Tc-knirps plays different roles in the specification of antennal and mandibular parasegment boundaries and is regulated by a pair-rule gene in the beetle Tribolium castaneum
- Andrew D Peel†1,
- Julia Schanda†2,
- Daniela Grossmann†2,
- Frank Ruge2,
- Georg Oberhofer2,
- Anna F Gilles2,
- Johannes B Schinko2,
- Martin Klingler3 and
- Gregor Bucher2Email author
© Peel et al.; licensee BioMed Central Ltd. 2013
Received: 1 April 2013
Accepted: 12 June 2013
Published: 18 June 2013
The Drosophila larval head is evolutionarily derived at the genetic and morphological level. In the beetle Tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. Unlike in Drosophila, a knirps homologue (Tc-kni) is required for development of the antennae and mandibles. However, published Tc-kni data are restricted to cuticle phenotypes and Tc-even-skipped and Tc-wingless stainings in knockdown embryos. Hence, it has remained unclear whether the entire antennal and mandibular segments depend on Tc-kni function, and whether the intervening intercalary segment is formed completely. We address these questions with a detailed examination of Tc-kni function.
By examining the expression of marker genes in RNAi embryos, we show that Tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments (i.e. the parasegmental boundaries). Moreover, we find that the role of Tc-kni is distinct in these segments: Tc-kni is required for the initiation of the antennal parasegment boundary, but only for the maintenance of the mandibular parasegmental boundary. Surprisingly, Tc-kni controls the timing of expression of the Hox gene Tc-labial in the intercalary segment, although this segment does form in the absence of Tc-kni function. Unexpectedly, we find that the pair-rule gene Tc-even-skipped helps set the posterior boundary of Tc-kni expression in the mandible. Using the mutant antennaless, a likely regulatory Null mutation at the Tc-kni locus, we provide evidence that our RNAi studies represent a Null situation.
Tc-kni is required for the initiation of the antennal and the maintenance of the mandibular parasegmental boundaries. Tc-kni is not required for specification of the anterior regions of these segments, nor the intervening intercalary segment, confirming that Tc-kni is not a canonical ‘gap-gene’. Our finding that a gap gene orthologue is regulated by a pair rule gene adds to the view that the segmentation gene hierarchies differ between Tribolium and Drosophila upstream of the pair rule gene level. In Tribolium, as in Drosophila, head and trunk segmentation gene networks cooperate to pattern the mandibular segment, albeit involving Tc-kni as novel component.
The insect head is composed of several segments and a non-segmental anterior region. However, the exact segmental composition of the insect head has long been a matter for debate [1–7]. The posterior gnathocephalon is made up of the mandibular, maxillary and labial segments that each bear a pair of appendages specialized for feeding. The anterior procephalon consists of anterior non-segmental parts and an antennal segment, which is separated from the mandibular segment by an appendage-free segment (the intercalary segment), whose development in insects is significantly delayed, as well as reduced in size.
The genetic mechanisms of head segmentation were first examined in the dipteran fruit fly Drosophila melanogaster. Its gnathal segments are patterned by the trunk segmentation gene cascade, involving maternal, gap, pair-rule and segment polarity genes [3, 8], while the patterning of the procephalic segments follows a different paradigm [3, 9–15]. Whilst segment polarity genes (i.e. en, wg, hh) are involved in establishing these segments, pair-rule genes are not [3, 10–15]. Four Drosophila head gap genes, orthodenticle (otd), empty spiracles (ems), buttonhead (btd) and sloppy paired are expressed in broad overlapping domains in the developing anterior head [9, 16]. Mutation of these genes leads to classic ‘gap phenotypes’ - the loss of all the adjacent segments covered by their domains of expression [9, 17]. However, mis-expression studies have shown that only otd affects segment polarity gene expression when expressed in ectopic domains, and only ems, with the help of btd, appears to confer identity to head segments [18–20]. Indeed, second order regulators have been identified that operate at levels in between the head gap genes and segment polarity genes: i.e. collier and cap ‘n’ collar[11, 12, 21, 22].
Drosophila exhibits an evolutionary derived mode of head development, in which the larval head is greatly reduced and undergoes ‘head involution’ during which head regions are folded into the body cavity . This situation is far from typical for insects and moreover, the reduced and experimentally inaccessible Drosophila larval head has limited the comprehensive identification and analysis of insect head development genes for technical reasons .
