 1B). A PhiC31 AttB integration sequence and a white marker were also added during the reduction step (Figure 1. B & C).
48 We first tested if expression derived from the sequences on this BAC was sufficient to rescue Abd-B mutant phenotypes. We integrated the Abd-B BAC into the 51C landing platform (Bischof, Maeda et al. 2007) and tested for complementation of two large deletions affecting Abd-B activity. We found that the presence of a copy of the BAC on the second chromosome rescues the mutant phenotypes of iab-6,7IH and iab-5,6J82 (Mihaly, Barges et al. 2006) (Figure 2.).
Figure 2. Rescue of iab-6,7IH by the Abd-B Bac. Panel A and B show male abdominal cuticle preparations from homozygous iab-6,7IH rescued with one copy of the Abd-B BAC (panel A) and homozygous iab-6,7IH (panel B). Male abdomens were cut along the dorsal midline and flattened on a slide. The dorsal surface of each
Note that the 5th and 6th tergites are pigmented. The ventral surface of abdominal segments is composed of soft cuticle called the pleura. On the ventral midline of the pleura there are small plates of harder cuticle called sternites.
In wild type (as well as in panel A), the 6th sternite, circled in red, can be easily distinguished from the more anterior sternites by its different shape and by the absence of bristles. Note also the absence of the 7th abdominal segment present in embryos and larvae, which does not contribute to any adult structures after metamorphosis. B In iab-6,7IH, A6 is completely transformed into a copy of A5 as revealed by the presence of a 6th sternite that completely resembles a more-anterior sternite, covered with bristles (circled in red in panel B). The striking appearance of a 7th tergite is indicative of a homeotic transformation into A6. The transformation is however only partial as seen by the shape of the 7th sternite that resembles the 6th, but harbors a few bristles (A5 character) see also reference (Mihaly, Barges et al. 2006)
Because the sequences preserved on the BAC seemed to drive appropriate Abd-Bm expression, we proceeded to modify the BAC by recombineering to replace the first codon of the first exon of Abd-B with the sequence encoding the Gal4 transcription factor, the mCherry fluorophore coupled to a nuclear localization signal (NLS) and the mCherry as N-terminal fusion to Abd-Bm. By inserting the mCherry sequence just before the stop codon of the last exon of Abd-B a C-terminal fusion was also created (Figure 3). In the two reporter constructs, the Gal4 or mCherry-NLS coding sequence ends with a stop codon, thus eliminating Abd-B expression from
49
Figure 3. BAC recombineering. A) Scheme of the initial recombineering to modify the original BACR24L18 by inserting the attB site (barred section of targeting construct), the white gene (red arrow section) needed for integrant selection after embryo injection and the kanamycin resistance gene (yellow arrow) to select clones positive for that is constructed after the recombineering procedure A. In the text this BAC is also referred as the rescue BAC since it has the intact Abd-Bm form and is able to rescue Abd-B mutants (see figure 2. B, C, & D) Panels are the scheme of modifying the rescue BAC into a reporter BAC by the use of a double selection recombineerng system for seamless insertion of the reporter sequences. SacB was used as the negative selector (in its presence on 5-10% sucrose plates E. coli does not grow) while Ampicillin was used as the positive selector in the double selection cassette.
In B in the first step of insertion of the double selection cassette in the N or C terminal end of Abd-B. In panel C. is the result of the recombineering done in B. and the continuation of the process to replace the double selection cassette with the reporter construct. In panel D is the result of C indicating the construction of the four BAC reporter constructs. The N-mCherry fusion, the C-mCherry fusion the C-mCherry reporter and the Gal4 reporter.
the BAC. As no sequences are removed for the region, we hoped to retain any sequences that are important for Abd-B gene regulation. The final BACs used in these experiments were approximately 109kb for the mCherry reporter and fusion constructs, and approximately 111kb for the Gal4 reporter construct (Figure.1B). All four constructs were integrated into the 51C landing platform on the second chromosome. It must be noted that none of the chromosomes carrying the BAC integrants are homozygosable. The reason for this homozygous lethality is
50 still not entirely clear, especially since Abd-B is not being expressed from half of our constructs.
However, we have noticed some strange transvection-like phenomena (see below) with the BACs that could account for some of this phenotype.
Figure 4. Embyonic expression patterns driven by the Abd-B-Gal4 Bac. Embryos were fixed and stained with antibodies directed against ß-galactosidase.(panels A,B,C and D) and Abd-B (panels E,F,G and H). Panel A,B and C show that the Abd-B-Gal4 BAC mimics Abd-B temporal activation during germband elongation shown in panels F,G and H (see ref (Simon, Chiang et al. 1992)). In panels D and E, stage 14 embryos were opened along the dorsal midline (through the amnioserosa) and flattened on a microscope slide: anterior is at the top, the ventral midline with the developing CNS is visible in the center. Panel E is stained for Abd-B. The parasegmental boundaries are indicated. Arrows in panel D point towards the anterior, ectopic expression already visible earlier in panels B and C. Note also the group of neuroblasts expressing lacZ in parasegments anterior to PS10.
