Ultrabithorax Is Essential for Bacteriocyte Development

Ultrabithorax is essential for bacteriocyte development

Yu Matsuuraa,b,c, Yoshitomo Kikuchid, Toru Miurab, and Takema Fukatsua,c,1

aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; bGraduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan; cGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan; and dBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Hokkaido Center, Sapporo 062-8517, Japan

Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved June 19, 2015 (received for review February 18, 2015) Symbiosis often entails the emergence of novel adaptive traits in transcription factors, in particular Ultrabithorax (Ubx), are involved organisms. Microbial symbionts are indispensable for diverse insects in the development of the host cells and organs specialized for via provisioning of essential nutrients, wherein novel host cells and harboring the symbiotic bacteria. organs for harboring the microbes, called bacteriocytes and bacteriomes, have evolved repeatedly. Molecular and developmental mech- Results and Discussion anisms underpinning the emergence of novel symbiotic cells and Bacteriocyte Development During Embryogenesis of N. plebeius. We organs comprise an unsolved question in evolutionary developmental performed a detailed description of the embryogenesis of N. ple- biology. Here, we report that a conserved homeotic gene, Ultra- beius with special focus on the dynamics of the bacteriocyte- bithorax, plays a pivotal role in the bacteriocyte differentiation in a associated gammaproteobacterial symbiont “Candidatus Schneideria hemipteran insect Nysius plebeius. During embryonic development, nysicola” (18) (Figs. 1 and 2, Table S1,andMovie S1). The sym- six pairs of aggregated presumptive bacteriocytes appear on both biotic bacteria were found as an aggregate at the anterior pole of sides of six abdominal segments, incorporate the symbiotic bacteria newly laid eggs (Fig. 1 A and B). After blastoderm formation at the stage of germband retraction, and fuse into a pair of lateral (12–24 h after oviposition; Fig. 2A) and germband elongation bacteriomes at the stage of germband flip, where bacteriocyte-asso- (24–33 h; Fig. 2 B–D), the symbionts were wrapped within ab- ciated Ultrabithorax expression coincides with the symbiont infec- dominal segments of the germband (36–60 h; Figs. 1 I and J and 2 tion process. Suppression of Ultrabithorax expression by maternal E–G). After germband retraction (∼72 h; Fig. 2H), the symbionts