In recent years the red flour beetle Tribolium castaneum has emerged as a powerful genetic insect model system  and offers an opportunity to study the genetic and cellular mechanisms underlying the development of a more insect-typical head . As in Drosophila, the Tribolium gnathal segments appear to be patterned using similar mechanisms to those operating in the trunk, including a central role for pair-rule gene homologues [24–30]. In the anterior head, second order regulators and the segment polarity genes might be relatively well conserved between Drosophila and Tribolium[27, 28, 31–35]. However, clear differences have been identified at the level of the head gap genes, and the maternally provided anterior protein gradients that establish their expression domains [1, 36–40]. For example, while the Tribolium homologue of orthodenticle (Tc-otd) apparently plays a broadly conserved role as a gap gene during head segmentation in Tribolium, it appears to be much more involved in axis formation than its Drosophila orthologue [36, 41, 42]. The expression of the Tribolium homologues of empty spiracles and buttonhead (Tc-ems and Tc-btd) is limited to single segment wide domains instead of large overlapping domains in the blastodermal head anlagen. Tc-ems is required to form parts of the antennal segment only and knockdown of Tc-btd does not lead to a head cuticle phenotype at all . This raised the question of what genes might control development of these head regions in Tribolium. Work by Cerny et al. suggests that the answer to this question is, at least in part, the single Tribolium homologue of the Drosophila genes knirps and knirps-related.
The Drosophila genes knirps and knirps-related encode steroid hormone receptor-like transcription factors [44–46]. Ancestrally, the insect knirps family consisted of two genes, eagle and knirps-related, while knirps arose via a recent gene duplication of the knirps-related gene in the higher Diptera . At the blastoderm stage, knirps and knirps-related are expressed in almost identical anterior and posterior domains [45, 48–50]. Drosophila knirps acts as a canonical gap gene during trunk segmentation [51–53]. In contrast, the anterior mandibular expression domain is not required for head segmentation, since segment polarity gene (i.e. engrailed) expression in the head is not affected in embryos that lack both paralogues  while a loss of the stomatogastric nervous system is observed .
Cerny et al. have shown that Tc-knirps (Tc-kni), the single Tribolium homologue of the Drosophila knirps-family paralogues, is also expressed in anterior and posterior domains during early development . However, the Tc-kni posterior domain is shifted anteriorly relative to its position in Drosophila, and knockdown of Tc-kni does not lead to a canonical gap phenotype in the trunk, but rather minor defects in the posterior abdomen. The anterior expression domain of Tc-kni is largely conserved. In contrast to Drosophila, the anterior domain does play an essential role in head patterning: Knockdown of Tc-kni leads to loss of both antennae and mandibles .
Cerny et al. found early loss of Tc-wg expression in the antennal segment in Tc-kni RNAi while the mandibular domain of Tc-wg expression disappeared at a later stage. Further, they showed that Tc-kni is not needed for correct Tc-wg expression in the intercalary segment. Finally, light abnormalities in the maturation and maintenance of the first pair-rule stripe of Tc-eve expression were observed, where the distance between the first segmental Tc-eve stripe (in the mandibular segment) and the ocular Tc-wg expression domain was reduced in Tc-kni RNAi blastoderms . It has remained unclear, however, whether antennal and mandibular segments are deleted completely and whether the intercalary segment is affected.
In this study we examined the effect of knocking down Tc-kni on a comprehensive set of genes that mark sub regions of the antennal, intercalary and/or mandibular segments. We show that Tc-kni is required for correctly specifying only posterior regions of antennal and mandibular head segments (i.e. the parasegmental boundaries). Interestingly, Tc-kni is essential for the initial specification of the antennal parasegmental boundary, while it is required only for the maintenance of the mandibular parasegmental boundary. The intercalary segment does not appear to be affected. Unexpectedly, we find that the trunk pair-rule gene Tc-even-skipped is required to set the posterior boundary of the mandibular Tc-kni expression. Unlike most RNAi studies, we have good evidence that we were investigating a Null situation due to our finding that antennaless, a Tribolium mutant arising from an EMS screen , is a likely regulatory Null-mutation of Tc-kni. Taken together, we show that Tc-kni is not a head gap gene, since its mutation does not lead to the complete deletion of several adjacent segments. Further, we provide a model, for how head and trunk patterning mechanisms cooperate to pattern the mandibular segment in Tribolium, and how these interactions differ between Drosophila and Tribolium.
