The Expression of the Proximodistal Axis Patterning Genes

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Developmental Biology 260 (2003) 97Ð112 www.elsevier.com/locate/ydbio

The expression of the proximodistal axis patterning genes Distal-less and dachshund in the appendages of Glomeris marginata (Myriapoda: Diplopoda) suggests a special role of these genes in patterning the head appendages

Nikola-Michael Prpic and Diethard Tautz*

Institut fu¨r Genetik, Universita¨t zu Ko¨ln, Weyertal 121, 50931 Ko¨ln, Germany Received for publication 23 January 2003, revised 1 April 2003, accepted 1 April 2003

Abstract The genes Distal-less, dachshund, extradenticle, and homothorax have been shown in Drosophila to be among the earliest genes that define positional values along the proximalÐdistal (PD) axis of the developing legs. In order to study PD axis formation in the appendages of the pill millipede Glomeris marginata, we have isolated homologues of these four genes and have studied their expression patterns. In the trunk legs, there are several differences to Drosophila, but the patterns are nevertheless compatible with a conserved role in defining positional values along the PD axis. However, their role in the head appendages is apparently more complex. Distal-less in the mandible and maxilla is expressed in the forming sensory organs and, thus, does not seem to be involved in PD axis patterning. We could not identify in the mouthparts components that are homologous to the distal parts of the trunk legs and antennnae. Interestingly, there is also a transient premorphogenetic expression of Distal-less in the second antennal and second maxillary segment, although no appendages are eventually formed in these segments. The dachshund gene is apparently involved both in PD patterning as well as in sensory organ development in the antenna, maxilla, and mandible. Strong dachshund expression is specifically correlated with the tooth-like part of the mandible, a feature that is shared with other mandibulate arthropods. homothorax is expressed in the proximal and medial parts of the legs, while extradenticle RNA is only seen in the proximal region. This overlap of expression corresponds to the functional overlap between extradenticle and homothorax in Drosophila. © 2003 Elsevier Science (USA). All rights reserved.

Keywords: Myriapods; Distal-less; dachshund; extradenticle; homothorax; Sensory organs; Gnathobasic mouthparts; Appendage development

Introduction provided by the AP, DV, and LR axes of the body itself (e.g., Cohen, 1990; Goto and Hayashi, 1997), but the out- Axis formation is one of the fundamental processes dur- growth away from the body requires an additional guidance ing embryogenesis of most multicellular animals. The body system along an axis from the tip of the growing appendage of higher metazoans is patterned along three axes, namely to its root on the body. the anteriorÐposterior (AP) axis, dorsalÐventral (DV) axis, In the fruitfly Drosophila genes have been identified that and leftÐright (LR) axis. Within this framework, many or- are involved in establishing the PD axis of the appendages. gans develop along additional, organ-specific axes. A bet- It has been shown that especially four genes set up the first ter-known example of the latter is the proximalÐdistal (PD) crude positional values on the PD axis of the fly legs. The axis of the appendages in arthropods. In general, develop- genes extradenticle (exd) and homothorax (hth) are cofac- ment of the appendages is guided by the coordinate system tors which together instruct proximal leg fates (Gonzalez- Crespo and Morata, 1996; Rieckhof et al., 1997; Abu-Shaar * Corresponding author. Fax: ϩ49-221-470-5975. et al., 1999; Wu and Cohen, 1999), whereas the gene Distal- E-mail address: [email protected] (D. Tautz). less (Dll) is indispensable for the development of distal leg

