Redundant enhancers in the iab-5 domain cooperatively activate Abd-B in the A5 and A6 abdominal segments of Drosophila

The homeotic Abdominal-B (Abd-B) gene belongs to Bithorax complex and is regulated by four regulatory domains named iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. It is assumed that male specific features of the adult cuticle in A5 is solely dependent on regulatory elements located in iab-5, while the regulatory elements in the iab-6 are both necessary and sufficient for the proper differentiation of the A6 cuticle. Unexpectedly, we found that this long held assumption is not correct. Instead, redundant tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our study of deletions shows that the iab-5 initiator is essential for the functioning of the iab-5 enhancers in A5, as well as for the correct differentiation of A6. This requirement is circumvented by deletions that remove the initiator and most of the iab-5 regulatory domain sequences. While the remaining iab-5 enhancers are inactive in A5, they are activated in A6 and contribute to the differentiation of this segment. In this case, Abd-B stimulation by the iab-5 enhancers in A6 depends on the initiators in the iab-4 and iab-6 domains. Summary Statement In Drosophila, the segmental-specific expression of the homeotic gene Abdominal-B in the abdominal segments is regulated by autonomous regulatory domains. We demonstrated cooperation between these domains in activation of Abdominal-B.


Introduction
In Drosophila melanogaster segment identity in the posterior 2/3rds of body is controlled by the three homeotic genes, Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B), which form the bithorax complex (BX-C) (Lewis, 1978). The specification of parasegments (PS)/segment identity depend upon the expression patterns of these three homeotic genes (Duncan, 1987;Karch et al., 1985;Karch et al., 1990;Maeda and Karch, 2015;Peifer et al., 1987). The genes are controlled by an array of nine regulatory domains, each of which is thought to be responsible for directing the expression of one of the homeotic genes in a spatio-temporal pattern appropriate for the particular PS/segment. The Abd-B gene is responsible for the specification and differentiation of PS10/A5, PS11/A6, PS12/A7, PS13/A8, in which the pattern of its expression is determined by four regulatory domains, iab-5, iab-6, iab-7 and iab-8 respectively (Fig. 1A).
Analysis of BX-C regulatory domains, including those controlling Abd-B indicate that they are composed of the same set of elements (Kyrchanova et al., 2015;Maeda and Karch, 2015). Each domain has an initiator element that sets the activity state (on or off) of the domain early in embryogenesis (Maeda and Karch, 2015;Mihaly et al., 2006;Peifer et al., 1987). Initiators responds to the maternal, gap and pairrule gene products that subdivide blastoderm stage embryos along the antero-posterior axis into 14 parasegments (Busturia and Bienz, 1993;Casares and Sánchez-Herrero, 1995;Drewell et al., 2014;Ho et al., 2009;McCall et al., 1994;Qian et al., 1991;Shimell et al., 1994;Starr et al., 2011). For example, in PS10/A5, the iab-5 initiator turns on the iab-5 domain, while the adjacent iab-6 and other more distal (relative to centromere) domains remain in the off state (Iampietro et al., 2010). In PS11/A6, the initiator in iab-6 turns the domain on. While iab-5 is also active in PS11, iab-7 and iab-8 are off. The gene products responsible for setting the activity state of the BX-C domains disappear during gastrulation and different mechanisms are deployed to remember the on or off state. The on state is maintained by Trithorax group proteins, while the off state is maintained by Polycomb group proteins (Busturia and Bienz, 1993;Kassis et al., 2017;Kuroda et al., 2020;Simon et al., 1992;Shimell et al., 2000;Ciabrelli et al., 2017;Müller and Bienz, 1992). These factors interact with special elements in each domain called Trithorax or Polycomb response elements (TREs or PREs). Finally, each domain has a stage and tissue specific enhancers which are responsible for activating patterns of homeotic gene expression that drive PS/segment differentiation (Maeda and Karch, 2015). Each domain is bracketed by chromatin boundary elements (Barges et al., 2000;Bender and Lucas, 2013;Bowman et al., 2014;Galloni et al., 1993;Gyurkovics et al., 1990;Hagstrom et al., 1996;Karch et al., 1994;Kyrchanova et al., 2020;Mihaly et al., 2006, 200;Zhou et al., 1996). The boundaries in the Abd-B region  have two important functions. The first is to block crosstalk between adjacent regulatory domains so that they can function autonomously. The loss of one of these boundaries leads to the ectopic activation/silencing of neighboring regulatory domains. For example, deletion of the Fab-6 boundary element can result in the ectopic activation of iab-6 and silencing iab-5 in PS10/A5 leading respectively to gain-of-function (GOF) and loss-of-function (LOF) transformation of PS/segment (Iampietro et al., 2010;Postika et al., 2021).
The second function is boundary bypass that enables enhancers in the Abd-B regulatory domains to bypass intervening boundaries and activate Abd-B (Kyrchanova et al., 2019a;Kyrchanova et al., 2019b;Postika et al., 2018).
While identity of PS10-PS13/A5-A8 is determined by the pattern of Abd-B expression in both sexes, the phenotype of the adult cuticle in segments A5 and A6 in Drosophila melanogaster differs in males and females (Jeong et al., 2006;Kopp et al., 2000;Massey and Wittkopp, 2016;Williams et al., 2008). In females, cuticle pigmentation and morphology in A5 and A6 are similar to that in more anterior segments whose identity is determined by abd-A. In these segments the tergite has a posterior stripe of dark pigmentation, while the sternite has a quadrilateral shape and has multiple bristles. The pigmented stripe in tergites A2-6 is generated by the yellow (y) and tan genes, which are regulated by the optomoter blind (omb) gene (Kopp and Duncan, 1997). The bric-a-brac (bab) complex encodes DNA-binding proteins that repress the expression of the genes responsible for cuticle pigmentation (Couderc et al., 2002;Kopp et al., 2000;Roeske et al., 2018). While female pupae express bab in abdominal segments A2-A6, bab expression in males is limited to segments A2-A4. The sex specific pigmentation pattern and cuticle morphology in A5 and A6 in males depend upon Abd-B and the male product of the double-sex gene (dsx M ), which together function to repress expression of bab genes in cells giving rise to the A5 and A6 (Kopp et al., 2000;Massey and Wittkopp, 2016;Wang et al., 2011).
The level of Abd-B expression in PS10/A5 and PS11/A6 is not the same and correlates with their distinctive morphology. The Abd-B expression in A5 is relatively low and this segment has morphological features of the A4 where abd-A is expressed: the A5 sternite has a quadrilateral shape and has multiple bristles, while the A5 tergite is covered by small trichome hairs. However, due to the expression of Abd-B in the A5 segment of males, differences are observed: sternite becomes wider, tergite is completely pigmented, and trichomes are less dense (Celniker et al., 1990;Maeda and Karch, 2015) (Fig.1). The higher levels of Abd-B in A6 are accompanied by specific morphological features in both the sternite and tergite. The A6 sternite lacks bristles and has a unique 'banana' shape, while the trichomes on the fully pigmented tergite are restricted to the anterior and dorsal margins instead of covering nearly the entire tergite.
Here, we have investigated the mechanisms responsible for regulating Abd-B expression during the differentiation of the male cuticle in segments A5 and A6. We have found that iab-5 and iab-6 domains share a common set of partially redundant cuticle enhancers located in iab-5 that are critical for male specific differentiation of the cuticle of A5 and A6 segments.