Results and discussion
Early Tc-kniexpression prefigures the appearance of the anterior head anlagen
Antennaless is likely a regulatory mutation at the Tc-knilocus
In most RNAi experiments outside Drosophila, it remains unclear to what extent a genetic Null-situation is phenocopied. We aimed at determining the Null situation by studying a genetic mutant.
Antennaless is a homozygous lethal mutation that was recovered from an EMS mutagenesis screen . The cuticular phenotypes of antennaless mutant larvae are highly reminiscent of Tc-kni RNAi phenotypes (Tc-kniRNAi) . Larvae of the antennaless mutant lack antennae and mandibles, and occasionally also display minor abdominal defects, very similar to those previously reported for weak Tc-kniRNAi larvae (Figure 1N, O and Cerny et al.). In order to check for defects in dorsal and lateral head tissues which are derived from pre-antennal and intercalary regions, respectively [32, 36, 56], we scored both sides of 20 antennaless larval cuticles for the head bristle pattern. All bristles were present, albeit shifted somewhat compared to the wildtype condition in these regions (Figure 1N, O). This is consistent with pre-antennal regions and much of the intercalary segment not being affected in the mutant [32, 43].
The development of both mandibles and antennae was more severely affected at a higher temperature in antennaless mutants. A larval cuticle was scored as an antennaless mutant if either the mandibles or antennae, or both, were reduced or absent. Most of the antennaless mutant larvae (67%, n = 43) lacked both the antennae and mandibles at 25°C. This increased to 75% (n = 32) at 32°C. In a few cases, rudiments of mandibles were found (5% at 25°C and 6% at 32°C). In other cases, antennae or antennal rudiments were found (28% at 25°C and 19% at 32°C). In contrast, and against the general observation that phenotypes tend to be more penetrant at higher temperatures, the frequency of abdominal defects was found to be higher at 25°C than at 32°C. At 25°C, 90.5% (n = 43) of phenotypic larvae displayed defects within segments A5-A8, compared to only 16% (n = 32) at 32°C. An inverse sensitivity with respect to temperature was also observed for defects in the urogomphi, dorsal outgrowths of the ninth segment. At 25°C this structure was defective in 79% of the phenotypic larvae examined (n = 43), compared to 44% at 32°C (n = 32).
In offspring from nine independent pairs of heterozygous parents, the antennaless cuticle phenotype is found in 25% (n = 364) and 27% (n = 267) of larvae at 25°C and 32°C respectively, levels consistent with a highly penetrant homozygous lethal mutation. A significant proportion of mutant larvae were able to hatch despite their lack of antennae and mandibles; 45% at 25°C (n = 91) compared to only 17% at 32°C (n = 72). The vast majority of these larvae died at the L1 stage. Only very rarely did individuals reach the L2 stage. When corrected for a background reduction in hatching rate associated with this shift in temperature (measured from wildtype larvae), these data reveal a 19% increase in sensitivity to temperature with regards to hatching in the antennaless mutant background.
In our Tc-kni RNAi experiments head defects were also found to be more severe at 32°C compared to 25°C (see Additional file 1, compare panel B to panel A). The severity of head defects decreased over time post-injection as expected for parental RNAi experiments  (see Additional file 1, panel C). However, as for antennaless, we found inverse temperature sensitivity with respect to defects in segments A5-A8 following Tc-kniRNAi (see Additional file 1, compare panel E to panel D). The frequency of abdominal defects also increased with time post-injection from about 20% (n = 10) at day eight post-injection, to 37% (n = 35) eleven days post-injection, and to 56% (n = 27) thirty-one days post-injection, before dropping again (For eggs at 25°C; see Additional file 1, panel D). This is not in line with the usual observation that in RNAi experiments phenotypic strength decreases over time and indicates a complex relationship between knockdown and phenotype, which we do not fully understand.
Since the antennaless cuticle defects described above are almost identical to the Tc-kniRNAi phenotypes both in terms of physical phenotype and sensitivity to temperature, we suspected Tc-kni as the gene affected by the mutation. We therefore independently sequenced the three Tc-kni exons from the genomic DNA of two first instar larvae that had been identified as homozygous mutant by their cuticle phenotype. We found that the coding sequence of Tc-kni is not altered in mutant beetles (data not shown).