0012-1606/03/$ Ð see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0012-1606(03)00217-3 98 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 parts (Sunkel and Whittle, 1987; Cohen et al., 1989). In sensory organs. This does not support a role in PD axis between these two domains is a third domain, governed by patterning in the mouthparts, but instead suggests involve- the gene dachshund (dac), which is required for the devel- ment in sensory organ development. Gm-dac is also ex- opment of medial leg parts (Mardon et al., 1994; Dong et pressed in sensory organ primordia in antennae and mouth- al., 2001). Apart from their function during PD axis forma- parts, but also at a medial PD position in the antenna and tion, all four genes have other functions as well. The genes probably is important for the specific tooth-like morphology exd and hth are expressed in most cells of the Drosophila of the mandibles, not only of myriapods, but also of other embryo and are involved in wing, eye, and salivary gland mandibulate arthropods. development, body tagmosis and segmentation, develop- The possibility that some PD axis patterning genes have ment of the peripheral and central nervous systems (PNS, a complex role during appendage development also has CNS), and several other developmental processes (Raus- ramifications for the use of these genes in evolutionary kolb et al., 1993, 1995; Rieckhof et al., 1997; Pai et al., developmental comparisons. The expression of the Dll gene 1998; Kurant et al., 1998; Henderson and Andrew, 2000; has been studied in a variety of arthropod species (e.g., Casares and Mann, 2000; Azpiazu and Morata, 2000; Popadic et al., 1998; Scholtz et al., 1998; Williams, 1998; Pichaud and Casares, 2000; Nagao et al., 2000; Bessa et al., Abzhanov and Kaufman, 2000), employing a cross-reacting 2002). The dac gene is involved in eye development, de- antibody (Panganiban et al., 1995). However, these studies velopment of the corpora pedunculata (“mushroom bod- have not all taken into account the possibility of additional ies”), and the development of the antennal lobe of the brain functions of Dll. We have therefore readressed this partic- (Shen and Mardon, 1997; Chen et al., 1997; Kurusu et al., ular possibility here and find indeed that some of the ho- 2000; Martini et al., 2000; Noveen et al., 2000; Jhaveri et mology assessments have to be revised. al., 2000). Furthermore, it is expressed in the ventral nerve cord and in the wing discs, but its function in these structures remains to be determined (Mardon et al., 1994). Fi- Materials and methods nally, Dll is involved in the development of specific cephalic sensory organs and thoracic mechanosensors Animals and embryo fixation (Keilin’s organs) (Sunkel and Whittle, 1987; Cohen and Ju¬rgens, 1989a). It is also expressed in the CNS (optic lobe, Glomeris marginata were collected in the city forest of glial cells of the ventral nerve cord) and the wing disc, but Cologne, Germany, kept in large petri dishes, and supplied its functions there are unclear (Gorfinkiel et al., 1997; regularly with earth, water, and food (decomposing beech Campbell and Tomlinson, 1998; Panganiban, 2000; Panga- leaves). Eggs were removed from the earth covers by hand niban and Rubenstein, 2002). Thus, for all four genes, PD and dechorionized with DanKlorix (Colgate-Palmolive; axis patterning is not the only function during development. equals a 4% sodium hypochloride solution) for 2 min. Eggs This opens up the possibility that these genes could have were then washed several times in water and fixed [1 ml additional functions also in the appendages apart of PD axis heptane, 100 ␮l formaldehyde (37%)] for up to4hatroom patterning. In fact, it is known that Dll function in the temperature and washed in methanol several times. appendages of Drosophila is not restricted to PD axis pat- Vitelline membranes were removed by hand using watch- terning alone. Dll also determines the sensory organs asso- maker forceps (Dumont 5). Embryos were stored in meth- ciated with the rudiments of labrum, antennae, maxillae, anol at Ϫ20¡C. labium, and thoracic legs in the larva (Sunkel and Whittle, 1987; Cohen and Ju¬rgens, 1989a). In addition, it has re- In-situ hybridisation cently been shown that Dll expression is associated with sensory bristles in the appendages in crustaceans, aptery- Treatment of the embryos followed the protocol by Tautz gote insects, and chelicerates (Mittmann and Scholtz, 2001; and Pfeifle (1989), using labeled riboprobes (Klingler and Williams et al., 2002). Thus, the function of the PD axis Gergen, 1993). All incubation/washing steps were pro- patterning genes in the appendages appears to be more longed, because of the bigger size of the Glomeris embryos. complex than previously thought, at least in the case of Dll. To reduce background, embryos were additionally treated Here, we report the isolation of the genes hth, exd, dac, with 2.5 ␮l acetic anhydride in 1 ml of 0.1 M TEA buffer and Dll from the diplopod Glomeris marginata (Gm), a (Sigma). A detailed step-by-step protocol is available upon representative of the myriapods. The myriapods are the only request. arthropod group from which data on these genes were hith- erto almost entirely missing. We show that the temporal and Cloning of cDNA fragments spatial expression patterns in the trunk legs are different from Drosophila, but are consistent with a conserved role in Total RNA from 60 selected G. marginata embryos was PD axis formation. In the head appendages, however, their extracted by using Trizol (Invitrogen). Messenger RNA was role appears more complex. It is demonstrated that Gm-Dll extracted by using the PolyATtract system (Promega) and in the mouthparts is expressed only in the primordia of the used to synthesize cDNA using the SuperScript II system N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 99

