Distinct genetic requirements for BX-C-mediated specification of abdominal denticles

Authors


Abstract

Background: Hox genes encode transcription factors playing important role in segment specific morphogenesis along the anterior posterior axis. Most work in the Hox field aimed at understanding the basis for specialised Hox functions, while little attention was given to Hox common function. In Drosophila, genes of the Bithorax complex [Ultrabithorax (Ubx), abdominalA (abdA), and AbdominalB (AbdB)] all promote abdominal identity. While Ubx and AbdA share extensive sequence conservation, AbdB is highly divergent, questioning how it can perform similar functions as Ubx and AbdA. Results: In this study, we investigate the genetic requirement for the specification of abdominal-type denticles by Ubx, AbdA, and AbdB. The impact of ectopic expression of Hox proteins in embryos mutant for Exd as well as of Wingless or Hedgehog signaling involved in intrasegmental patterning was analyzed. Results indicated that Ubx and AbdA do not require Exd, Wg, and Hh activity for specifying abdominal-type denticles, while AbdB does. Conclusions: Our results support that distinct regulatory mechanisms underlie Ubx/AbdA- and AbdB-mediated specification of abdominal-type denticles, highlighting distinct strategies for achieving a similar biological output. This suggests that common function performed by distinct paralogue Hox proteins may also rely on newly acquired property, instead of conserved/ancestral properties. Developmental Dynamics 243:192–200, 2014. © 2013 Wiley Periodicals, Inc.

INTRODUCTION

Although animal species are distinct in their gross morphologies, they use homologous genes to establish their body plans during development. Hox genes form an important part of this common gene toolkit, and through restricted expression, provide positional information that in turn serves selecting distinct developmental programs responsible for diversifying animal body plans (Costa et al., 1988; Lewis, 1978; Sánchez-Herrero et al., 1985; Krumlauf, 1994 ; Gehring et al., 2009; Duboule, 2007). Hox genes encode homeodomain (HD) containing transcription factors, which control developmental fates by regulating the transcription of downstream target genes (McGinnis and Krumlauf, 1992; Graba et al., 1997).

Specialised Hox protein function requires the help of cofactors, of which the best-characterised examples are HD-containing proteins belonging to TALE family, including PBC (PBX in vertebrates and exd in Drosophila) and Meis proteins (Homothorax (Hth) in Drosophila (Mann and Chan, 1996; Moens and Selleri, 2006). These cofactors physically interact with Hox proteins, and have been shown to improve the DNA binding affinity and specificity of Hox proteins. In addition to performing distinct functions, Hox proteins also execute common functions. Specialised and common functions likely reflect the phylogeny of Hox proteins that derived through duplications from a unique or unique set of ancestral genes, leading up to 13 paraloguous groups in vertebrates (Sharkey et al., 1997). Following duplication, conservation of protein sequences likely allows for common function, while sequence divergence likely creates the frame for the acquisition of novel and distinct functions. Common functions are well illustrated by the finding that most Drosophila Hox proteins can specify the tritocerebral commissure in embryonic brain (Hirth et al., 2001), and that central and posterior Hox genes can repress the head-specific gene optix (Coiffier et al., 2008).

The phylogeny of Hox paralogue groups suggests that common function likely results from retained ancestral function. If this is the case, then similar functions performed by distinct Hox proteins should rely on the use of similar regulatory mechanisms. Hox proteins from the Bithorax complex (BX-C), Ultrabithorax (Ubx), AbdominalA (AbdA), and AbdominalB (AbdB) provide a suitable paradigm to investigate how common function is achieved by distinct paralogue proteins. Ubx and AbdA are central class Hox proteins. The HDs of Ubx and AbdA show significant conservation (92%, Fig. 1A) and in addition to this Ubx and AbdA also share hexapedtide (HX) and UbdA motifs, lying upstream and downstream of the HD, respectively, which contribute to recruit the Hox cofactor Exd (Merabet et al., 2007; Saadaoui et al., 2011). Biochemical and structural analysis showed that the HD of PBX/Exd makes contact with its Hox partner through the TALE insertion, which associates with the Hox HX motif (Lu et al., 1995 ; Passner et al., 1999). HX motifs are found in most Hox proteins and are defined by a central W residue that provides a key contribution to the contact made with Exd, as well as a canonical environment that presents the W in a favorable environment for the interaction. How the paralogue-specific motif UbdA, only found in Ubx and AbdA, provides contacts towards Exd remains to be defined. AbdB is a posterior class Hox protein, displaying a high degree of divergence when compared to Ubx and AbdA. The AbdB Hox protein differs significantly in the HD (only 59% of identity) and also lacks a canonical HX and UbdA motifs (Fig. 1A).