RNA interference results in disappearance of the bacteriocytes and migrated from the abdominal population to presumptive bacter- EVOLUTION the symbiont localization therein, suggesting that Ultrabithorax is iocytes that appeared on both sides of abdominal segments A2–A7 involved in differentiation of the host cells for symbiosis. Suppression as six pairs of clusters (72–84 h; Figs. 1 K and L and 2 I and J). of other homeotic genes abdominal-A and Antennapedia disturbs Then, during the process of drastic embryonic flip called katatrepsis, integrity and positioning of the bacteriomes, affecting the configura- the six bacteriocyte clusters on each side fused into a coherent tion of the host organs for symbiosis. Our findings unveil the molec- bacteriome located at abdominal segments A2–A4 (84–96 h; ular and developmental mechanisms underlying the bacteriocyte Figs. 1 M and N, and 2 K–M and Q–R). After the symbiont in- differentiation, which may have evolved either via cooption of the fection, the bacteriocytes accumulated red pigment and became transcription factors for inducing the novel symbiotic cells, or via re- easily recognizable (∼84 h; Figs. 1 C and 2 R–T). The red pig- vival of the developmental pathway for the bacteriocytes that had mentation provided a visible marker useful for tracing the existedintheancestralhemipterans. bacteriocytes, although it is unclear whether the red pigment was derived from the symbiont or the host. bacteriocyte | homeotic gene | transcription factor | evolution | symbiosis Significance ymbiosis is the source of novel adaptive traits, thereby con- Stributing to organismal evolution and diversification (1, 2). A Among the most fundamental questions in developmental bi- variety of insects are obligatorily dependent on microbial sym- ology is how novel cell types have emerged in the metazoan bionts via provisioning of essential nutrients lacking in their diets evolution. Among the most challenging questions in evolu- (3, 4), wherein novel host cells and organs for harboring the mi- tionary biology is how sophisticated symbiotic associations crobes, called bacteriocytes and bacteriomes, have evolved repeat- have evolved through less intimate interorganismal in- edly in such insect groups as hemipterans (aphids, whiteflies, – teractions. These fundamental biological issues are crystalized mealybugs, leafhoppers, spittlebugs, etc.) (5 9), dipterans (tsetse in the evolution and development of insect’s bacteriocytes flies, bat flies, etc.) (10, 11), coleopterans (weevils, etc.) (12, 13), specialized for harboring symbiotic bacteria. Here, we report and many others (14). Despite a considerable body of embryo- that a conserved transcription factor Ultrabithorax is essential logical descriptions (14), molecular mechanisms underlying the for bacteriocyte development in an insect, thereby uncovering bacteriocyte differentiation have been a long-lasting enigma in a molecular mechanism underlying the emergence of the novel evolutionary developmental biology (15, 16). Although cellular host cells for symbiosis. Our finding highlights the importance and developmental aspects of the bacteriocyte formation have of developmental cooption of preexisting transcription factors Acyrthosiphon pisum been best documented for the pea aphid, and sheds new light on a long-lasting enigma in evolutionary Nysius plebeius (5, 15, 17), the seed bug and allied heteropteran developmental biology. bugs of the superfamily Lygaeoidea have recently emerged as a promising model system for investigating the development, evo- Author contributions: Y.M. and T.F. designed research; Y.M., Y.K., and T.M. performed lution, and origin of the bacteriocytes, on the grounds that research; Y.M. analyzed data; and Y.M. and T.F. wrote the paper. (i) heteropteran bugs are generally associated with gut symbiotic The authors declare no conflict of interest. bacteria without bacteriocytes; (ii) thus, the bacteriocytes in these This article is a PNAS Direct Submission. lygaeoid species are regarded as a novel trait whose evolution was Freely available online through the PNAS open access option. iii N. plebeius a relatively recent event (18, 19); and ( )in and allied Data deposition: The nucleotide sequences determined in this study have been deposited lygaeoid species, RNA interference (RNAi) works efficiently, in the DNA Data Bank of Japan database (accession nos. LC010622–LC010625). which enables functional analysis of genes involved in the bac- 1To whom correspondence should be addressed. Email: [email protected]. teriocyte formation (20, 21). Here we demonstrate that, by making This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. use of the emerging model insect N. plebeius, conserved homeobox 1073/pnas.1503371112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1503371112 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Symbiont localization and bacteriocyte differentiation during the development of N. plebeius.(A and B) Newly laid eggs. (C and D) Embryos 5 d after oviposition. (E and F) First-instar nymphs. (G and H) Adult insects. (I and J) Embryos ∼48 h after oviposition. (K and L) Embryos of 72–84 h. (M and N) Embryos of ∼96 h. (O and P) Embryos of ∼120 h. A, C, E, and G are light-microscopic images; I, K, M, and O are schematic illustrations of symbiont localization (red) in the embryos; and B, D, F, H, J, L, N,andP are fluorescence microscopic images in which blue and green signals show the host nuclei and the symbiotic bacteria, respectively. Arrowheads depict bacteriocytes, and arrows indicate aggregated symbionts within the embryos. A1, A2, and A3, first, second, and third abdominal segments, respectively; ob, ovarial bacteriocytes in adult female; T3, third thoracic segment.