(Invitrogen). To amplify a fragment of the Gm-Dll ho- Gm-hth). All clones of each gene contained identical se- meobox, the primers eDP fw (GGN AAR GGN AAR AAR quences, suggesting that no paralogous genes are present in ATN MG) and eDP bw (TTY TGR AAC CAD ATY TTN the Glomeris genome. The fragment of Gm-dac is 940 bp AC) were used. A nested PCR was performed by using the long and encodes a partial protein with high similarity to primers iDP fw (ATN MGN AAR CCN MGN ACN ATH DAC proteins from other arthropods. The Gm-DAC se- TA) and iDP bw (AAC CAD ATY TTN ACY TGN GTY quence contains parts of the DD1 and DD2 domains that are TG). The resulting fragments were subjected to standard conserved in DAC/DACH proteins in nematodes, arthro- molecular cloning and sequenced on an ABI 377 automated pods, and vertebrates (Hammond et al., 1998; Kozmik et al., sequencer (Perkin Elmer). Using this sequence, species- 1999; Davis et al., 1999; Caubit et al., 1999). specific primers were designed. In a primary PCR, the The fragment of Gm-exd is 529 bp long and encodes a primer eGSP Gm (CGT GAG ACC CAA AGA AGC TGC partial protein with very high similarity to EXD proteins C) was used together with the degenerate primer DP dlxm1 from other arthropods. It contains a part of the PBC-A (AAR WSN GCN TTY ATN GAR HTN CAR CAR C) domain, a PBC-B domain, and a partial homeodomain. The directed against the DLX-1 motif (Aspo¬ck and Bu¬rglin, amino acid conservation is very high within the PBC-A and 2001). A nested PCR using the primers iGSP Gm (TTC PBC-B domains with 96, 89 and 70% sequence identity to AGC ACG CTC TGG TAG AGC C) and DP dlxm1 spe- Drosophila EXD, mouse PBX1, and nematode CEH-20, cifically amplified Gm-Dll cDNA fragments. respectively. The fragment of Gm-hth is 817 bp long and The primers used for amplification of Gm-dac have been encodes a partial protein with very high similarity to HTH described before (Prpic et al., 2001). For Gm-exd, the prim- proteins from other arthropods. It contains a partial MEIS ers were exd-fw1 (YTN AAY TGY CAY MGN ATG AAR domain and a partial homeodomain. Amino acid sequence CC) and exd-bw1 (TTN CCR AAC CAR TTN SWN ACY conservation within the MEIS domain ranges from 95, 91, TG). In a nested PCR, the primers exd-fw2 (GTN YTN and 63% sequence identity to Drosophila EXD, mouse TGY GAR ATH AAR GAR AAR AC) and exd-bw2 (GCN MEIS1, or nematode UNC-62, respectively. The MEIS and ARY TCY TCY TTN GCY TCY TC) were used. For PBC domains mediate the heterodimerization of the HTH/ Gm-hth, the primers hth-fw1 (GAY AAR GAY GCN ATH MEIS and EXD/PBX proteins, which is necessary for their TAY GRN CAY CC) and hth-bw1 (YTG RTC DAT CAT joint translocation to the nucleus where they bind target NGG YTG NAC DAT) were used in the initial PCR, hth- DNA sequences (e.g., Abu-Shaar et al., 1999; Berthelsen et fw1 and hth-bw2 (GC RTT DAT RAA CCA RTT RTT al., 1999; Jaw et al., 2000). The high degree of amino acid NAC YTG) were used in a nested PCR. GenBank Acces- sequence conservation suggests that, also in Glomeris, the sion nos. are as follows: Gm-Dll (AJ551276), Gm-dac Gm-HTH and Gm-EXD proteins form heterodimers before (AJ551277), Gm-exd (AJ551278), Gm-hth (AJ551279). they are able to enter the nucleus.

Correlation between embryonic and adult appendages in Results Glomeris marginata

Cloning and sequence analysis We have studied the expression of these genes in several embryonic structures, focusing on the developing append- A PCR strategy combining degenerate and gene-speciﬁc ages of the head and trunk. Therefore, we begin with a brief primers was used to amplify sequences homologous to Dll/ description of the correlation of embryonic appendages with Dlx genes. Eleven identical clones with similarity to Dro- the appendages in adult G. marginata in order to introduce sophila Dll were recovered. We designate the gene from the speciﬁc terms for myriapod appendage morphology. which these cDNA fragments derive as Gm-Dll. The align- The trunk legs are simple outgrowths of the body and, thus, ment of the deduced amino acid sequence of Gm-DLL and the embryonic leg appears to correlate directly to the adult all other known arthropod DLL homologues is shown in leg (Fig. 2A). The same is true for the antennae (Fig. 2D). Fig. 1. Apart from the homeodomain, there are only short In the adult, this appendage has four sensory organs at its stretches of sequence which are conserved to some extent in tip, the primordia of which can already be discerned in the all species. The Glomeris sequence shows an insertion of an embryo (Fig. 2D; sc). The mandible in the embryo is a stout alanin and histidin stretch directly following the DLXM1 structure with two lobes (Fig. 2C; il, ol). These two lobes motif. The insertion before the DLXM2 motif that is seen in differentiate into the gnathal parts of the mandibles (Dohle, the spider Cupiennius salei (Schoppmeier and Damen, 1964) (Fig. 2C; lower part): the inner lobe will give rise to 2001) is not present in Glomeris. The amino acid sequence the pectinate lamella, intermediate piece, and molar plate. of the homeodomain part is fully conserved between the The outer lobe will give rise to both the external and the different species. internal part of the tooth. Finally, the lower lip in adult The same strategy was applied to clone Gm-dac, Gm- Glomeris, the gnathochilarium, is a complex structure. It exd, and Gm-hth. For each gene, a number of clones were clearly develops from the appendages of the maxillary seg- sequenced (13 for Gm-dac, 16 for Gm-exd, and 29 for ment (Fig. 2B; upper part). Already at late embryonic stages 100 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112