Figure 1.

Sequence conservation and specification of abdominal-type denticles by BX-C Hox proteins. A: Sequence alignments of HD and flanking regions from Ubx, AbdA, and AbdB proteins are shown. The HD (marked by a thick brown horizontal line) is highly conserved in Ubx and AbdA, while AbdB shares far less amino acids conservation. Non-conserved amino acids are colored. The two Exd recruiting motifs in Ubx and AbdA, the HX (red letters) and UbdA (green letters) are highlighted by orange boxes. The AbdB Hox protein lacks these motifs but still possess a conserved tryptophan “W” upstream of the HD (marked by a star). B: Morphology of a third instar larvae and functional domain of BX-C protein activity. The arrow points to the boundary between the thorax (T) and abdomen (A). C: Magnification of A1 and T3 segments. Distinction between abdominal-type and thoracic-type denticles is illustrated by the comparison of denticles found in the abdominal A1 (large thick and refringent) and thoracic T3 (small and poorly refringent) segments.

While most work in the Hox field aimed at understanding the basis for specialised Hox functions, little attention was given to Hox common functions. We recently started addressing the issue of how common Hox function is achieved using the repression of the limb-promoting gene Distalless (Dll) by the abdominal Hox proteins of the BX-C (Sambrani et al., 2013). Absence of leg in the Drosophila abdomen results from the repression of Dll by Ubx, AbdA, and AbdB in their respective abdominal expression domains. In repressing Dll, some aspects of AbdB activity are similar to Ubx and AbdA: AbdB uses the same cis regulatory sequences and the same compartment-specific corepressors, Engrailed and Sloppy paired. However, other aspects of AbdB activity in repressing Dll are distinct from Ubx and AbdA. AbdB likely binds Dll cis regulatory sequences as a monomer without the aid of the Exd and Hth Hox cofactors. These results demonstrate that, at least in the case of Dll repression, AbdB and Ubx/AbdA use different molecular mechanisms to achieve a similar function. This suggests that common function performed by distinct paralogue Hox proteins may also rely on newly acquired property.

In this study, we aimed at further investigating the basis for common functions performed by Ubx/AbdA and AbdB. Epidermal cells secrete cuticles that harbor specialised structures termed denticles (Lohs-Schardin et al., 1979). The identity and spatial organisation of these denticles reflects distinct segmental identities: Ubx specifies a pattern unique to segment A1, AbdA a pattern shared by segments A2–A5, and AbdB patterns specific to A5–A9 (Fig. 1B). Although BX-C-controlled cuticular patterns are distinct, they all use abdominal-type denticles, large and highly refringent, which are easily distinguishable from thoracic-type denticles that are smaller and less refringent, and only specified in the absence of BX-C proteins (Fig. 1C). Here we used the specification of abdominal-type denticles as a BX-C-mediated common trait to investigate the genetic requirements for BX-C common protein activity.

RESULTS

Depletion of Maternal and Zygotic exd Function Suggests a Distinct Requirement for Ubx/AbdA- and AbdB-Mediated Specification of Abdominal-Type Denticles

