Journal of Biotechnology and Sericology 78, 99-105 (2009)

Embryonic RNAi analysis in the , domestica: Distal-less is required to form caudal filament

Takahiro Ohde, Mika Masumoto, Toshinobu Yaginuma and Teruyuki Niimi＊

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan (Received March 9, 2009; Accepted April 15, 2009)

Ametabolous are important for understanding the mechanism of insect evolution based on their phylo- genetic position. Thus, the development and application of an effective gene functional analysis using the RNA interference (RNAi) method is an important step in research on ametabolous insects. We tested RNAi utility in the firebrat, Thermobia domestica (, ) by focusing on the homeobox gene, Distal-less (Dll), based on its conserved sequence and obvious loss-of-function phenotype. Thermobia nymphs that were injected with Dll double-stranded RNA at an early embryonic stage displayed truncated appendages, and thus, we concluded that the RNAi method is useful for analyzing gene function in Thermobia. Remarkably, Dll RNAi in- duced truncation of the caudal appendage, cerci and the caudal non-appendage outgrowth, caudal filament. It is known that although these two caudal structures look similar, they have different origins. Our data suggests that these two types of outgrowths may be formed by similar developmental program, at least with respect to Dll, de- spite their different origins and that Dll even plays a role in a non-appendage structure. Keywords: embryonic RNAi, Thermobia domestica, Distal-less, cerci, caudal filament

not only in model system, but also in non-model systems INTRODUCTION (Agrawal et al., 2003). Ametabolous insects show direct development to the We tested the utility of RNAi using dsRNA based on adult stage with almost no morphological changes. These the sequence of Distal-less (Dll) homolog from Thermo- insects are indispensable for understanding several of the bia. Dll encodes a transcription factor that includes a ho- most important aspects in development in insects, such as meodomain as a DNA-binding domain (Cohen et al., evolution of wing and metamorphosis. However, previous 1989) and has a surprisingly well-conserved sequence studies have concentrated on holometabolous or hemime- among (Panganiban et al., 1995; Prpic and tabolous insects based on the availability of well-estab- Tautz, 2003; Niimi et al., 2005). In Drosophila, Dll pro- lished genetic analysis tools. Since the genetic analysis in tein is required for the formation of ventral appendages, ametabolous insects may be key to understanding molecu- legs, antennae, mouthparts, genitalia, and analia (Cohen lar mechanism underlying insect evolution, gene function- and Jürgens, 1989; Gorfinkiel et al., 1997, 1999; Camp- al analysis tools in ametabolous insects are needed. bell and Tomlinson, 1998; Moreno and Morata, 1999). Here, we applied RNA interference (RNAi) as a gene This function of appendage formation has also been re- functional analysis tool in the ametabolous insect, the fir- ported in other insects and non-insect arthropods, such as ebrat, Thermobia domestica (Fig. 1A). RNAi is a post- the spider mite, Tetranychus urticae (Khila and Grbić, transcriptional gene silencing process that allows rapid 2007), the spider, Cupiennius saler (Schoppmeier and Da- analysis of unknown gene function (Agrawal et al., 2003). men, 2001), the milkweed bug, Oncopeltus fasciatus (An- In this process, double-stranded RNA (dsRNA) is degrad- gelini and Kaufman, 2004), the ladybird beetle, Harmonia ed into small RNAs of ~20 to 30 nucleotides and is fur- axyridis (Niimi et al., 2005), and the red flour beetle, Tri- ther unwound into single-stranded small RNAs. The target bolium castaneum (Beermann et al., 2001; Bucher et al., single-stranded RNA (guide strand) is loaded onto the nu- 2002; Suzuki et al., 2009). Since Dll loss-of-function phe- clease complex, designated RISC (RNA-induced silencing notype appears obvious truncations of the distal regions of complex), and then the RISC cleaves specific mRNA - de appendages, it is a good marker to examine the utility of pending on the sequence of the guide strand (Siomi and RNAi method. Corresponding to its function, Dll expres- Siomi, 2009). Since the first discovery of RNAi in Cae- sion in the distal end of limbs has been observed in a norhabditis elegans (Fire et al., 1998), this technique has wide range of organisms (Panganiban et al., 1995, 1997; been developed in various eukaryotic organisms and has Grenier et al., 1997). Remarkably, Dll expression in the been applied as a powerful reverse-genetic analysis tool caudal filament of Thermobia was reported by Rogers et  al. (2002). In the caudal region of Thermobia, there are *To whom correspondence should be addressed. two types of outgrowth: cercus and caudal filament. Fax: +81-52-789-4036. Tel: +81-52-789-5504 Although these outgrowths have similar morphology Email: [email protected] (Fig. 1B), the developmental origin is different. The cau- 100 Ohde et al.

that Dll is required to form both cerci and the caudal fila- ment. Further, this is an interesting example of two body parts, each with different origins that are formed by a similar developmental program, at least for a gene, Dll.