Fig. 1. Alignment of all known arthropod DLL sequences. Dashes are gaps introduced to improve the alignment. Bars below the alignment denote the conserved regions (DLXM1, DLXM2; Aspo¬ck and Bu¬rglin, 2001). Bars on top denote newly identified variable regions. Sequence underlaid gray: partial homeodomain. Sequence underlaid black: nonhomologous homopolymeric amino acid stretches. Question marks replace primer sequences. GenBank Accession nos. are as follows: BaDLL (AAL69325), JcDLL (AAK97630), TcDLL (AAG39634), DmDLL (AAB24059), CsDLL (CAC34380), GmDLL (CAD82905). Abbreviations: HPAAS, homopolymeric amino acid stretch; Ba, Bicyclus anynana (butterfly); Jc, Junonia coenia (butterfly); Tc, Tribolium castaneum (beetle); Dm, Drosophila melanogaster (fruitfly); Gm, Glomeris marginata (milliped); Cs, Cupiennius salei (spider). Fig. 2. Schematic representation of embryonic and adult appendages of G. marginata. (A) Stage 6 embryonic (top) and adult (bottom) trunk leg. (B) Ventral view of the maxillary segment (light gray) with the developing maxillae. At stage 4, the maxillae are still separate (top). At stage 6, the maxillae have approached each other and the internal tissue contributing to the intermaxillary plate (black) has already fused (middle figure). In the adult, the maxillae form the lower lip (gnathochilarium) (black, intermaxillary plate; dark gray, stipes; medium gray, cardo). (C) Stage 6 embryonic (top) and adult (bottom) mandible. Corresponding tissues have the same shade of gray/black. (D) Stage 6 embryonic (top) and adult (bottom) antenna. Abbreviations: li, lobi interiori; sp, sensory palps; il, inner lobe; ol, outer lobe; ch, cheek; mp, molar plate; ip, intermediate piece; pl, pectinate lamella; i/ot, inner/outer; sc, sensory cones. N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 101

Fig. 3. Expression of Gm-Dll in Glomeris embryos. (A) Embryo at stage 1. (B) Higher magniﬁcation of the head segments of the embryo in (A), showing the expression of Gm-Dll in the premandibular segment (asterisk). (C) Stage 2 embryo. (D) Head of a stage 2 embryo. Expression in the premandibular segment is vanishing (arrows). (E) Stage 3 embryo. (F) Stage 4 embryo. (G) Stage 5 embryo. The inset demonstrates the natural position of the gnathal appendages: antenna (top), mandible (middle), and maxilla (bottom). Compare to the preparations in Fig. 4. (H) Early stage 6 embryo. (I) Stage 6 embryo. The animals in (AÐG) are shown in ventral aspect. The embryos in (H) and (I) are lateral views. Anterior is toward the top in all panels, except for (D), where anterior is to the left. Abbreviations: ac, anterior cap; ant, antenna; av, anal valves; lbr, labrum; pmx, postmaxillary segment. Trunk segments are indicated by 1Ð8.