Given its generic role in specifying Hox protein function, we examined the requirement of Exd activity for proper specification of abdominal-type denticles. exd is a gene expressed maternally and zygotically. Depleting its maternal contribution while leaving its zygotic contribution intact (mz+), or depleting its zygotic contribution while leaving its maternal contribution intact (m+z), does not impact on the specification of abdominal-type denticles by BX-C proteins (see Supp. Fig. S1, which is available online; Peifer and Wieschaus, 1990). When depleting both maternal and zygotic contribution (mz) by generating germ line clones (Chou and Perrimon, 1992), the ventral cuticle exhibits strong patterning defects, including fusion of abdominal segments with loss of naked cuticle and reduced or absence of denticles in the thorax (Peifer and Wieschaus, 1990; Fig. 2A). Segment fusions were previously shown to result from inappropriate regulation of genes required for proper segmentation (Peifer and Wieschaus, 1990; Kobayashi et al., 2003). The anterior-most abdominal segments can unambiguously be identified and display abdominal-type denticles, suggesting that Exd is not required for Ubx and AbdA function. By contrast, in most of the exd m−z− embryos, it is not possible to unambiguously identify the posterior-most segments, including A8, which questions the requirement of Exd for AbdB-mediated specification of abdominal-type denticles. Given this difficulty, we used a gain-of-function approach to examine the capacity of BX-C proteins to impose abdominal-type denticles in the thorax.

Figure 2.

Ubx and AbdA gain-of-function in zygotic and maternal exd mutant. The complete and enlarged anterior region of embryos is displayed for each genotype. Arrow, boundary between thorax and abdomen; A, abdomen; T, thorax. The display remains the same in all subsequent figures where cuticle preparations are shown, and transformation of thoracic to abdominal-type denticles is highlighted by the curved arrow on the right of each panel when appropriate. A: Cuticle of an exd m−z− embryo. B: Effect of arm-Gal4-driven Ubx ubiquitous expression. Segments anterior to A1 are transformed towards an abdominal A1 identity. C: Effects of arm-Gal4-driven Ubx in exd m−z− homozygote embryos. Embryos show abdominal-type denticles in the thoracic region. D: Effect of arm-Gal4-driven AbdA ubiquitous expression. Segments anterior to A2 are transformed towards an abdominal A2 identity. E: Effects of arm-Gal4-driven AbdA in exd m−z− homozygote embryos. Embryos show abdominal-type denticles in the thoracic region.

Ubx and AbdA Do Not Require Exd for the Specification of Abdominal-Type Denticles

We first examined the requirement of Exd for Ubx and AbdA control of abdominal-type denticles by ubiquitously expressing Ubx or AbdA using the armadillo-Gal4 driver (arm-Gal4) and examining the effect of ubiquitous expression in the thoracic region. Previous work showed that thoracic expression of Ubx transforms thoracic segments to an A1 fate (González-Reyes and Morata, 1990; Mann and Hogness, 1990; Fig. 2B), a fate normally promoted by Ubx, and recognisable by a linear denticle belt. In the absence of zygotic Exd, Ubx promotes instead an A2 identity, characterised by a trapezoidal denticle belt (Peifer and Wieschaus, 1990). Thus in both contexts (WT or exdm+z−), Ubx promotes abdominal-type denticles. However, the effect in gain of function experiments in the complete absence of Exd was not examined. In exdm−z− embryos, Ubx was still able to induce abdominal-type denticles in the thoracic region (Fig. 2C). The exact identity of abdominal belts was difficult to identify as segmentation and intrasegmental patterning was severely affected.

Ubiquitous expression of AbdA results in transformation of thoracic segments into A2-like segments (Sanchez Herrero et al., 1994, Fig. 2D). As for Ubx, the exact identity that thoracic segments take following ubiquitous AbdA in exdm−z− embryos is not easy to determine. However, whatever their exact identity is, these denticles are clearly of the abdominal type, being large and refringent (Fig. 2E). Thus, Ubx and AbdA do not require Exd for specification of abdominal-type denticles.

AbdB Requires Exd for the Specification of Abdominal-Type Denticles

The AbdB gene encodes two isoforms, AbdBm, also called morphogenetic isoform, and AbdBr referred to as the regulatory isoform. The two isoforms share most of the protein-coding sequence including the HD, and differ in the extent of their N-terminal sequences: AbdBm is 223 amino acids longer than the AbdBr isoform. AbdB isoforms also differ in their expression patterns, with AbdBm expressed in segments A5–A8 and AbdBr in A9 (Celniker et al., 1989). Ubiquitous expression of AbdBm promotes ectopic posterior spiracle development (Castelli-Gair et al., 1994), a structure normally exclusively found in A8. Remarkably, when continuously expressed using the Gal4/UAS system, it always promotes, irrespective of the temperature (18°C, 22°C, or 29°C), suppression of ventral denticles (Fig. 3A). This effect is distinct from heat-shock-driven transient expression previously reported (Kuziora, 1993; Lamka et al., 1992).