Bacteriocyte-Associated Expression of Ubx in Embryogenesis of abdominal segments A2–A7 after the germband retraction (Fig. 3 N. plebeius. The homeobox genes encode transcription factors B–D and Fig. S1D), which agreed with the locations of the pre- that assign segment identities and specify functional body parts sumptive bacteriocytes (Figs. 1 K and L and 2J). Meanwhile, abd-A in the development of insects and other animals (16, 22). In the was expressed broadly across the abdominal segments without embryogenesis of the pea aphid A. pisum, some homeobox gene specific association with the presumptive bacteriocytes (Fig. 3 E products—including an appendage-patterning transcription fac- and F and Fig. S1 H–J). tor Distal-less (Dll), homeotic proteins Ubx or Abdominal-A (Abd-A), and a segment polarity protein Engrailed (En)—were Disappearance of Bacteriocytes by Ubx Suppression. In heteropteran shown to localize to the bacteriocytes, although their functions in species including O. fasciatus, injection of double-stranded RNA the symbiotic cells have been elusive (15). We investigated the (dsRNA) into mother insects was reported to efficiently suppress spatial expression patterns of some of these homeobox genes their offspring’s gene expression during embryogenesis (20, 26). during the development of N. plebeius (Fig. 3 A–F and Fig. S1). Using the maternal RNAi technique, we suppressed the embry- Dll was prominently expressed at the distal portion of append- onic expression of these genes and observed the development of ages in antennal, labial, and thoracic segments and faintly detected N. plebeius. Dll suppression affected neither the bacteriocyte for- in the maxillary segments, but not expressed in the areas of the mation nor the symbiont localization (Fig. 3 G and H). Strikingly, presumptive bacteriocytes (Fig. 3 A and A′), which agreed with the Ubx suppression resulted in disappearance of the red-pigmented expression patterns of Dll in a related lygaeid species without bacteriocytes and the symbiont localization associated with them bacteriocytes, the milkweed bug Oncopeltus fasciatus (23, 24). (Fig. 3 I and J). Detailed observations of the Ubx-suppressed em- Notably, Ubx exhibited remarkable expression patterns in the bryos revealed that, in contrast to the control embryos wherein the abdominal regions where the presumptive bacteriocytes were symbionts aggregating in the abdomen were migrating to the bac- supposed to differentiate: Although the strong expression at the teriocytes (Fig. 3 M and M′) and strictly localized therein (Fig. 3O), abdominal segment A1 and the milder expression in the following the symbionts were dispersing within the embryonic body (Fig. 3 N abdominal segments were observed during the germband elon- and N′) and lost subsequently (Fig. 3P). Meanwhile, abd-A sup- gation and retraction (Fig. S1 A–C), which are typical of the Ubx pression did not lead to disappearance of the bacteriocytes (Fig. 3 expression in O. fasciatus and other insects (24, 25), Ubx was also K, L, Q,andR), although the spatial organization of the bacter- strongly expressed as six pairs of clusters on both sides of iocytes was affected as detailed later.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1503371112 Matsuura et al. Downloaded by guest on September 30, 2021 Fig. 2. Embryogenesis of N. plebeius.(A–P) Fluorescence microscopic images in which blue and green signals reflect the host nuclei and the symbiotic bacteria, respectively. Unless otherwise described, the embryos are oriented such that the anterior pole of the egg is to the left and the dorsal surface of the egg is upward.

Arrows indicate the symbiont cells aggregating within the embryos, and arrowheads show the symbiont cells localized to the bacteriocytes/bacteriomes. (A)The EVOLUTION blastoderm stage (12–24 h after oviposition). (B) The early invagination stage (24–27 h). (C) Elongation of the germband (∼30 h). (D) Further elongation of the germband (∼33 h). (E) The late invagination stage (∼36 h) by which segmentation occurs so that embryonic head, thorax, and abdomen are recognizable. (F) The germband with growing appendages, whose tail is curving around the dorsal surface of the egg and wrapping the symbionts within the abdominal segments (∼48 h). (G) The germband whose appendages are growing further (∼60 h). (H) The germband retraction stage (∼72 h), at which the embryonic body thickens. (I) The retracted embryonic body with shrunk abdominal segments, on which all of the appendages are folded toward the longitudinal center (∼84 h). (J) An enlarged image of the thoracic and abdominal segments, where the symbiont infection to the embryonic body is taking place (∼84 h). The symbionts aggregating on the abdominal segments (arrow) and the symbionts incorporated into six clusters of presumptive bacteriocytes on each side of the abdomen (arrowheads) are observed. (K) The katatrepsis, or embryonic flip, stage (84–96 h). During the katatrepsis, the embryo turns backward along the ventral surface of the egg, and the six clusters of the bacteriocytes on each abdominal side fuse into a coherent bacteriome. (L and M) Lateral and dorsal views of a postkatatrepsis embryo (∼96 h), which have flipped and escaped the yolk, and left serosal fold above its head. (N and O) Embryos after dorsal closure (108–120 h), in which the bacteriomes are oval in shape and located at both sides of the abdominal segments A2–A3 or A2–A4. (P) A mature embryo about to hatch (∼145 h). (Q–T)Light- microscopic images of developing embryos. (Q) An embryo at the germband retraction stage (∼72 h). The abdominal region where the symbionts are localized before migrating to the presumptive bacteriocytes is recognizable by reddish hue (arrow). (R) An embryo in which symbiont migration to the presumptive bacteriocytes is taking place (∼84 h). The symbiont localizations before and after the migration are seen as red-colored abdominal regions (arrow and arrowhead, respectively). (S) An embryo after dorsal closure (∼108h),whosebacteriomesarecoloredindeepred.(T) A mature embryo about to hatch (∼145 h). A1, A2, A3, and A4, abdominal segments 1, 2, 3, and 4, respectively; An, antenna; Gb, germband; Hl, head lobe; Lb, labium; Mn, mandible; Mx, maxilla; Sf, serosal fold; T1, T2, and T3, thoracic segments 1, 2, and 3, respectively. (Scale bars: 100 μm.)