In order to test the hypothesis that antennaless is a regulatory mutation at the Tc-kni locus, we carried out in situ hybridization on embryos from heterozygous mutant parents with a mix of probes targeting Tc-kni and Tc-caudal as positive control. We failed to detect Tc-kni expression in 15% of the offspring examined (n = 100), whereas Tc-caudal was well stained in the same colour reaction in all cases (see Additional file 2). This is consistent with embryos homozygous for the antennaless allele not expressing Tc-kni at levels detectable via in situ hybridization.
Taken together, our results are consistent with the hypothesis that antennaless is a regulatory mutation of Tc-kni, and as such we now refer to it as Tc-kniatl. However, we cannot rule out that antennaless is a mutation in a gene that acts rather exclusively upstream of, or is a required interaction partner of, Tc-kni. Further studies will be necessary to confirm our hypothesis by identifying the cis-regulatory regions that are affected by the mutation.
The Tc-kni Null phenotype is revealed by both Tc-kniatl and Tc-kniRNAi
The lack of Tc-kni expression in homozygous Tc-kniatl embryos implies that Tc-kni function is strongly reduced in the mutant. However, it remains possible that some residual function remains. In order to test whether Tc-kniatl constitutes a complete Null phenotype for Tc-kni, we performed Tc-kni RNAi in the Tc-kniatl background, assuming that the added effects should not lead to stronger phenotypes if Tc-kniatl is a Null mutant. The frequency of the Tc-kni phenotype in the pooled offspring from 30 independent pairs of Tc-kniatl heterozygous animals was 11% (n = 149) at 25°C and 17% (n = 82) at 32°C. As expected, after injecting the same females with Tc-kni dsRNA, the frequency of the Tc-kni phenotype strongly increased, to 97% (n = 32; 25°C) and 81% (n = 32; 32°C). Crucially, we did not find evidence for an increase in the severity of the cuticle phenotype: i.e. there were no larvae with phenotypes more severe than those seen in Tc-kniatl or Tc-kniRNAi alone. Hence, unlike in most RNAi experiments, we can be rather confident that the most severe RNAi phenotypes we observe fully phenocopy a Null situation. Therefore, we use results from both mutant and RNAi embryos for this work.
Tc-kniis differentially required for specifying antennal, intercalary and mandibular parasegment boundaries
The mandibular stripe of Tc-en expression is initiated in early Tc-kniRNAi germband embryos, albeit abnormally; the stripe of expression is broken and in extreme cases only patches of expression are seen (compare panels B and C with panel A in Figure 2; mandibular stripe marked by black arrow). In older Tc-kniRNAi germband embryos, Tc-en expression associated with the mandibular segment is often missing completely (compare panels F, I and O, with panels D, G and M respectively in Figure 2), suggesting a failure to properly maintain Tc-en expression in this segment in strong knockdowns of Tc-kni expression. Similarly, Tc-hh expression in the mandibular segment is greatly reduced in young Tc-kniRNAi embryos, but a thin broken stripe is still detected medially in early germbands (compare black arrows in panels B and D to panels A and C respectively in Figure 3). Later, this Tc-hh expression disappears in contrast to wildtype (compare panels F and H to panels E and G respectively in Figure 3). Note that Tc-en mandibular expression appears to be more sensitive to the loss of Tc-kni expression in lateral regions of the germband (Figure 2E, H, K and N).
Tc-en and Tc-wg expression in the intercalary segment appears relatively late and at a quite variable time point during mid germband elongation in wildtype embryos. Whilst we cannot completely rule out minor disruptions of intercalary Tc-en/Tc-hh expression initiation and/or maintenance in Tc-kniRNAi embryos, we do detect wildtype expression of these genes in late elongation (i.e. post 10 Tc-en stripe embryos) and early fully elongated Tc-kniRNAi embryos (black asterisk in panels K, L, N and O in Figure 2 and black asterisk in panel F in Figure 3).
In conclusion, the disruption of Tc-kni function leads to the complete failure to initiate segment polarity gene expression in the antennal segment, failure to properly maintain segment polarity gene expression in the mandibular segment, and most likely has no effect on segment polarity gene expression in the intercalary segment .