MATERIALS AND METHODS Insects A colony of Thermobia domestica was maintained at 34 to 35°C and provided with water and an artificial cricket food (Rep-Cal). Eggs were collected from tissue paper placed in the cage and incubated at 37°C in a plastic case. Embryos were staged according to Woodland (1957).

Cloning Total RNA was extracted from a whole body of Ther- mobia with TRIzol (Gibco BRL) according to the manu- facturer’s instructions. First-strand cDNA was synthesized from total RNA with a SMART PCR cDNA Amplification Kit (Clontech). The Td-Dll cDNA fragment was amplified using the following degenerate primer set for Dll designed Fig. 1. (A) Thermobia domestica adult. (B) Higher magnifi- from highly conserved amino acid sequences found in D. cation image of the Thermobia caudal region. An, antenna; Ce, cercus; Cf, caudal filament; Mp, maxillary palp; St, stylus; melanogaster Dll (S47947), zebrafish Dlx-8 (U67846), T1-T3, thoracic legs. mouse Dlx-1 (U51000), and human Dlx-2 (U51003): Dll-1: 5′-ATGMGIAARCCIMGIACIATHTA-3′ dal filament is not an appendage structure while cerci are Dll-2: 5′-YTTISWICKICKRTTYTGRAACCA-3′ true appendages of the eleventh segment. The origin of (M=C+A, R=A+G, H=A+T+C, Y=C+T, S= the caudal filament is still controversial. One classical the- G + C, W = A + T, K = T + G, I = Inosine) ory is that the caudal filament is an elongation of the The PCR was performed using 2.5 μl of the 10-fold-dilut- eleventh abdominal tergum (Snodgrass, 1935). Alterna- ed first-strand cDNA, the designed primer pair, - andAm tively, it has also been proposed, from the studies of the pliTaq Gold (Perkin Elmer). bristletail (Machida, 1981) and the mayfly (Tojo and Machida, 1997), that the caudal filament is a structure that Sequencing and sequence analysis is independent from the eleventh segment. In a previous PCR products were subcloned into the EcoR V site of report, Dll expression was demonstrated in both cerci and the pBluescript KS+ vector (Stratagene). The nucleotide caudal filament (Rogers et al., 2002). Examining the simi- sequences of the PCR products and the flanking regions larities in the developmental program of two body parts around the restriction enzyme-digested fragments, which with different origins and similar morphology should be were inserted into the vectors, were confirmed using the instructive. dideoxy chain-termination method on an automated DNA In this study, we first cloned the Dll homolog from sequencer 3130 genetic analyzer (Applied Biosystems). Thermobia (Td-Dll). Then, we examined the expression Sequence analysis was carried out using a DNASIS pattern of Dll in embryos of Thermobia by antibody stain- system (Hitachi Software Engineering). Deduced amino ing. Dll showed similar expression patterns in both the acid sequences were aligned with ClustalW2. The cerci and the caudal filament. This result suggests the DDBJ/EMBL/GenBank accession number for Td-Dll is similar function of Dll in these structures. To examine the AB489202. role of Td-Dll in Thermobia, we then performed an RNAi experiment by injecting Td-Dll dsRNA into an early em- Antibody staining and imaging bryo. Consequently, the nymphs treated with Td-Dll Thermobia embryos were dissected in ice-cold phos- dsRNA displayed a lack of distal region of appendages. phate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl,