(stage 6; for staging see Dohle, 1964), the two maxillae are lary plate in the adult gnathochilarium (Fig. 2B; lower part, fused along their innermost margins (Fig. 2B; middle part). black filling). Both the external parts of the gnathochilarium This fused part will contribute to the so-called intermaxil- (stipes; Fig. 2B; dark gray filling) and the intermaxillary 102 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 plate bear sensory organs (Fig. 2B; li, sp). The premandibu- also detected in other structures. Throughout development, lar segment, which is homologous to the intercalary seg- expression is seen in the labrum and the developing anal ment in insects, and the postmaxillary segment, which is valves (Fig. 3; lbr and av, respectively). Expression in the homologous to the labial segment in insects, do not develop CNS is restricted to the brain. At stage 1, diffuse staining appendages. Dohle (1964) found no evidence for a contri- fills the entire anterior part of the germ band (anterior cap; bution of remnants of possible postmaxillary appendages to Fig. 3A), similar to what has been reported for Drosophila the gnathochilarium, as it is sometimes claimed (Kraus, (Kumar and Moses, 2001). At stage 2, anterior staining is 2001). This is confirmed by our results described below. reduced to several spots in the head lobes (Fig. 3D). These spots persist through stages 3 and 4 (Fig. 3E and F). The pattern increases in complexity at stage 5 (Fig. 3G) and Gm-Dll expression dissolves into a rather diffuse staining at stage 6 (Fig. 3H and I). The position of the spots is consistent with the Gm-Dll transcripts are detected in all segments of the expected location of the optic lobes and the corpora pedun- head and trunk. At stage 1 (for staging see Dohle, 1964), culata, respectively. However, further studies are necessary mRNA expression is seen in segmental pairs of spots in the to corroborate the function of Gm-Dll in the CNS. antennal, premandibular, mandibular, maxillary, and the first three trunk segments (Fig. 3A and B). Except for the premandibular segment, which does not develop append- Dynamic expression of Gm-Dll in the mouthparts ages, this segmental expression prefigures the sites of appendage formation in later stages of development. It is The expression pattern of Gm-Dll is dynamic and therefore referred to as premorphogenetic expression. No changes with the developmental stage (Fig. 4). This is most signal is detected in the postmaxillary segment at this stage. prominent in the gnathal appendages. In the maxillary seg- At stage 2, however, expression of Gm-Dll is detected also ment, premorphogenetic Gm-Dll expression fades as soon in this segment at a position that is apparently serially as the maxillary limb buds start to form (stage 3; Fig. 3E) homologous to the remaining segmental spots (Fig. 3C; and is replaced at stage 4 by expression in the primordia of pmx). In the premandibular segment, the signal has van- the two maxillary sensory palps (Fig. 4D; arrows). Subse- ished at this point (Fig. 3D; arrows) and a small spot of quently, the maxillae are displaced toward the ventral mid- expression has appeared in the fourth trunk segment (Fig. line. This, at stage 5, results in the close proximity of the 3C). Expression in trunk segment 1Ð3 at this stage extends two maxillary appendages. Expression of Gm-Dll is now dorsally. This changes as soon as limb buds appear on these seen also in the primordium of the lobus interior (Fig. 4E; segments at stage 3. Now expression is restricted to the arrow). At stage 6, the maxillae have started to fuse to form forming buds and does not extend toward the dorsal side the gnathochilarium (lower lip) and have to be torn apart for anymore (Fig. 3E). Expression in the postmaxillary segment preparation (Fig. 4F; arrowhead). The lateral (outer) part of meanwhile has ceased. Coincident with the appearance of each half of the primordial gnathochilarium will give rise to limb buds on the mandibular and maxillary segment expres- cardo and stipes (see Fig. 2B). The primordia of the two sion strength of Gm-Dll decreases in these segments. sensory palps of the stipes still express Gm-Dll (Fig. 4F). At stage 4 (Fig. 3F), the homogeneous mRNA expres- The middle (inner) part of each half of the primordial sion in the buds of the mandibles and maxillae has been gnathochilarium will contribute to the intermaxillary plate. replaced by a more complex pattern, which will develop The primordia of the lobi interiori on the developing inter- further complexity at stages 5 and 6 (see below). In the maxillary plate express Gm-Dll (Fig. 4F). trunk, expression marks primordial and developing trunk In the mandibles, the strong premorphogenetic expres- legs (Fig. 3F and G). Clearly, the legs on the first three trunk sion at stage 2 (Fig. 3C) decreases at stage 3 with the onset segments develop simultaneously and ahead of the remain- of the formation of mandibular buds (Fig. 3E) and at stage ing legs, which develop in a regular temporal sequence. 4 is replaced by expression in three small clusters of cells This is most obvious at stage 6 when the first three trunk (Fig. 4G). The innermost two clusters are within the inner legs have grown considerably, but the other legs are still at lobe, whereas the lateral cluster is in the outer lobe. At stage the limb bud stage—the oldest on trunk segment 4, the 5, expression in the outer lobe increases and in the inner youngest on trunk segment 8 (Fig. 3H and I). lobe three clusters of cells express Gm-Dll (Fig. 4H). At Apart from the segmental expression, Gm-Dll mRNA is stage 6, expression is weaker and diffuse. Prominent ex-