Molecular markers show that Tc-kniis not a canonical head gap gene
Tc-lim1 is a similar marker for the posterior of the ocular segment and anterior compartment of the antennal segment , albeit shifted slightly posteriorly relative to Tc-gsc. In early embryos, expression is partially overlapping the ocular Tc-wg domain and extending posterior to it (Figure 4G). In elongating germbands, Tc-lim1 expression forms a wedge shaped domain that covers all cells between the ocular and antennal Tc-wg stripes (Figure 4I). In the Tc-kniRNAi background, the posterior boundary of this domain is irregular and the domain is somewhat narrower indicating posterior reduction of the Tc-lim marked tissues (Figure 4H). In wild-type elongating germbands, additional Tc-lim1 segmental expression arises that laterally overlaps the segmental Tc-wg domains in gnathal and thoracic segments (Figure 4K). At these stages, the ocular-antennal domain of Tc-lim1 expression splits into a posterior domain that overlaps the antennal Tc-wg stripe, and an anterior domain positioned between the ocular and the antennal Tc-wg stripes (Figure 4K; white arrowheads). Expression in the ocular-antennal region remains also in Tc-kniRNAi germbands, but the domain is not split as in wildtype (upper red arrowhead in Figure 4L) and appears to be fused with the mandibular domain of lateral Tc-lim1 expression (lower red arrowhead in Figure 4L). In extreme cases this domain is also fused to the maxillary Tc-lim1 expression domain (Figure 4L and discussed below). This confirms that anterior antennal tissue is properly specified while the parasegmental boundaries of the antennal and mandibular segments are not formed correctly.
Tc-sloppy-paired-1 (Tc-slp-1) is also expressed in a segmentally reiterated pattern in the developing Tribolium head (Figure 5C, F and [25, 32]). Tc-slp-1 expression domains overlap the Tc-wg stripe in each head segment, but also extend further into the anterior compartment, as well as a little across the parasegmental boundary into the posterior compartment [25, 32]. Tc-slp-1 expression therefore predominantly marks a posterior portion of the anterior segment compartment. Tc-slp-1 is expressed in a stripe in the antennal and each gnathal head segment in both Tc-kniRNAi and Tc-kniatl embryos (Figure 5D, E and G, H), as in wild-type embryos (Figure 5C, F). However, the antennal stripe often appears to broaden (white arrowhead in Figure 5D, E, H), and the distance between the mandibular and the maxillary Tc-slp-1 stripes is often decreased, completely fusing in lateral regions (red arrows in Figure 5D, E and G, H). These data indicate that overall the Tc-slp-1 stripes are not dramatically affected by Tc-kni RNAi. However, the decreased distance between the mandibular and maxillary stripes could indicate incorrectly specified tissue in the intervening posterior compartment of the mandibular segment.
The Tc-kniphenotype is due to a failure to correctly specify cell fates
We decided to check whether missing head regions in Tc-kni knockdown and mutant embryos were lost due to a failure to maintain already specified tissue - which might be indicated by high levels of cell death - or through the failure to specify cells to their correct developmental fate. Using TUNEL staining, we did not detect any apoptosis in either the blastoderm (see Additional file 3) or early germband stages (see Additional file 4) in Tc-kniRNAi embryos. In later germband stages there were a few apoptotic cells detectable in the head (as well as the posterior growth zone), but no more than in wild-type embryos, and there was no specific pattern of apoptosis that would indicate the loss of the tissues in question, namely the posterior portions of the antennal and mandibular segments (see Additional files 3 and 4). Thus, the lack of antennae and mandibles in Tc-kniRNAi and Tc-kniatl larvae is not due to tissue degeneration, but rather the failure to specify the respective segmental regions.
The posterior border of the anterior Tc-kni expression domain is regulated by Tc-even-skipped
Head and trunk segmentation gene networks cooperate to establish the mandibular segment in a different way in Tribolium and Drosophila
In this study we show that anterior regions of the antennal and mandibular segment, as well as the intervening intercalary segment, are correctly specified in the absence of Tc-kni function. Moreover, we identify key differences in the role of Tc-kni in setting up the non-adjacent antennal and mandibular parasegment boundaries. We identify the pair-rule gene Tc-even-skipped as a potential positive regulatory input that enables the initial specification (but not maintenance) of the mandibular parasegmental boundary in the absence of Tc-kni function. We also present the first evidence that, as in Drosophila, head and trunk gene regulatory networks cooperate to specify the mandibular segment. However, our data, and those of others, point to significant divergence in the molecular interactions involved in mandibular patterning between Drosophila and Tribolium.