Thus, we conclude that the RNAi method is valid to ana- 10.14 mM Na2HPO4, pH 7.2), fixed with PLP (4% para- lyze gene function in Thermobia. Importantly, the caudal formaldehyde, 30 mM NaPO4, 10 mM NaIO4, and 75 mM filament of Td-Dll RNAi nymph exhibited a truncated lysine, pH 6.8) for 2 h on ice, and then washed 5 times in phenotype similar to that in the cerci. Our data indicate 100% methanol at room temperature and stored at −20°C Embryonic RNAi in Thermobia 101 until use. Working at 4°C, embryos were rehydrated in ered with Voltalef 10S oil (Elf Atochem). The embryos PBT (PBS, 0.3% Triton X-100), blocked in 2% PBTS were injected at either the anterior or posterior pole. In- (PBT, 2% BSA) for 6 h, and then incubated overnight in jected embryos were incubated in a moist chamber at 1:200 dilution of a rabbit polyclonal anti-Dll antibody (a 37°C. kind gift from G. Panganiban; generated against Drosoph- ila Dll) as a primary antibody. After 6 washes in PBT, Scanning electron microscope (SEM) analysis embryos were incubated overnight in 1:300 dilution of Al- Hatched nymphs were fixed in 70% EtOH, dehydrated exa flour 488 conjugated anti-rabbit antibody (Molecular in a graded ethyl alcohol series, and then transferred to Probes) as a secondary antibody. Finally, embryos were acetone. The nymphs were dried in a critical-point dryer, washed 6 times in PBT and mounted in VECTASHIELD coated with platinum, and observed under an S-3000N mounting medium (Vector Laboratories). Images were col- electron microscope (Hitachi). lected using fluorescence microscopy BZ-9000 (KEY- ENCE). RESULTS AND DISCUSSION Embryonic RNAi Cloning of Thermobia Dll homolog Preparation of double-stranded (ds) RNA and microin- Dll homologs isolated from a variety of organisms jection into embryos were performed as previously de- show remarkable sequence conservation, especially in the scribed (Niimi et al., 2005; Kuwayama et al., 2006). The homeodomain. Such high homology is also seen among plasmids containing Td-Dll and egfp (Kuwayama et al., Dll homologs (Panganiban et al., 1995; Prpic 2006) cDNA were used as templates for dsRNA synthesis. and Tautz, 2003; Niimi et al., 2005). To investigate se- The dsRNA was dissolved in RNase free water and ad- quence conservation among Dll homologs from an ame- justed concentration to 2 μg/μl. Thermobia embryos were tabolous insect, such as Thermobia and other insects, we collected within 12 h of oviposition to perform dsRNA in- cloned Thermobia Dll homolog (Td-Dll) using a pair of jection during the syncytial blastoderm stage (Woodland, degenerate primers based on the well-conserved region of 1957). To protect against desiccation, embryos were cov- the Dll homeodomain. We obtained an 118-bp cDNA frag-