Fig. 4. Expression of Gm-Dll in the developing appendages of Glomeris. (AÐC) Expression in the trunk leg (trunk segment 1) at stage 5, stage 6, and late stage 6, respectively. At late stage 6, expression is heterogeneous (stronger expression is marked with arrowheads in C). (DÐF) Expression in the maxilla at stage 4 (arrows: primordia of the sensory palps), stage 5 (arrow: primordium of the lobus interior), and stage 6, respectively. Note that at stage 6 (F) the maxillae have fused via the tissue of the future intermaxillary plate and have to be torn apart for preparation. This causes the rupture of tissue at the inner margin (arrowhead). (GÐI) Expression in the mandible at stage 4, stage 5, and stage 6, respectively. (J, K) Expression in the antenna at stage 5 and stage 6, respectively. Abbreviations: max, maxilla; mdb, mandible; ant, antenna. Stages are indicated by “st” followed by a number. N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 103 104 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 pression is seen in two fuzzy stripes, one in the outer lobe premorphogenetic signal is detected in nonappendage bear- and one in the inner lobe (Fig. 4I). ing head segments or any of the trunk segments. In the legs In the antenna and trunk legs 1Ð3, expression of Gm-Dll on trunk segments 1Ð3, Gm-dac is first expressed at stage 3 is found in the distal portion of the appendages. Expression when the limb buds start to form (Fig. 5E). At this stage, in the antenna is very strong (Fig. 4J) but decreases at stage expression encircles the leg buds slightly above the limb 6 (Fig. 4K). Expression in the trunk legs is also strong (Fig. base. At stage 4, Gm-dac is clearly confined to medial parts 4A) but is getting slightly heterogeneous at stage 6 (Fig. of the PD axis of the legs. However, at stage 5, expression 4B). In embryos shortly before the secretion of the embry- starts to spread proximally (Fig. 6A) and at stage 6 Gm-dac onic cuticle (late stage 6, the upper age limit accessible for is also expressed in the proximal leg, albeit visibly weaker in situ hybridization), expression of Gm-Dll has decreased than in the medial leg (Fig. 6E). Additionally, a small in terminal and medial cells but is still strong in supraproxi- cluster of dorsal PNS cells near the leg tips expresses mal and subterminal cells (Fig. 4C; arrowheads). Gm-dac at stage 6 (Fig. 6E; arrowhead). In the antenna, Gm-dac is also expressed in a ring at a medial level on the PD axis. At first, this ring is narrow Gm-dac expression (stage 3) (Fig. 5F), but soon broadens (stage 4) and even starts to separate into two incompletely separated rings Expression of Gm-dac is seen in a number of different (stages 5 and 6) (Fig. 6D and H). In contrast to the trunk organ primordia. The most prominent expression occurs in legs, proximal parts of the antenna always remain free from the central nervous system, which is also highly dynamic. Gm-dac expression. Similar to the trunk legs, Gm-dac is The earliest CNS expression is in the brain (stage 2) in a expressed in the PNS in the antenna. Starting at the end of spot roughly corresponding to the prospective visual centers stage 3, prominent expression of Gm-dac is found in the (Fig. 5A; oc). Expression in the brain rapidly becomes more primordia of the four sensory cones on the tip of the anten- complex (Fig. 5E), and at later stages, many proto- and nae (Fig. 6D and H; arrows). deutocerebral cells express Gm-dac (Fig. 5H and I). In the In contrast to the trunk legs and the antennae, in the ventral nerve cord, expression starts in the mandibular and mouthparts, Gm-dac is never expressed in a fashion that premandibular segment at early stage 3 (Fig. 5C and D), but would suggest a role in the definition of medial fates on soon thereafter is initiated also in the remaining head and their PD axis. At stage 3, expression is strong in the buds of first three trunk segments (Fig. 5E), with the exception of mandibles and maxillae (Fig. 5F). At stage 4, this homoge- the postmaxillary segment, in which expression is delayed neous expression is replaced by a more complex pattern in until stage 4 (Fig. 5G). Each of the trunk segments as it both appendages. In the maxilla, Gm-dac is expressed matures goes through the same temporalÐspatial sequence strongly but diffusely in the primordial stipes, and a separate of Gm-dac activation in a steadily increasing number of expression domain is present in the tissue that will contrib- neuroectodermal cells, that is reminiscent of the discrete ute to the intermaxillary plate (not shown). At stage 5, the pulses of neuroblast formation in Drosophila (Goodman expression in the stipes becomes slightly bipartite (Fig. 6B) and Doe, 1993). However, the nature of these neuroecto- and at stage 6 the entire expression pattern resolves into dermal cells is not directly comparable to neuroblasts (Dove several smaller clusters of cells within the primordia of the and Stollewerk, 2003). sensory palps of the stipes and the lobi interiori of the Prominent expression is also seen in the anal valves (Fig. intermaxillary plate (Fig. 6F). In the mandibles, expression 5A; av) and, in later stages, extending from there along the at stages 4 and 5 is very strong in the outer lobe. In the inner lateral edges of the posterior zone (Fig. 5E). This expression lobe, several smaller clusters of cells show expression (Fig. may be correlated with the formation of the proctodeal part 6C). The strong expression in the outer lobe persists through of the digestive system. Weaker expression is seen laterally stages 5 and 6, while expression in the inner lobe becomes in the postmaxillary segment and all trunk segments as soon increasingly more diffuse toward stage 6 (Fig. 6G). as they are separated from the posterior zone (Fig. 5C). At stage 6, however, this lateral expression is restricted to the Gm-exd and Gm-hth expression primordium of the heart (Fig. 5J). Finally, expression is seen also in the labrum with increasing intensity from stage 3 to Both Gm-exd and Gm-hth are expressed in most cells of 6 (Fig. 5D; lbr). the germ band (Fig. 7). In general, judging from the in situ signal, Gm-exd seems to be expressed significantly weaker Differential Gm-dac expression in the trunk legs and than Gm-hth; however, no quantitative comparative mea- mouthparts surements have been performed. Gm-exd is expressed only weakly in many areas of the developing brain (Fig. 7BÐF), Similar to Gm-Dll, premorphogenetic expression prefig- whereas Gm-hth is expressed strongly in most cells of the uring the location of the appendage buds is also observed proto- and deutocerebrum (Fig. 7HÐL). Gm-hth is strongly for Gm-dac. However, this only applies to the antennal, expressed in the lateral plates of the segments (Fig. 7J and mandibular, and maxillary segment (Fig. 5A and B). No K), while expression of Gm-exd in the lateral plates is weak N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 105