Although the majority of the control embryos hatched normally mature embryos exhibited separate bacteriocyte clusters (Fig. 3 with a pair of red-pigmented bacteriomes in the abdomen (Fig. 4 K, L, Q, and R), as observed in the control younger embryos (Fig. A–C and Table S2), considerable proportions of the RNAi-treated 3M). Whereas most of 329 abd-A–suppressed hatchlings and 68 embryos failed to hatch with morphological abnormalities typical embryos retained the bacteriomes, all 23 hatchlings with strong of the homeobox gene defects (23, 25) (Table S2). Dll suppression phenotypes whose abdominal segments A2–A8 were transformed produced the hatchlings without distal regions of the appendages, into thorax-like identity, and also some embryos, exhibited un- in which a pair of bacteriomes formed normally (Fig. 4 D–F, Fig. fused bacteriomes, ranging from two to six in number, in the ab- S2 A–D,andTable S2). Of 160 Ubx-suppressed hatchlings, all 42 domen (Fig. 4 J–L, Fig. S2 I–L,andTable S2). These results hatchlings with strong phenotypes, whose abdominal segment A1 suggest that abd-A may be involved in the integrity of the symbiotic grew extra appendages, lacked the bacteriomes (Fig. 4 G–I and organs via, presumably, homeotic regulation of segment identity in Table S2), whereas 48 hatchlings with moderate phenotypes the abdomen of N. plebeius. lacked the symbiotic organs partially (Fig. S2 E and F and Table S2). Of 130 Ubx-suppressed unhatched embryos, 111 embryos Translocated Bacteriomes by Antp Suppression. Additionally, we lacked the bacteriomes (Fig. S2 G and H and Table S2). The ab- performed maternal RNAi of Antennapedia (Antp), which is a sence of the bacteriomes in the Ubx-suppressed hatchlings and homeotic gene expressed at thoracic and anterior abdominal embryos was evidently and significantly more frequent than in the segments in O. fasciatus and other insects (24, 25). Antp sup- Dll-suppressed, abd-A–suppressed, and control hatchlings and pression also affected the bacteriome localization. Of 211 Antp- embryos (Table S3). suppressed hatchlings, all 13 hatchlings with strong phenotypes, whose six legs were deformed with swollen femurs and shortened Unfused Bacteriomes by abd-A Suppression. Notably, abd-A sup- tibiae, exhibited the bacteriomes at the T3 thoracic segment and pression affected the spatial organization of the bacteriocytes: In the A1–A2 anterior abdominal segments. The bacteriomes were contrast to a pair of fused bacteriomes in the control mature found not only in the body trunk but also inside the hind legs (Fig. embryos (Figs. 1 O and P and 2 S and T), the abd-A–suppressed 4 M–O, Fig. S2 M–P,andTable S2). These phenotypes look like