Figure 3.

AbdBm and AbdBr gain of function in zygotic and maternal exd mutant. A: Effects of arm-Gal4-driven AbdBm. Embryos show lack of ventral denticles and ectopic accumulation of posterior spiracles (asterisks). B: Effects of arm-Gal4-driven UAS-AbdBm in exd m−z− homozygote. The absence of Exd partially suppresses the deleterious effect of AbdBm on ventral denticles. The thoracic domain does not contain abdominal-type denticles. C: Effects of arm-Gal4-driven AbdBr. Embryos show abdominal-type denticles in the thoracic region. D: Effects of arm-Gal4-driven AbdBr in exd m−z− homozygote embryos. In the absence of Exd, AbdBr fails to induce abdominal-type denticles in the thoracic region.

In contrast, ubiquitous expression of AbdBr does not suppress ventral denticles and at 29°C promotes A8 identity, which is recognisable by a trapezoidal arrangement of denticles in all segments anterior to A9, including thoracic segments (Fig. 3C). This likely indicates that the A8 morphogenetic function of AbdB relies on sequences shared by the two protein isoforms, and that this morphogenetic function is suppressed in A9 by a local not yet identified determinant. Thus, anteriorly expressed AbdBr promotes the same developmental program as AbdBm without displaying the deleterious effect on the ventral cuticle. Ubiquitous expression of AbdBr in exdm−z− embryos shows that AbdBr does not promote abdominal denticle specification in the thorax (Fig. 3D). We next investigated the impact of AbdBm expression in the absence of Exd. Results show that the deleterious effect of AbdBm on ventral denticles is partially released in the absence of Exd, and in these conditions, abdominal-type denticles do not form in the thorax (Fig. 3B). We concluded that AbdB, in contrast to Ubx and AbdA, shows a strict requirement for Exd for imposing abdominal denticle fate.

Wg and Hh Signaling Are Dispensable for Ubx and AbdA-Mediated Specification of Abdominal-Type Denticles

Each epidermal cell needs to sense where broadly it is in the embryo, i.e., to which segment it belongs, and also more precisely where within the segment it resides. The first relies on axial positional information delivered by Hox proteins, while the latter uses intrasegmental positional cues delivered by segment polarity genes, including wingless (wg) and hedgehog (hh) that encode, respectively, Wnt and Hh class signaling molecules (Alexandre et al., 1999). Wg and Hh signaling provide key regulatory input to discriminate between the naked and denticle harboring fate, and also acts to provide a subtle distinction between denticles' identity and organisation. Consequently, signaling pathways and Hox genes function synergistically to specify epidermal fate. In order to further assess if BX-C encoded Hox proteins have similar genetic requirements for specifying abdominal-type denticles, we investigated if Ubx, AbdA, and AbdB similarly or distinctly require Wg and Hh signaling using the gain-of-function strategy defined for Exd requirement.

Loss of Wg signaling in embryos results in the loss of naked cuticle (Fig. 4A). Effects of ubiquitous expression of Ubx or AbdA in a context homozygote for wgCX4, a wg null allele, were analysed. Results indicate that both Ubx and AbdA retain the capacity to promote abdominal-type denticles in the thorax (Fig. 4B, C), indicating that Wg signaling is dispensable for both Ubx- and AbdA-mediated specification of abdominal-type denticles.

Figure 4.

Ubx, AbdA, and AbdBr gain-of-function in wg mutant. A: wgcx4 homozygote mutant embryos display fused belts of abdominal denticles with no naked cuticle and a thoracic domain predominantly devoid of any denticles. B: Effects of 69B-Gal4-driven Ubx in wgcx4 homozygote embryos. Embryos show formation of abdominal-type denticles in the thoracic region. C: Effects of 69B-Gal4-driven AbdA in wgcx4 homozygote embryos. Embryos show formation of abdominal-type denticles in the thoracic region. D: Effects of 69B-Gal4-driven AbdBr in wgcx4 homozygote embryos. Embryos do not show formation of abdominal-type denticles in the thoracic region.