Fig. 2. Multiple alignment of arthropod Dll homeodomain. Shading indicates sequence homologous with Td-Dll. Since Dll homologs of some species are not yet fully determined, unknown residues are shown as dashes. Arrows in- dicate the primer set used for Td-Dll cDNA amplification. Abbreviations: Td, Thermobia domestica; Sa, Schistocerca americana (grasshopper, AY566290); Of, Oncopeltus fasciatus (milkweed bug, AY584472); Ha, Harmonia axyridis (la- dybird beetle, AB200969); Tc, Tribolium castaneum (red flour beetle, AF317551); Aa, Aedes aegypti (yellow fever mos- quito, EAT47119); Dm, Drosophila melanogaster (fruit fly, S47947); Jc, Junonia coenia (peacock butterfly, AF404110); Ms, Manduca sexta (tobacco hornworm, AY616435); Ar, Athalia rosae (coleseed sawfly, AB378321); Am, Apis mellifera (honey bee, AB365073); Tl, Triops longicaudatus (tadpole shrimp, DQ923136); Gm, Glomeris marginata (pill millipede, AJ551276); Cs, Cupiennius salei (spider, AJ278606); Tu, Tetranychus urticae (two-spotted spider mite, DQ442864). 102 Ohde et al. ment that encodes 39 amino acids. Sequence comparison 40% of embryogenesis is not uniformly expressed, and al- revealed almost perfect homology among Thermobia and though the pattern is weak, it resembles conventional other arthropods (Fig. 2) and, thus, we concluded that the proximal ring and distal sock expression patterns (Fig. 3C) cDNA isolated in this study is part of the Dll homolog (Panganiban et al., 1994). This Dll expression pattern is from Thermobia. almost identical in both the cerci and caudal filament. Even though the cerci and caudal filament have been pro- Td-Dll expression pattern in Thermobia embryo posed to have different developmental origins (Snodgrass, Dll protein expression patterns have been investigated 1935; Machida, 1981; Tojo and Machida, 1997), based on in a wide range of organisms using a broadly cross-react- the similar expression patterns in the cerci and caudal fila- ing antibody. The expression of Dll in Thermobia was ex- ment, Dll is required to form both structures. amined previously, and remarkably, Dll accumulated in the caudal region (Rogers et al., 2002). To confirm Dll Td-Dll functional analysis using embryonic RNAi expression in the Thermobia embryo, we performed anti- Embryonic RNAi is one of the effective RNAi tech- body staining using anti-Dll antibody. In embryos at 30% niques in arthropods, and it was carried out by microin- of embryogenesis, Dll was detected in the labrum, anten- jection of dsRNA into embryos at the syncytial blastoderm na, maxillary and labial appendages, and thoracic legs, stage. To investigate the function of Td-Dll, we tested the and relatively weak expression was detected in the pro- embryonic RNAi method with Td-Dll dsRNA. Nymphs cepharon, pleuropodia (transitory appendages on the first injected with Td-Dll dsRNA displayed defects in forma- abdominal segment), cerci, and caudal filament, but not in tion of outgrowths similar to the previously reported Dll the mandibular segment (Fig. 3A). These Dll-expressing loss-of-function phenotypes (Fig. 4) (Cohen and Jürgens, regions are consistent with a previous report by Rogers et 1989; Beermann et al., 2001; Schoppmeier and Damen, al (2002). Further observation of Dll expression in the 2001; Bucher et al., 2002; Angelini and Kaufman, 2004; caudal region showed weak staining to 30% of embryo- Niimi et al., 2005; Khila and Grbić, 2007; Suzuki et al., genesis (mid-stage of blastokinesis) (Fig. 3B) and more 2009). In the head, the antennae and the maxillary and la- intense expression at 40% of embryogenesis (completed bial palps were significantly shortened by Td-Dll dsRNA blastokinesis, before eye pigmentation) (Fig. 3C). Dll at injection (Fig. 4C). Although the labrum showed relatively minor defects, it was almost missing in several of Td-Dll RNAi nymphs (data not shown). Such defects in the la- brum are consistent with previous reports in the spider mite, Tetranychus urticae (Khila and Grbić, 2007), the spider, Cupiennius saler (Schoppmeier and Damen, 2001), Tribolium (Beermann et al., 2001), and Drosophila (Cohen and Jürgens, 1989). In thoracic legs of nymphs with the most severe phenotype, only the most proximal segment, the coxa was present. (Fig. 4C). This severe defect in the legs resembles the phenotype of Triborium mutant larvae (Beermann et al., 2001) and suggests that Td-Dll is re- quired to form the region distal to the trochanter in Ther- mobia. Significantly, a similar defect was observed in the cerci (Fig. 4E) and caudal filament (Fig. 4G). The caudal filament of Td-Dll RNAi nymph exhibited prominent re- duction, while only a slight deletion of the distal tip was observed in the cerci. Tribolium has a non-appendage structure in the caudal region, termed urogomphi, which is regarded as an outgrowth of the dorsum of the ninth Fig. 3. Expression pattern of Dll in Thermobia embryo. (A) segment. As a Dll loss-of-function phenotype, the urogom- Embryos stained with the Dll antibody at about 30% of em- phi shows dramatic reduction, and is almost completely bryogenesis. Dll accumulates in the procephalon (Pc), labrum (Lr), antenna (An), maxillary (Mx) and labial (Lb) appendag- missing (Beermann et al., 2001; Suzuki et al., 2009). This es, thoracic legs (T1, T2, T3), the pleuropodia of the first ab- phenotype also indicates Dll function in the caudal non- dominal segment (Pl), cercus (Ce) and caudal filament (Cf) appendage structure. Interestingly, suggestive expression but not in mandibular segment (Mn). The caudal region of has been observed in the crustacean telson (Scholtz et al., embryos at (B) 30% and (C) 40% of embryogenesis. In (C), Dll staining appeared conventional proximal ring (arrowhead) 1998) and the posterior end of the spider (Schoppmeier and distal sock (parenthesis) pattern. Scale bars are 100 μm. and Damen, 2001), although their functions are unknown. Embryonic RNAi in Thermobia 103