Inactivation of the iab-5 domain affects expression of Abd-B in the A6 segment
In contrast to the boundaries of the Abd-B region, that have blocking and bypass activity (Kyrchanova et al., 2016;Kyrchanova et al., 2019a;Postika et al., 2018) the heterologous boundaries, for example, DNA fragment consisting of five binding sites for the multi-Cys2His2 zinc finger protein Pita (Pita ×5 ), have only blocking activity. To test whether the regulation of Abd-B by iab-5 requires the bypass boundary activity, we took advantage of F6 1attP replacement platform, in which a 1389 bp sequence spanning the Fab-6 boundary was substituted by an attP site (Postika et al., 2021). We have replaced Fab-6 with Pita ×5 . In order to assess the activity of cuticle enhancers in iab-5 and iab-6, we included a miniyellow (mini-y) reporter which we placed either upstream of Pita ×5 or downstream so that it would be located in the iab-5 (mini-y Pita ×5 ) or iab-6 (Pita ×5 mini-y) domains, respectively (Fig. S1). The reporter consists of a yellow (y) cDNA fused to the 340 bp y promoter. As it lacks the enhancers of the endogenous y gene, its expression depends upon nearby enhancers. Expression of mini-y was examined in a y 1 background. In flies carrying the null y 1 allele, the tan gene is appropriately expressed in A5 and A6 reflecting the Abd-B activity, and the resulting pigmentation in the tergite is light brownish, not black (Camino et al., 2015;Rebeiz and Williams, 2017).
F6 1attP deletion mutant males differ from wild type (wt) in that segment A5 has an incomplete GOF and LOF transformation (Fig. 1). The A5 sternite has a shape like that normally observed in A6, but with several bristles, the A5 tergite has patches of cuticle that lack trichomes, which is indicative of a GOF transformation towards A6 identity. On the other hand, large portions of the A5 cuticle also lack tan pigmentation, indicating that the cells have an A4 identity. There are also LOF phenotypes in A6 including bristles on the sternite and regions of the tergite that are depigmented or have ectopic trichomes.
In the Pita ×5 replacements with or without mini-y the GOF transformations of A5 are eliminated ( Fig. 1). Since Pita ×5 does not support bypass, A5 resembles A4: the sternite has a quadrilateral shape, and is covered in bristles, while the tergite is covered in trichomes, and instead of being covered in pigmentation, there is only a posterior stripe. This result shows that iab-5 is unable to activated Abd-B in A5, and that the cells in this segment have an A4 identity. However, there is also an unexpected result: the differentiation of A6 is altered compared to wt. The defects are most clearly evident when mini-y is excised and y + allele is introduced. Fig. 1 shows that pigmentation of the A6 tergite resembles A4: there is only a stripe of pigment along the posterior margin of the tergite. In addition, the A6 tergite is covered in trichomes just like A4. While the A6 sternite has a nearly normal shape, there are multiple bristles. These LOF transformations indicate that the Pita ×5 insulator disrupts Abd-B dependent cuticle differentiation not only in A5, but also in A6. These results suggest that iab-5 is required for the proper activation of Abd-B in the cuticle in both A5 and A6.
Further support for this conclusion comes from analysis of mini-y expression in the y 1 background.
In Pita ×5 mini-y males the reporter located in the iab-6 domain it is not turned on in neither in A5, nor in A6. Instead, only the tan gene is expressed in an A4 like pattern. This result would indicate that enhancers in iab-5 are required to drive expression of mini-y inserted in iab-6 in the A6 tergite. When the mini-y reporter is in iab-5 we observe a mosaic pattern of pigmentation in the posterior stripes of the A4, A5 and A6 segments.