Fig. 5. Expression of Gm-dac in Glomeris embryos. (A) Stage 2 embryo. (B) Embryo at later stage 2. (C D) Embryo at early stage 3. Only the head is shown in (D). The asterisk denotes beginning expression in the neuroectoderm of the premandibular and mandibular segment. (E) Stage 3 embryo. Arrowheads point to the staining in the primordium of the heart at the lateral edges of the germ band. (F) Embryo at late stage 3. (G, H) Embryo at stage 4. Only the head is shown in (H). (I) Stage 5 embryo. (J) Stage 6 embryo. Anterior is to the top in all panels, except for (D) and (H) where anterior is to the left. All embryos are shown in ventral view, except for the animals in (F) and (J) which are in lateral aspect. Abbreviations: oc, primordia of the ocular lobe; mdb, mandible; max, maxilla. For further abbreviations, see Fig. 3. during early stages (Fig. 7BÐD) and absent towards stage 6 Dll, dac, and hth/exd in the fruitﬂy set up the ﬁrst crude (Fig. 7F). Expression of Gm-exd appears evenly distributed, positional values along the PD axis of the legs: distal, whereas expression strength of Gm-hth is clearly heteroge- medial, and proximal, respectively (Abu-Shaar and Mann, neous, indicating tight spatial regulation of the expression 1998; Dong et al., 2001). Although the expression of the of this gene. homologous Glomeris genes differs from the Drosophila In the trunk legs, expression of Gm-exd is restricted to patterns in several aspects, they are expressed in a similar proximal cells (Fig. 8A), whereas Cs-hth is expressed in proximal-to-distal order, which suggests that their function proximal and medial cells (Fig. 8E). The tip of the legs is of subdividing the developing leg into three distinct units is free from expression in both cases. In the antenna, expres- conserved. In the head appendages, however, their function sion of Gm-hth appears uniformly distributed in the entire is apparently more complex. The Gm-dac gene is expressed appendage (Fig. 8H). In contrast, Gm-exd in the antenna is in two distinct domains in the antenna: at a medial level on expressed in a more complex pattern consisting of high the PD axis and in the sensory cones, suggestive of a role in expression in proximal cells followed distally by two nar- PD axis patterning as well as sensory organ development. row rings of low and high expression, respectively (Figs. 7E The strong expression of Gm-dac in the part of the mandible and 8D). Cells at the tip of the antenna do not express that will develop a tooth-like morphology suggests a third Gm-exd. In the mouthparts, expression of Gm-hth and Gm- role for Gm-dac, that of specifying mandible identity. In the exd is ubiquitous, but nonhomogeneous (Figs. 7E, 8B and mouthparts, expression of Gm-Dll is correlated with form- C, and 8F and G). ing sensory organs, but not with distal elements.

Premorphogenetic expression of Gm-Dll is independent of Discussion later appendage development

In order to study proximodistal axis formation in the In Drosophila embryos, Dll preﬁgures the sites where the appendages of the pill millipede G. marginata (Myriapoda: imaginal disc primordia will arise (Cohen et al., 1991, Diplopoda), we have isolated homologues of four genes 1993). Similar to the situation in Drosophila, also in Glom- known from Drosophila to be most relevant in this process. eris, expression of Gm-Dll preﬁgures the sites where head 106 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112

Fig. 6. Expression of Gm-dac in the developing appendages of Glomeris.(AÐD) Expression at stage 5 in first trunk leg (A), maxilla (B), mandible (C), and antenna (D). The inset in (D) shows an antenna’s tip (cut away and viewed from above) in order to demonstrate expression in all four sensory cones. (EÐH) Expression at stage 6 in first trunk leg (E), maxilla (F), mandible (G), and antenna (H). The arrowhead in (E) points to a cluster of PNS cells expressing Gm-dac. Arrows in (D) and (H)indicate expression in the primordia of the antennal sensory cones. The asterisk in (B, D, F, G and H) denotes neuroectodermal tissue (not part of the appendages). For abbreviations, see Fig. 4. and trunk appendages will form. However, in contrast to difference between myriapods and insects. However, be- Drosophila, we also detect expression of Gm-Dll in seg- cause Gm-Dll expression in the premandibulary and post- ments which do not develop appendages, namely in the maxillary segment is only transient and short-lived, it may premandibular and the postmaxillary segment. The location be a nonfunctional rudiment of an ancestral state. of the Gm-Dll expression in these segments nevertheless Gm-dac shows also premorphogenetic expression, but suggests that they are serially homologous to those in the only in the antennal, mandibular, and maxillary segment. appendage-bearing segments. This demonstrates that pre- Premorphogenetic expression of dac has not been found in morphogenetic Gm-Dll expression is maintained irrespec- other arthropods so far. Rather, reports from Drosophila, tive of whether a segment will develop appendages or not. Tribolium, and Cupiennius (Abu-Shaar and Mann, 1998; Given that there is no premorphogenetic expression of Dll Prpic et al., 2001; unpublished observations) had suggested in the nonappendage-bearing segments in either Drosophila that the temporal sequence of dac activation following after or the more basal insect Tribolium (Beermann et al., 2001), the activation of Dll is a conserved feature in the arthropods. premorphogenetic expression may represent a significant In fact, this temporal sequence is also found in the trunk legs N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 107