Matsuura et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fig. 3. Expression patterns and RNAi phenotypes of Dll, Ubx, and abd-A during the embryogenesis of N. plebeius.(A and A′) Lateral and dorsal views of Dll expression pattern (48–72 h after oviposition). (B and B′) Lateral and ventral views of Ubx expression pattern (48–72 h). (C and D) Dorsal and lateral views of Ubx expression pattern (∼84 h) just after the symbiont infection to the bacteriocytes. (E and E′) Lateral and dorsal views of abd-A expression pattern (48–72 h). (F) A dorsal view of abd-A expression pattern just after the symbiont infection to the bacteriocytes. In A–F, arrowheads indicate specific expressions of the homeobox genes in the abdominal segments. Smaller arrowheads indicate bacteriocyte-associated expressions, where numbers 1–6 correspond to the regions of the abdominal segments A2–A7. Larger arrowheads show the other specific expressions in the abdominal region. An, antenna; Lb, labium; Pp, pleuropodium; T1, T2, and T3, thoracic segments 1, 2, and 3, respectively. (G and H) Lateral views of Dll-suppressed embryos just after katatrepsis (∼100 h). (I and J) Lateral views of Ubx-suppressed embryos just after katatrepsis (∼100 h). (K and L) Lateral views of abd-A–suppressed embryos just after katatrepsis (∼100 h). (M and M′)Dorsaland z-stacked images of a control embryo (∼84 h). (N and N′) Dorsal and z-stacked images of a Ubx-suppressed embryo (∼84 h). (O–Q) Dorsal views of control, Ubx-, and abd-A–suppressed embryos just after katatrepsis (∼100 h). (R) An enlarged lateral view of an abd-A–suppressed embryo (∼100 h). In G–R, G, I,andK are light- microscopic images in which the abdominal region for bacteriome formation is highlighted by a white circle, whereas the others are florescence microscopic images in which blue and green signals indicate the host nuclei and the symbiotic bacteria, respectively. (Scale bars: 100 μm.)

invasion of posterior segmental identity into the thoracic segment, the preexisting and conservative homeobox transcription factors suggesting the possibility that, although speculative, Antp may for acquisition of the novel cells and organs for symbiosis. Fig. 5 negatively regulate the bacteriocyte differentiation at the T3 schematically illustrates expression patterns of homeotic genes and A1 segments during the embryogenesis of N. plebeius. during the embryogenesis of N. plebeius. The mechanism gov- erning the bacteriocyte-associated expression pattern of Ubx is Conclusion currently unknown and deserves future study. As reported in The disappearance of the bacteriocytes by Ubx suppression, in fruit flies (27), it seems plausible, although speculative, that combination with the bacteriocyte-associated Ubx expression, Ubx might have acquired a novel cis-regulatory element for the strongly suggests that Ubx plays a pivotal role in the bacteriocyte unique regional expression in an ancestor of N. plebeius. differentiation in N. plebeius. In addition, abd-A and Antp may affect the bacteriome integrity and positioning. Our findings shed Perspectives light on the long-lasting enigma as to what molecular mechanisms It should be noted that the bacteriocyte-specific Ubx/Abd-A lo- underlie the development of insect’s bacteriocytes and bacter- calization was also observed in the aphid A. pisum (Sternor- iomes, which unveil the importance of developmental cooption of rhyncha:Aphididae) (15), which is phylogenetically distinct from

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1503371112 Matsuura et al. Downloaded by guest on September 30, 2021 EVOLUTION

Fig. 4. Strong RNAi phenotypes of Dll, Ubx, abd-A, and Antp in newborn nymphs of N. plebeius.(A–C) Control nymphs. (D–F) Dll-suppressed nymphs, whose appendages are severely truncated. (G–I) Ubx-suppressed nymphs, whose abdominal segment A1 exhibits thorax-like pigmentation (arrow) and grows appendage-like structures. (J–L) abd-A–suppressed nymphs, whose abdominal segments grow appendage-like structures on both sides. (M–O) Antp-suppressed nymphs, whose legs are deformed and swollen (circle). A, D, G, J, J′, and M are light-microscopic images. B, E, H, H′, K, K′, and N are scanning electron microscopic images. C, F, I, L, and O are fluorescence microscopic images in which blue and green signals indicate the host nuclei and the symbiotic bacteria, respectively. Asterisks show cuticle-derived autofluorescence. Filled arrowheads indicate the bacteriomes, and open arrowheads highlight the absence of the bacteriomes. ap, appendage-like structure; vm, vitelline membrane. (Scale bars: 100 μm.) For mild and moderate RNAi phenotypes, see Fig. S2.