The iab-5 domain contains a set of redundant cuticle enhancers that can drive yellow expression outside BX-C
To map enhancers in the iab-5 regulatory domain responsible for Abd-B expression, we linked 1-3 kb overlapping DNA sequences from the iab-5 domain (i5 1 -i5 7 and i5 ini ) to a y reporter in a transgene that also carries a mini-white (w) (Fig. S2). To reduce potential position effects, we placed Pita ×5 upstream of the iab-5 DNA fragments. Using phiC31-mediated recombination (49), we integrated a collection of eight i5 transgenes into a well characterized 86Fb platform. Of these i5 fragments, only three, i5 1 (1013 bp), i5 2 (2145 bp) and i5 7 (2524 bp), activated y expression in the cuticle. For all three, pigmentation was observed in the A5 and A6 tergites. Interestingly, we found that the i5 7 fragment was only able to activate y in the forward (genomic) orientation. We tested two i5 7 sub-fragments from the proximal (i5 S5 ) and distal (i5 S6 ) ends relative to the centromere. Of these, only i5 S6 , activated y. Thus, in larger i5 7 fragment, the enhancer in i5 S6 must be located next to the promoter to function. Since the i5 1 and i5 2 overlapped, it seemed possible that they share the same enhancer. To test this, we generated three smaller fragments (i5 S1 , i5 S2 , and i5 S3 ) spanning most of i5 1 and i5 2 . Of these only i5 S2 which includes the overlap between i5 1 and i5 2 activates mini-y.