A recent study of the expression and function of head patterning genes in the hemimetabolous insect Oncopeltus fasciatus suggests that the developmental gene networks operating in Tribolium castaneum more closely resemble the ancestral insect condition, perhaps not surprisingly given the highly derived Drosophila larval head . Indeed, recent data from a myriapod suggest that the developmental genetic basis of Tribolium larval head development might even closely resemble the ancestral arthropod condition [60, 61]. It will be interesting to see whether studies on other arthropods reveal an ancestral role for knirps-family homologues in head segmentation, which would imply ems and btd have usurped the role of knirps, and potentially other genes, in the lineage leading to Drosophila.
The antennaless mutant line was maintained as described by Berghammer et al. and Maderspacher et al.. The separation of offspring from single pair crosses was performed after three and six days of egg laying at 32°C and 25°C respectively. Cuticle preparations of mutant larvae were prepared as previously described . Genomic DNA was extracted from individual L1 antennaless mutant and wildtype larvae by first removing flour and chorion by bleaching egg collections two times for four minutes in 100% bleach. Embryos were washed three times with extraction buffer (without proteinase K - see below), put individually into 0.5 ml cups and frozen at −20°C. 10 μl of extraction buffer was added (25 mM NaCl, 10 mM Tris pH8.0, 1 mM EDTA, 200 μg/ml proteinase K) and the embryos macerated with a pipette tip. After 90mins at 42°C, the preparations were shortly spun down and proteinase K was inactivated by three minutes at 95°C. Debris was spun down for two minutes at 14000 rpm and 8 μl of supernatant was transferred to new tubes. 4 μl were used for a 20 μl PCR reaction. The three Tc-kni exons were amplified by PCR using the following four primer combinations: ex1 fw 5′-ACATTCCCCACCATTGAAATCACA-3′; ex1 rev 5′-GGGTTAAGTTTCTCGGTATTGGGACTA-3′; ex2 fw 5′-CCTGTAATGTGTACAGTCACGAGCAG-3′; ex2 rev 5′-ATTCTTGCATCGGCCGAAGTTTACGT-3′; ex3A fw 5′-CGGAAGCTCTGTCAAACAATAATCTCA-3′; ex3A rev 5′-TCCAGGAACACCCGCTTGTTGA-3′; ex3B fw 5′-CGCCGACGTTTCTACCTCCTCA-3′; ex3B rev 5′-TCGACGCTAATAGCTGCCATCATC-3′. Sequencing of the PCR products was performed by Macrogen (Korea).
Fixation of embryos and enzymatic single and double in situ hybridizations were carried out according to established protocols . For double in situ hybridizations Fastred ® (Sigma) was sometimes used (e.g. for Tc-wg) in place of INT/BCIP (Roche 11-681-460-001) . Probes for in situ hybridization were prepared using either the Digoxigenin RNA Labeling Kit or Fluorescein RNA Labeling Kit (Roche Applied Science, Mannheim) following established protocols and the manufacturers instructions . The DNA templates used for RNA probe production were in some cases (i.e. for probes detecting Tc-ems, Tc-slp-1, Tc-wg, Tc-eve, Tc-gsc, Tc-hh, Tc-lab, Tc-lim1, Tc-odd, Tc-run) produced by PCR-amplifying DNA fragments of the gene of interest from clones using appropriate vector primers (T3, T7, SP6). In other cases (i.e. for probes detecting Tc-en, Tc-col, Tc-Dfd) clones containing a fragment of the gene of interest were used directly as templates following 5′ linearization using an appropriate restriction enzyme. Clones and information are available on request. Following in situ hybridizations, nuclei in blastoderm and early germband embryos were sometimes counterstained using Hoechst 33258 (Additional file 2), Hoechst 33342 (Additional file 3) or DAPI (Figures 1, 2 and 7).