To study the Abd-B expression pattern, a line was established containing the Abd-B-Gal4 BAC and a UAS-GFP reporter on the same chromosome. While the mCherry constructs could also have been used (and indeed were checked for pattern confirmation), we chose to use the Gal4 reporter due to the signal amplification from the Gal4 transcription factor. Initial examination of the embryonic expression pattern in these lines confirms that the Abd-B-Gal4 BAC appears to recapitulate most of the wild-type expression pattern of Abd-Bm in early embryos (Fig.4). However, we do observe some evidence of ectopic expression from the BAC, particularly in the ventral nerve cord (Figure 4D). Even with the slight level of ectopic
51 expression, the Abd-B-Gal4 BAC seems to be a useful tool, as it recapitulates the known patterns of B expression even in adult and larval tissues (Figure 5). The other three constructs Abd-B-mCherry-N, Abd-B-mCherry-C and Abd-B-mCherry reporter exhibited the same pattern of expression as the Gal4 BAC (Figure 6 A-E) in all areas that were checked, however at a much weaker level.
Figure 5. Larval and adult expression patterns driven by the Abd-B-Gal4 BAC. A and B: pictures of live larvae and adult expressing GFP under the control of the Abd-B-Gal4 BAC. A third instar larva is shown in A. The arrow points towards the abdomen emanates from the accessory gland and the fat body.
Previously unknown location of Abdominal B expression in adult flies.
Using the new Abd-B-Gal4 BAC reporter, we identified the adult male accessory gland (Figure 7) as a location of Abd-B expression (Figure 7B). More specifically, based on the expression of our Abd-B-Gal4 BAC, Abd-B appears to be specifically expressed in the secondary cells (Figure 7B,C, and D).
The accessory glands of Drosophila melanogaster are part of the male reproductive system, somewhat similar to the prostate gland and seminal vesicles in mammals. Much of the male reproductive system develops during the pupal stage from the ectodermal cells of the male genital disc. The cells that give rise to the accessory glands are not initially part of the genital disc. Instead they are recruited to the genital disc, with the help of the fibroblast growth factor (FGF), from mesodermally derived cells expressing the fibroblast growth factor receptor (FGFR) during the late third instar stage. Upon recruitment, these cells lose their mesodermal marker
52 twist and start expressing the epithelial marker Coracle, suggesting that they transform from mesodermal to epithelial cells (Ahmad and Baker 2002).
Figure 6. Expression pattern exhibited from the Abd-B mCherry fusion BAC constructs. A) Antibody Abd-B staining in wild type embryo; B) Antibody Abd-B staining in embryo carrying the Abd-B-mCherry-C , a mild expression of Abd-B anterior to PS10 can be detected (compare with A); C) Antybody RFP staining in embryo carrying the Abd-B-mCherry-C , strong anterior RFP signal can be detected; D) Antibody Abd-B staining in wild type larval CNS; E) Abd-B-mCherry-N construct expressing mCherry, strong expression in the posterior part of the brain (presumptive ventral cord), ectopic expression can be detected in the more anterior parts compared to D (wild type) where there is no B expression in the anterior part of the larval brain; F & G) B-mCherry-N & Abd-B-mCherry-C fusion constructs expressing mCherry in the secondary cells of the accessory glands.
The male accessory glands are composed of two types of binucleated cells: the main cells and secondary cells. The main cells have a hexagonal shape and are the majority of the cells of the gland, making up 96% of the gland (~1000 cells). The remaining 4% of the gland is made up of secondary cells (~40 cells) that are located at the distal tip of the gland, interspersed between the main cells. The secondary cells are much larger than the main cells and are pear shaped, with multiple large vacuoles (Figure 7C & D). The primary purpose of the accessory glands seems to be the production of accessory gland proteins (Acps) to be transferred to the female upon copulation. Within the female, these Acps (over 180 Acps) elicit numerous behavioral and physiological changes in the female including an increased rate of egg laying and ovulation, a
A B C
D E
F G
53 reduction in receptivity to secondary courting males, and the formation of a mating plug (Wolfner 2009).
Figure 7. Expression patterns driven by the Abd-B-Gal4 BAC. A) Cartoon depicting the male reproductive apparatus with testis, the paired accessory glands, the ejaculatory duct and ejaculatory bulb. Each accessory gland contains two secretory cell types, the main cells which make up the majority of the gland (top insert) and the secondary cells which are located at the distal tip of the gland interspersed among the main cells (bottom insert) Drawing by J. L. Sitnik; B) Picture of the male reproductive system from flies carrying the Abd-B-Gal4 BAC crossed to a UAS-GFP reporter with the secondary cells of the accessory glands showing GFP expression. The different organs composing the system are marked. C) Magnification of three secondary cells from flies carrying the
Figure 7. Expression patterns driven by the Abd-B-Gal4 BAC. A) Cartoon depicting the male reproductive apparatus with testis, the paired accessory glands, the ejaculatory duct and ejaculatory bulb. Each accessory gland contains two secretory cell types, the main cells which make up the majority of the gland (top insert) and the secondary cells which are located at the distal tip of the gland interspersed among the main cells (bottom insert) Drawing by J. L. Sitnik; B) Picture of the male reproductive system from flies carrying the Abd-B-Gal4 BAC crossed to a UAS-GFP reporter with the secondary cells of the accessory glands showing GFP expression. The different organs composing the system are marked. C) Magnification of three secondary cells from flies carrying the