Functioning of the iab-5 enhancers in the iab-6 domain
We next determined whether iab-5 sequences are able to regulate Abd-B in A6 when placed in iab-6. We used the F6 1attP landing platform to insert the same collection of iab-5 sequences into the iab-6 regulatory domain (Fig. S3). The starting transgene included Pita ×5 to block crosstalk with iab-5 and excisable mCherry and mini-y reporters arranged so that they are located in iab-6 in the replacements (Fig.   2, Fig. S1).
Three of the iab-5 sequences, i5 1 , i5 3 , and i5 7 are able to stimulate mini-y expression to different extents in the A6 segment (Fig. S3). While both i5 1 and i5 7 also stimulated y when inserted in 86Fb platform, i5 3 did not. Conversely, i5 2 failed to function when placed in iab-6, while it is active in the 86Fb platform. It seems likely that "position effects" are responsible for the differences in the activity of the iab-5 sequences when linked to the y reporter in 86Fb or inserted in iab-6. As would be expected from their placement in iab-6, i5 1 , i5 3 and i5 7 do not activate the reporter in more anterior segments.
Surprisingly, insertion of the i5 7 fragment in the reverse orientation (i5 7R ) stimulates mini-y expression in posterior stripes not only in the A6 tergite, but also in the A5 and A4 tergites (Fig. S3). Since the distal part of i5 7 , i5 S6 , induces a much stronger activation of the mini-y, it would appear that sequences elsewhere in i5 7 contain a silencer.
As the reporters compete with Abd-B for enhancer activity we assessed the cuticle phenotypes after removing the reporters and introducing a y + allele (Fig. 2). When i5 1 , i5 3 or i5 7 are included in the Pita ×5 replacements the phenotype of A6 is close to wt. Even though i5 S2 and i5 2 activate mini-y at 86Fb, neither could rescue the LOF phenotypes induced by Pita ×5 . On the other hand, i5 S1 and i5 3 , which does not stimulate mini-y at 86Fb, completely rescues the Pita ×5 induced LOF phenotypes in A6 (Fig. 2). The remaining fragments that are active when introduced into iab-6 are i5 ini and i5 7 . The former is not active at 86Fb, while the latter is. Both partially rescue the Pita ×5 induced defects in A6. The fragment of i5 ini , i5 S4 , restore A6 identity with variable efficiency. As was the case in 86Fb, the i5 7 enhancer activity is orientation dependent and it is not observed in i5 7R . Thus, there are several enhancers in iab-5 that could help drive Abd-B expression in the cuticle and generate morphological features that are characteristic of A6.
These results suggest that enhancers in iab-5 are important for the proper differentiation of the adult cuticle in A6.

Deletion of the iab-5 initiator disrupts morphology of the A5 and A6 segments
According to the previous results deletion of the iab-5 initiator will disrupt the development of the adult cuticle not only in A5 but also in A6. We used CRISPR/Cas9 to delete a 1975 bp genomic DNA segment that spans the iab-5 initiator and replace it with an attP site and an excisable dsRed reporter under control of the 3×P3 hsp70 promoter (Fig. S1). As expected for an initiator deletion, the A5 segment in i5 attP males resembles A4 (Fig. 3, Fig. S4 and S5). Critically, this is not the only phenotypic alteration in i5 1attP males: the A6 sternite has an intermediate quadrilateral shape and also has bristles, while the A6 tergite has an irregular and variable pigmentation. In addition, trichome hairs are found in large patches often coinciding with areas of depigmentation. These results show that deletion of the iab-5 initiator affects Abd-B expression in both the A5 and A6 segments. To confirm that iab-5 is not properly activated in i5 1attP we integrated a mini-y reporter using the attP site. As expected, the mini-y reporter introduced into iab-5 is off in A5. In A6, black pigmentation is restricted to several patches on the tergite, while tanonly dependent pigmentation occupies a somewhat larger area (Fig. 3, Fig. S5).
To confirm that the observed effects on mini-y and A6 morphology are induced by deletion of the iab-5 initiator, we introduced a 1025 bp i5 ini fragment together with mini-y into i5 1attP . The resulting flies have wt morphology except for 1-2 bristles on the A6 sternite, and the mini-y reporter is expressed throughout the tergite in A5 and also A6 (Fig. 3). The presence of bristles on the A6 sternite is likely due to competition between the mini-y and Abd-B promoters.