N. plebeius (Heteroptera:Lygaeidae) in the Hemiptera. Consid- the common ancestor of the Hemiptera, lost in the ancestor of ering that the heteropterans without the bacteriocytes/bacter- the Heteroptera, and evolved again in the Lygaeoidea, to which iomes [with exceptions of bedbugs (Cimicidae) and some seed N. plebeius belongs (Fig. S3B) (14, 18, 31). Hence, the bacter- bugs (Lygaeoidea)] (18, 19, 28) constitute a monophyletic group iocytes/bacteriomes of N. plebeius may have evolved either via nested within the Hemiptera and all of the other hemipteran cooption of the homeobox transcription factor for inducing the groups possess the bacteriocytes/bacteriomes (Fig. S3A) (29, 30), novel cells for symbiosis or via revival of the developmental it is assumed that the bacteriocytes/bacteriomes were present in pathway for the bacteriocytes/bacteriomes that had existed in the ancestral hemipterans but been disrupted in the lineage of the heteropteran bugs. Whether the regain of the bacteriocytes/ bacteriomes in association with the localized Ubx expression in the stinkbug lineage is regarded as reversal, parallelism, or con- vergence is an intriguing issue in evolutionary developmental biology (32, 33), which should be addressed by comparative studies on molecular mechanisms underlying the development of symbiotic cells and organs in A. pisum, N. plebeius, and other hemipteran species. It is still an enigma what type of cells com- prises the developmental origin of the bacteriocytes. It is also of interest, but totally unknown, what mechanisms underlie the specific targeting of the bacterial symbiont to the newly formed bacteriocytes at the stage of germband retraction in N. plebeius (Figs. 1 K and L and 3 M and M′). Although Ubx starts specific expression in the presumptive bacteriocytes at this stage, it seems likely that not Ubx itself but some factor(s) downstream of Ubx Fig. 5. Schematic illustration of expression patterns of homeotic genes in must be responsible for the host–symbiont interplay, like flavo- the embryogenesis of N. plebeius. Note that we inspected the detailed ex- Rhizobium pression patterns of Ubx and abd-A only, and the expression patterns of the noids in the legume- nitrogen-fixing symbiosis (34) other genes are according to previous studies on a related lygaeid bug and chitin oligosaccharides in the squid–Vibrio luminescent O. fasciatus (24, 25). symbiosis (35, 36). These evolutionary and developmental issues

Matsuura et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 would be addressed by transcriptomics of the presumptive bac- labeled RNA probes, which were synthesized by T7 RNA polymerase (TaKaRa) teriocytes microdissected from embryos of N. plebeius or other and digoxigenin-11-UTP (Roche) using DNA templates amplified with primers hemipteran insects. listed in Table S4. The dissected embryos were fixed with 4% paraformaldehyde, stored in absolute methanol, permeabilized, and hybridized with the probes at Materials and Methods 61 °C. Antidigoxigenin antibody conjugated with alkaline phosphatase was used Insect Material. N. plebeius was maintained at 25 °C under a long-day regime for enzymatic visualization of the bound probes. (16-h light, 8-h dark) on sunflower seeds, whole wheat, and distilled water supplemented with 0.05% ascorbic acid. RNAi. Approximately 10 ng of dsRNA for each target gene was injected into each adult virgin female within a few days after eclosion. The eggs laid by the In Situ Hybridization of Symbiont. Whole-mount fluorescence in situ hybrid- injected mothers after 3 d and on were allowed to develop at 25 °C for 7–10 d ization targeting bacterial 16S rRNA was performed to visualize the symbiont until hatching. Some of the embryos were subjected to phenotypic observa- localization in embryos and whole insects of N. plebeius as described (18) tions, whereas other embryos were fixed and analyzed by scanning electron with a probe listed in Table S4. microscopy and in situ hybridization. See SI Materials and Methods for complete details on the materials Cloning of Homeobox Genes. Total RNA was extracted from embryos of and methods. N. plebeius, reverse-transcribed to yield cDNA, and subjected to PCR amplification of the homeobox genes with degenerate primers listed in Table S4.The PCR products were cloned and sequenced, and the complete or partial cDNA ACKNOWLEDGMENTS. We thank R. Futahashi, M. Moriyama, and T. Harumoto for technical advice; R. Koga, S. Koshikawa, and M. Matsunami for comments sequences were determined by rapid amplification cDNA end methods. on the manuscript; and C. Ueda for embryo illustrations. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant In Situ Hybridization of Host’s Homeobox Genes. Whole-mount in situ hybrid- 25221107 (to T.F.). Y.M. was supported by the JSPS Fellowship for Young ization of the homeobox gene transcripts was performed with digoxigenin- Scientists.

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