Loss of Hh signaling in embryos results in a phenotype very similar to loss of Wg signaling, with an absence of naked cuticle in between denticle belts (Fig. 5A). Impairing Hh signaling using a hh21 mutant while expressing Ubx or AbdA ubiquitously does not prevent their capacity to specify abdominal-type denticles in the thorax (Fig. 5B, C). We thus concluded that as for Wg signaling, Hh signaling is also dispensable for both Ubx- and AbdA-mediated specification of abdominal-type denticles.

Figure 5.

Ubx, AbdA, and AbdBr gain of function in hh mutant. A: hh21 homozygous embryos display fused belts of abdominal denticles and a thoracic domain predominantly devoid of any denticles. B: Effects of arm-Gal4-driven Ubx in hh21 homozygote embryos. Embryos show formation of abdominal-type denticles in the thoracic region. C: Effects of arm-Gal4-driven AbdA in hh21 homozygote embryos. Embryos show formation of abdominal-type denticles in the thoracic region. D: Effects of 69B-Gal4-driven AbdBr in hh21 homozygote embryos. Embryos do not show formation of abdominal-type denticles in the thoracic region.

AbdB Requires Wg and Hh Signaling for Specification of Abdominal-Type Denticles

To investigate signaling requirement for AbdB function, we used the AbdBr isoform that lacks the ventral denticle suppressive capacity and permits assessing the denticle-inducing capacity of AbdB. Wg and Hh signaling was impaired either using mutants for wg or hh, or by ubiquitous expression of a dominant-negative form of dTCF, a nuclear effector of the Wg signaling pathway (dTCFDN; van de Wetering et al., 1997), or a dominant-negative form of Cubitus interuptus (Ci), a nuclear effector of the Hh signaling pathway (CiDN; Glazer and Shilo, 2001). The effect of AbdBr ubiquitous expression was scored in the thorax. Results showed that impairing either Wg or Hh signaling prevents AbdBr from specifying abdominal-type denticles (Figs. 4D and 5D, respectively, and Supp. Fig. S2 for DN experiments). We thus concluded that in contrast to what was observed for Ubx and AbdA, Wg and Hh signaling are essential for AbdB-mediated specification of abdominal-type denticles.

DISCUSSION

The specification of abdominal-type denticles by BX-C proteins was investigated in mutants for the Hox cofactor Exd, as well as in mutants for signaling molecules known to provide intrasegmental positional information. To understand how the highly divergent central Ubx/AbdA and posterior AbdB Hox proteins perform an identical function, we focused on the distinction between abdominal and thoracic type denticles, a trait controlled by all three BX-C proteins. We did not include in our analysis distinctions between abdominal denticle types, neither differences in number and spatial organization, which is difficult to assess in the exd, wg, and hh mutant embryos, and which constitute traits that contribute to distinguish abdominal denticle belts according to the axial position of the segment.

Results obtained highlight that Ubx/AbdA and AbdB display distinct genetic requirements for specifying abdominal-type denticles: Ubx and AbdA perform this function without the aid of Exd, Wg, and Hh signaling, while AbdB requires all three. These distinct genetic requirements for specification of abdominal-type denticles suggest that the molecular mechanisms used by the central Ubx/AbdA and the posterior AbdB Hox proteins are distinct.