In contrast to Td-Dll dsRNA, no detectable effect was ob- Further analysis of genes involved in the appendage for- served in hatched nymph that had been injected with egfp mation is necessary to understand differences in formation dsRNA (Fig. 4B, D, F). Therefore, phenotypes induced by mechanisms underlying these two types of outgrowth. Td-Dll dsRNA injection are specific for Td-Dll. Our data reveal that Dll not only plays a role in appendage forma- RNAi efficiency in Thermobia tion but also functions in non-appendage outgrowth. Although nymphs that were injected with Td-Dll

Fig. 4. RNAi of Td-Dll. (A) Lateral view of the wild-type first instar Thermobia nymph. A pair of palpi is present on both the maxilla (Mp) and labium (Lp). Wild-type legs consist of five segments: coxa (Cx), trochanter (Tr), femur (Fe), tibia (Ti) and tarsus (Ta). The name of the injected dsRNA is shown at the right-bottom of the panel in B-G. (C) Trun- cation of the head and thoracic appendages was observed in hatched nymphs that were injected with Td-Dll dsRNA. Only the most proximal segment, the coxa, remained in the second and third thoracic legs. This phenotype was not in- duced by egfp dsRNA injection (B). A1, the first abdominal segment; T1, prothorax; T2, mesothorax; T3, metathorax. (E) Td-Dll RNAi nymph displayed a minor reduction of the distal tip of the cerci compared to that of nymphs injected with egfp dsRNA (D). (G) A similar but more significant reduction was observed in the caudal filament (compared withF). Nymphs which had one of the most severe phenotypes are shown for Td-Dll RNAi. Scale bars are 100 μm. 104 Ohde et al.

Table 1. Phenotype of Thermobia injected with dsRNA Phenotype of nymphs dsRNA Injected pole of embryo N** Dll-specific phenotype no detectable effect anterior 27 9 (33.3%) 18 Td-Dll posterior 35 13 (37.1%) 22 egfp –* 54 0 54 *Injected pole of embryo was not distinguished for egfp dsRNA injection. **Number of injected embryos that hatched or developed until just before hatching. The latter was dissected to observe phenotype. dsRNA exhibited truncated outgrowth structures (Fig. 4C, D. and Jürgens, G. (2001) The Short antennae gene of Tri- E, G), no detectable effect was induced by egfp dsRNA bolium is required for limb development and encodes the injection (Fig. 4B, D, F). The specific phenotype of ap- orthologue of the Drosophila Distal-less protein. Develop- ment, 128, 287-297. pendage reduction was observed in 33.3% and 37.1% of Bucher, G., Scholten, J. and Klingler, M. (2002) Parental hatched nymphs that were injected with Td-Dll dsRNA RNAi in Tribolium (Coleoptera). Curr. Biol., 12, R85-R86. into the anterior and posterior pole of embryo, respective- Campbell, G. and Tomlinson, A. (1998) The roles of the ho- ly (Table 1). This data suggests that injection site has no meobox genes aristaless and Distal-less in patterning the significant effect on RNAi efficiency. In the previous legs and wings of Drosophila. Development, 125, 4483- 4493. study of embryonic RNAi in the ladybird beetle, Harmo- Cohen, S.M., Brönner, G., Küttner, F., Jürgens, G. and Jäckle, nia axyridis showed that specific phenotype was observed H. (1989) Distal-less encodes a homoeodomain protein re- in 20% of Dll homolog dsRNA injected embryos (Niimi quired for limb development in Drosophila. Nature, 338, et al., unpublished data). Compared to this data, 37.1% 432-434. RNAi efficiency in this study is relatively high. In this Cohen, S.M. and Jürgens, G. (1989) Proximal-distal pattern formation in Drosophila: cell autonomous requirement for experiment, a small number of hatched nymphs exhibited Distal-less gene activity in limb development. EMBO J., 8, a strong phenotype. Dll has been reported to be involved 2045-2055. in embryonic neurogenesis in Drosophila (Panganiban, Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. 2000). It is likely that Thermobia embryo with severe Td- and Mello, C.C. (1998) Potent and specific genetic interfer- Dll RNAi effect died during early embryogenesis because ence by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806-811. of an unknown defect in the nervous system, and thus, it Gorfinkiel, N., Morata, G. and Guerrero, I. (1997) The homeo- was difficult to observe severe phenotypes in the hatched box gene Distal-less induces ventral appendage development nymphs. From these results, we concluded that the func- in Drosophila. 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