Adult or pupal parental RNAi was carried out using established protocols . dsRNA was produced using the T7 and SP6 MEGAscript High Yield Transcription Kits (Ambion). Template DNA was either PCR-amplified using the vector insert flanking primers T7: 5′-TAATACGACTCACTATAGG-3′ and T7-Sp6: 5′-TAATACGACTCACTATAGGATTTAGGTGAACACTATAGA-3′) or by using a stock of the linearized plasmid. In this case the antisense and sense strands were amplified separately, and later the ssRNA combined in equimolar amounts. A concentration of between 2 and 4.3 μg/μl of Tc-kni dsRNA was injected in each experiment, since this concentration range has been previously shown to consistently produce fully penetrant Tc-kni RNAi phenotypes . To knockdown Tc-eve, Tc-odd and Tc-runt concentrations of between 2 and 3.5μg/μl of dsRNA were used.
Fixed embryos stored in methanol at −20°C were gradually rehydrated by washing in 70% methanol/PBT, then 50% methanol/PBT, then 30% methanol/PBT and finally 100% PBT (PBS + 0.1% (v/v) Tween-20). Embryos were then incubated for six minutes in 1 ml of PBT + 0.5 μl proteinase K (15 mg/ml) and subsequently washed several times in PBT. Embryos were then post-fixed in 1 ml of PBT + 125 μl of formaldehyde (37%) for 20 minutes, before being washed three times in PBT. At this point, positive control embryos were washed three time in DNaseI buffer (40 mM Tris–HCl, pH7.5, 0.1 mM dithiothreitol, 6 mM MgCl2), incubated for 30 minutes at 37°C in DNaseI buffer with 0.06 U DNaseI per microlitre of buffer, before being washed several times in PBT. All embryos were then incubated for 20 minutes in 0.1% sodium borohydride. During incubation, the embryos were gently shaken several times. The sodium borohydride was then removed by repeated washing with TdT buffer (140 mM cacodylic acid, 1 mM cobalt chloride, 30 mM Tris–HCl, pH7.2). Embryos were then incubated at 37°C in TdT buffer containing 20 μM DIG-UTP and 0.3 U/μl terminal deoxynucleotidyl transferase (TdT) (Sigma). In the case of negative control embryos, the TdT was omitted. All embryos were then washed three times for five minutes in TBST (140 mM NaCl, 2.7 mM KCl, 25 mM Tris–HCl pH7.4, 0.1% Tween-20) at room temperature, before being incubated at 70°C for 20 minutes in TBST. Embryos were then washed three times for five minutes in PBT, before being incubated in PAS (PBT with 10 mg/ml bovine albumine (BSA) and 2% sheep serum) for one hour. Embryos were then incubated in PAS with anti-Dig antibody (1:2000) for one hour. This was followed by several washes in PBT for a total of two hours. Embryos were stored overnight at 4°C in PBT before being washed for 30 minutes at room temperature in PBT prior to NBT/BCIP staining. The staining was stopped by repeated washing in PBT and the embryos stored at 4°C in 1 ml of PBT + 125 μl of formaldehyde (37%).
Most in situ hybridization stained preparations were imaged on an Axioplan 2 photomicroscope (Carl Zeiss Vision GmbH, Jena) using a polarized light (DIC) filter with low Normaski contrast (ImageProPlus, Version 5.0 .2.9, MediaCybernetics). For clear detection of the fluorescence signal of the Fastred® color reaction the filter set No. 43 (Cy3) from Zeiss, and a mercury vapor lamp HBO100 as a light source, was used. For the detection of Hoechst 33258 and Hoechst 33342 signal the filter set No. 49 from Zeiss (DAPI filter), and a mercury vapor lamp as the light HBO100 source, was used. A few in situ hybridization stained preparations were imaged on a Leica MZ16F epifluorescence stereoscope with a DFC300FX digital camera (images in Figures 2 and 7). Larval cuticles were imaged using a confocal laser-scanning microscope (LSM 510, Zeiss) and processed as described .
Availability of supporting data
The data set supporting the results of this article is included within the article (and its additional files).
We thank Michalis Averof for the use of reagents and laboratory space and Ernst A. Wimmer for support. This work was funded by Deutsche Forschunggemeinschaft DFG (BU-1443/3-1). MK’s screen for segmentation mutants was funded by DFG grant Kl656/2.
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