Our approach relies on a gain-of-function strategy, scoring phenotypes in the thorax, where none of these three BX-C proteins are expressed, but where Exd, Wg, and Hh are expressed. This allows circumventing the difficulty resulting from the incapacity to unambiguously identify the posterior-most abdominal segments, where AbdB acts, and to avoid complications in interpreting results that would arise from cross-regulation between BX-C genes in the abdomen. However, the approach also questions if our conclusion applies for BX-C proteins activity in their endogenous expression domains. Regarding Exd requirements, maternal and zygotic loss results in abdominal segment fusion, where segments are fused by pairs: A1/A2–3/A4–5/A6–7/A8. Although denticle belts are highly disorganized, denticles are clearly of the abdominal type in anterior abdominal segments. When present in A8, the belt of denticles is very much reduced, and in most embryos is absent. This indicates that Ubx and AbdA do not require Exd for the specification of abdominal-type denticles in their endogenous expression domain, while AbdB does. The complete segment fusion resulting from loss of Wg and Hh signaling make it impossible to unambiguously identify the A8 segments, and therefore does not allow addressing if AbdB also requires Wg and Hh signaling in its endogenous expression domain. Denticles are found in continuous lawn that encompasses most abdominal segments. Therefore, denticles in the Ubx and AbdA expression domains are clearly of the abdominal type, indicating that Ubx and AbdA do not require Wg and Hh signaling for the specification of abdominal-type denticles. Thus, whenever possible, resident BX-C Hox protein activity in loss of exd, wg, and hh function is consistent with the conclusion raised in the gain-of-function approach, further supporting that Ubx/AbdA and AbdB use distinct regulatory mechanisms for achieving a common function (Fig. 6).

Figure 6.

Summary of abdominal-type denticles specification by Ubx, AbdA, and AbdB. Ubx/AbdA and AbdB have distinct genetic requirements for specifying abdominal-type denticles: Ubx/AbdA do not require exd, wg, and hh, while AbdB does.

Such a conclusion was recently reached by studying the molecular mechanisms underlying repression of the limb-promoting gene Dll by Ubx and AbdB. It was shown that the cofactor requirement and intrinsic protein domain requirement for Ubx versus AbdB repression of Dll was distinct. Ubx represses Dll by binding DNA cooperatively with the Exd and Hth cofactors, which relies on the UbdA domain, a domain specific to Ubx and AbdA and located C-terminal to the HD (Sambrani et al., 2013). Surprisingly, Ubx DNA binding is dispensable, probably due to cooperative binding to DNA with Exd and Hth, DNA-binding proteins that likely compensate for Ubx loss of DNA binding. By contrast, AbdB represses Dll without the help of the Exd and Hth, and DNA binding of AbdB is strictly required for repression. It was further established that in specifying posterior spiracles and regulating empty spiracles expression, Exd/Hth antagonize AbdB activity, showing that the AbdB/Exd partnership depends on the biological context (Rivas et al., 2013). Mechanisms at the origin of cooperativity/antagonism are still to be discovered.

The present study corroborates the conclusion reached by the analysis of Dll repression by Ubx and AbdB and extends it in several ways: first by using a distinct Hox biological activity as functional readout; second by including in the analysis the AbdA Hox protein; and third by examining additional genetic requirements (Wg and Hh signaling). The work, therefore, provides further support for the view that distinct molecular strategies underlie an apparent unicity in BX-C protein controlled biological function.

Given the observation that Ubx and AbdA are very similar, sharing a highly conserved HD as well as additional protein domains such as the HX and UbdA motifs, while AbdB lacks these domains and has a highly divergent HD, it is not surprising that the genetic requirements are similar for Ubx/AbdA and distinct for AbdB. More unexpected was the finding that Ubx and AbdA do not require Exd for specifying abdominal-type denticles, while AbdB does. This indeed contrasts with the known and previously described Exd requirement for Ubx in A1 segment identity specification and Dll repression, and also contrasts with the dispensability of Exd for A8 segment identity specification, posterior spiracle specification (Peifer and Wieschaus, 1990), and Dll repression (Sambrani et al., 2013). This highlights that requirement of Exd for Hox activity depends on the specific function examined, rather than being a general and universal requirement.

A salient difference between the central Ubx/AbdA and posterior AbdB Hox proteins is the mode of Hox DNA binding. Posterior paralogue Hox proteins have usually a stronger affinity for DNA when binding as monomer than central class Hox proteins. This difference mainly stems from the ability of posterior but not central class Hox proteins to make extensive contacts with the DNA backbone (LaRonde-LeBlanc and Wolberger, 2003). These differences provide a frame to understand the requirement of Exd/Pbx cofactor for central class Hox proteins, which upon interaction with Hox proteins raises their DNA-binding affinity. In the case of specification of abdominal-type denticles, the contribution of Exd is likely different, as required for AbdB and not Ubx/AbdA activity. This suggests that Exd may be involved in regulating the activity, rather than DNA binding, a function previously suggested in the regulation of Deformed Hox protein function (Li et al., 1999).