Deletions of the iab-5 regulatory domain: impact on segment specification
To further assess the functional role of the i5 enhancers in both A5 and A6, we deleted most of iab-5 sequence and then reintroduced i5 enhancers in various combinations. For this purpose, we used Cre-mediated recombination between lox sites located in the i5 attP and an Mcp boundary deletion, M 3attP , in which a 3,333 bp sequence spanning the region around the Mcp boundary was substituted by attP and lox sites (Fig. 3, Fig. S6 and S7). After Cre recombination, the final deletion, M-i5 attP , is 10935 bp. It extends from the centromere proximal side of the Mcp boundary through the iab-5 initiator, leaving the 2126 bp i5 7 sequence (Fig.2) and single attP and lox sites. M-i5 attP males have a pigmented A4 segment and display other signs of GOF transformation of not only A4 and A5, but also A3: the sternites have two lobes somewhat like the A6 sternite, while there is a depletion of the trichomes on the tergites (Fig. 3, Fig.   S4).
Aiming to prevent the iab-4 domain from activating Abd-B, we re-introduced a minimal M 413 boundary, characterized previously (Kyrchanova et al., 2007), with the mini-y reporter using the phiC31 integration system (Fig. S1). The resulting M 413 mini-y replacement contains only the i5 7 sequence. As would be expected since there is no initiator in iab-5, the domain is inactive in A5 and mini-y is not expressed in this segment. However, in spite of the fact that the iab-5 domain is inactive, the phenotype of A6 resembles wt and the mini-y reporter, which is located in the inactive iab-5 domain, is expressed throughout the A6 tergite (Fig. 4). When the reporters are excised, the minimal Mcp 413 boundary is not able to prevent iab-4 from activating the enhancer in i5 7 , or Abd-B directly. In addition to having a wt A6 segment, the tergites in A4 and A5 are nearly covered in pigmentation indicating that the Abd-B gene active in both of these segments (Fig. S8).
To test the role of the iab-4 regulatory region in the GOF transformation of A4 in Mcp 413 , we deleted a 4,401 bp sequence (iab-4  ) including the iab-4 initiator as described previously (Postika et al., 2018). The deletion of these iab-4 sequences not only reverts the GOF transformations in M 413 , but also results in a dramatic LOF transformation of both A5 and A6 (Fig. 3). While A5 resembles A4 in M 413 iab-4  males, the pigmentation patterns in the A6 tergite range from a few dark spots to almost ubiquitous pigmentation (Fig. S9). The A6 sternite is also misshapen and covered in bristles. Since A6 appears wt when the iab-4 domain is intact, it would appear that in the M 413 platform sequences in iab-4 are able to collaborate with elements in iab-6 to activate enhancers in i5 7 , and direct the proper expression of Abd-B in A6.

Reconstructing a minimal iab-5 domain
We next used the M-i5 attP platform to reconstruct a minimal iab-5 regulatory domain. Since Mcp 413 in combination with the two reporters is more effective in insulating against elements in iab-4, we will first consider the functioning of different iab-5 sequences in the presence of the reporters. In the first set of experiments, we tested i5 S2 and i5 3 . Since the M-i5 attP deletion retains the i5 7 , it is included in all of the replacements we tested. Thus, the three combinations are i5 S2 +i5 7 ; i5 3 +i5 7 and i5 S2 +i5 3 +i5 7 (Fig. 4). In both i5 7 and i5 S2 +i5 7 the mini-y reporter is expressed in a mosaic pattern along the posterior margin of A4 and A5. In contrast, we observed only rare spots of dark pigmentation in the A5 segment in combinations containing i5 3 . Thus, the i5 3 region has a negative effect on mini-y expression in cis. In all three combinations, the anterior 2/3rds of A5 tergite is largely devoid of pigmentation, indicating that the tan gene is also not expressed in much of the tergite. At the same time, the A6 segment has a nearly wt phenotype.
We next tested the same combinations of i5 enhancers with the initiator, i5 ini . The i5 ini +i5 7 combination expands the expression domain of mini-y in A5, while having minimal effect on expression in A4. However, there are regions in the anterior of the A5 tergite where mini-y is not expressed (Fig. 4).
While adding i5 S2 has little effect on the pattern of mini-y expression (i5 S2 +i5 ini +i5 7 ), there is a noticeable expansion in the expression area in A5 when i5 3 is combined with the initiator (i5 3 +i5 ini +i5 7 ) (Fig. 4). This is the opposite of what was observed for i5 S2 +i5 S7 and i5 3 +i5 S7 combinations without the initiator sequence.
However, even in this case, mini-y expression is not observed throughout the A5 tergite. On the other hand, when the initiator is combined with all three sequences (i5 S2 +i5 3 + i5 ini +i5 7 ), mini-y is expressed throughout the entire A5 tergite as is tan, while the ectopic activation in A4 is absent. (Fig. 4). Thus, this combination appears to be sufficient for full domain function.
We also examined the activity of the iab-5 enhancers after reporter excision (Fig. S8). The GOF transformations (misshapen sternites and loss of trichome hairs) in the morphology of segments A3-6 in the starting M-i5 attP platform are largely rescued by the introduction of the Mcp boundary, M 413 . However, as mentioned above, the pigmentation patterns in A4 and A5 are still abnormal. The former has patches of ectopic pigmentation, while the latter is not fully pigmented. The pigmentation patches in anterior of A4 and A5 mostly disappear in the i5 S2 +i5 7 combination. When i5 S2 +i5 7 are combined with i5 3 there is a further suppression in A5 pigmentation, and a loss of pigmentation in A4. Thus, the i5 3 and i5 7 sequences, in cooperation i5 S2 , can block the activation of Abd-B expression mediated by sequences in the iab-4 domain. However, as was observed when mini-y is present, the iab-5 enhancers in i5 S2 , i5 3 and i5 7 are unable to direct the proper development of A5 unless the iab-5 initiator is also present. Addition of i5 ini to i5 7 , or i5 S2 +i5 7 , or i5 3 +i5 7 substantially expands the area of pigmentation not only in the A5 tergite, but also in A4 (Fig. S6). As was observed for the mini-y reporter, combining i5 ini with i5 S2 +i5 S3 +i5 S7 gives what appears to be a fully wt pattern of pigmentation in both A4 and A5. Thus, the i5 S2 +i5 S3 +i5 S7 combination blocks incorrect activity of the iab-4 and iab-5 initiators in the A4 segment and is sufficient in cooperation with i5 ini for the proper stimulation of Abd-B in the A5 segment.

Discussion
In Drosophila melanogaster, segments A5 and A6 in males are completely pigmented, while the cuticle morphology of A6 is distinctive from that in more anterior segments. Unexpectedly, we find that the regulatory elements in iab-6 are not in themselves sufficient to direct the proper differentiation of the cuticle in males. Instead, the iab-5 domain contains several enhancers that are required for male pigmentation not only in A5 but also A6. These iab-5 enhancers also play a role in generating the proper distribution of trichome hairs in the A6 tergite, and in ensuring that the sternite is devoid of bristles.
According to the generally accepted model (Maeda and Karch, 2015), the most likely initiator function is turning the iab-5 domain on so that the cuticle enhancers are active. Deletion of the iab-5 initiator results in a transformation of A5 into A4. We find that the iab-5 initiator is also required for the proper differentiation of A6.
Functional dissection of iab-5 suggests that at least four distinct DNA sequences (i5 S2 , i5 3 , i5 7 and the iab-5 initiator i5 ini ) are required for proper differentiation of A5. With respect to the cells in the cuticle in which these enhancers are active, it would appear that they have overlapping rather than completely distinct activities. Several lines of evidences (orientation or position dependent activation of the yellow reporter) suggest that the i5 S2 , i5 3 and i5 7 sequences contain not only tissue-specific enhancers but also silencers. The i5 sequences also seem to help the Mcp 413 boundary block interactions between the iab-4 and iab-5 initiators/regulatory elements.
While our results demonstrate that the proper differentiation of A6 depends upon enhancers in iab-5 to drive Abd-B expression in the appropriate manner, previous studies showed that in addition to the initiator the iab-6 domain has at least two regions that are required for the proper differentiation the A6 segment in the males (Iampietro et al., 2010). The deletion of the iab-6 initiator results in a clean LOF transformation of A6 into A5. Thus, the iab-5 enhancers that appear to complement the cuticle enhancers in iab-6 are not able to function in A6 unless the enhancers in iab-6 are also active and can communicate with iab-5. Further studies will be needed to refine and extend these findings.

Generation of transgenic lines carrying different insertions in the attP-platforms.
The replacement vector was a plasmid with the mini-yellow and mCherry reporters as shown in Fig. S1.
Integration of the plasmids in the landing platforms was achieved by injecting the plasmid and a vector expressing the фC31 recombinase into embryos of yw; i5 1attP /i5 1attP , or yw; M 3attP /M 3attP , or yw; M-i5 attP /M-i5 attP lines. The successful integrations were selected on the basis of expression of mini-y in abdominal segments. The integration of the replacement DNA fragments was confirmed by PCR.
The yellow and mCherry reporters were excised by Cre-mediated recombination between the lox sites.
All stocks are available upon request.

Cuticle preparations
Cuticle preparations were carried out as described in (Postika et al., 2018). The localization of trichomes on the A5 and A6 tergites are shown in dark field.  Designations are the same as in Fig. 1. reporters are present. Designations are the same as described in Fig. 1.