In summary, this work together with the study of Dll repression by BX-C proteins, highlights that distinct regulatory mechanisms and molecular strategies underlie common Hox protein functions. Thus, while sequence divergence following gene duplication promotes functional divergence, it also generates novel gene regulatory mechanisms and molecular strategies that promote a common biological output.

EXPERIMENTAL PROCEDURES

Fly Stocks

UAS-Ubx, UAS-AbdA, UAS-AbdB m and r (received from J. Castelli-Gair-Hombria), arm-Gal4 (Sanson et al., 1996), 69B-gal4 (Brand and Perimonn, 1993), exdxp11-FRT18D (Wieschaus et al; 1984), OVOD2 FRT 18D, (Bloomington stock No: 2980), hh21 (Bloomington stock no: 5338), wgCx4 (Bloomington stock No: 2980), UAS-dTCFDN (van de Wetering et al., 1997), UAS-CiDN (Glazer and Shilo, 2001).

Cuticle Preparation for Embryos/First Instar Larvae and Phenotypic Analyses

Embryos from overnight collection were dechorionated and washed with tap water. The desired genotypes (phenotypically recognisable) were later transferred to a clean slide with sufficient amounts of 1:1 lactic acid: Hoyer's-based medium and a cover slip was placed over the embryos or first instar larvae. Phase contrast and bright field images were obtained on a Zeiss axioskop using Zeiss digital camera and images were further processed using Adobe PhotoShop software.wgCX4 and hh21 and exdm−z− embryos display gross morphological alteration. They, however, all display a thoracic region that can be recognised by being predominantly naked. This allows following Hox ubiquitous expression to unambiguously identify transformation in the thoracic domain, by an extension of the abdominal domain at the expense of the thoracic domain. The thoracic to abdominal kind of denticle transformations were studied using different UAS and Gal4 lines (see fly stocks above). In the experimental setting used in the study (22°C for AbdBm and 29°C for AbdBr), the phenotypes were fully penetrant.

Preparation of Hoyer's Medium

Thirty grams of gum arabic (Sigma-Aldrich, UK) was added to 50 ml of distilled water. It was stirred overnight until completely dissolved, and then gradually 200 g of chloral hydrate (anhydrous) (Sigma-Aldrich) was mixed. To that 20 g of glycerol was added. The mixture was centrifuged for 2 hr at 25,000g. The mixture of Hoyer's-based medium was diluted with lactic acid in 1:1 ratio for cuticle preparations.

Generation of Germ Line Clones for exd

To generate exd maternal mutant embryos carrying exdXP11 FRT 18D/FM7-B1, females were crossed with OVOD2 FRT 18D; hs-Flp1 males to generate exdXP11 FRT 18D/OVOD2 FRT 18D; hs-Flp1/+ females (non-B), which were heat shocked during the wandering larval stage (1 hr at 37°C every 24 hr for 2days) to induce mitotic recombination. These females with homozygous mutant germ-line clones were then crossed with wildtype (control) males. To generate exd maternal mutant embryos carrying UAS-Ubx or UAS-AbdA or UAS-AbdB, exdXP11 FRT 18D/FM7-B1; UAS-Hox stocks were generated. Then, crossing the females from the exdXp11; UAS-Hox stocks to males of genotype OVOD2 FRT 18D; hs-flp to generate exdXP11 FRT 18D/OVOD2 FRT 18D; hs-Flp1/+; UAS-Hox females (non-FM7), which were heat shocked as above to induce mitotic recombination. These females were then crossed with arm-Gal4 males.

ACKNOWLEDGMENTS

We thank R. Mann, E. Sanchez Herrero, J. Castelli-Gair Hombria, S. Kerridge, and the Bloomington Stock Center (Bloomington, IN) for providing fly stocks. We also thank Samir Merabet for an initial contribution to the project and all lab members for discussions.

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