Fig. 7. Expression of Gm-exd and Gm-hth in Glomeris embryos. (AÐF) Expression of Gm-exd in embryos at stage 1 (A), stage 3 (B), early stage 4 (C), early stage 5 (D), stage 5 (E), and late stage 5 (F). (GÐL) Expression of Gm-hth in embryos at stage 2 (G), stage 3 (H), stage 4 (I), stage 5 (J), early stage 6 (K), and stage 6 (L). Arrowheads in (J) point to staining in the lateral plates of the segments. All embryos are shown from the ventral side and anterior to the top, except for (E), (F), and (L), which are lateral aspects. For abbreviations, see Figs. 3 and 5. of Glomeris. The fact that in antennae, mandibles, and quently, a third unit is introduced between these ﬁrst two, maxillae this temporal sequence is broken and Gm-Dll and which is determined by the activity of the dac gene (Mardon Gm-dac are activated more or less simultaneously even et al., 1994; Dong et al., 2001). Thus, the leg at this stage before limb buds are formed suggests that their regulation in comprises three distinct and antagonistic units, which form these appendages may be different from the regulation in the basis for the subsequent subdivision of the PD axis into the trunk legs. ﬁner positional values. In Glomeris, the expression patterns in the trunk legs are Proximodistal patterning functions in the trunk legs compatible with conserved functions in PD axis patterning. resemble those in other arthropods Expression of Gm-Dll in a domain comprising the entire distal leg is consistent with a function of this gene in Results from Drosophila indicate that the early leg is determining distal leg structures. Expression of Gm-dac in a subdivided into two fundamental compartment-like units, medial domain is consistent with a function of Gm-dac as governed by two independent genetic pathways. The prox- instructor of medial leg fates. The genes hth and exd encode imal unit expresses both hth and exd, whereas the distal unit cofactors and thus have to be regarded together. In Dro- expresses Dll (Gonzalez-Crespo and Morata, 1996; Abu- sophila, EXD is expressed in the entire leg, but exd function Shaar and Mann, 1998; Wu and Cohen, 1999). Subse- is restricted to those parts of the legs where the protein can 108 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112

Fig. 8. Expression of Gm-exd and Gm-hth in the developing appendages of Glomeris. (AÐD) Expression of Gm-exd in stage 6 embryos in first trunk leg (A), maxilla (B), mandible (C), and antenna (D). Arrow and arrowhead in (D) point to the two rings of weak and strong Gm-exd expression, respectively, that follow distal to the proximal expression domain of Gm-exd in the antenna. (EÐH) Expression of Gm-hth in stage 6 embryos in first trunk leg (E), maxilla (F), mandible (G), and antenna (H). For abbreviations, see Fig. 4. bind its cofactor HTH, i.e., the proximal leg parts where the of the leg gap genes have been found in the woodlouse hth gene is active (Rauskolb et al., 1995; Rieckhof et al., Porcellio scaber (Abzhanov and Kaufman, 2000) and the 1997). In Glomeris, expression of Gm-exd and Gm-hth is spider Cupiennius salei (unpublished observations). How- very different from Drosophila, but coexpression is also ever, the expression dynamics in the woodlouse and the restricted to the proximal leg. Thus, the PD sequence of spider are different from each other and those in Drosoph- exd/hth, dac, and Dll expression is similar to Drosophila ila. Assuming a conserved relevance of the dynamics for the and suggests that also in Glomeris the developing leg is number and positioning of the leg joints, the differences separated into three units that are governed by the activities observed between Porcellio, Cupiennius, and Drosophila of hth/exd, dac, and Dll, respectively. could explain the different segment composition of their In addition to their role in establishing distinct develop- legs (Drosophila: six primary leg segments plus the tarsal mental domains along the PD axis of the Drosophila leg, segments; Porcellio and Cupiennius: seven leg segments). hth, dac, and Dll, also collectively termed “leg gap genes,” The present study shows dynamic expression profiles for the have been shown to be also involved in leg segmentation leg gap genes also in the myriapod Glomeris. The dynamics (Rauskolb and Irvine, 1999; Rauskolb, 2001). Their expres- in Glomeris are different from those in Drosophila and sion profiles are dynamic and these dynamics are responsi- Porcellio, indicating that the seven leg segments in Glom- ble for the formation of the leg joints in a complex temporal eris are not directly comparable to the leg segments in the sequence (Rauskolb, 2001). Thus, modifications in the dy- fly and the woodlouse. Interestingly, the pattern dynamics in namics likely will change the number and location of leg Glomeris are virtually identical to those in Cupiennius. This joints. In noninsect arthropods, dynamic expression profiles indicates that the number and location of joints in spiders N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 109 and diplopods may be directly comparable and suggests that Porcellio scaber and the insect Tribolium castaneum the leg segments in these arthropod groups are homologous. (Abzhanov and Kaufman, 2000; Prpic et al., 2001). It is interesting to note that very strong mandibular dac expression in all three cases correlates with the tooth-like mor- Gm-Dll and Gm-dac apparently play a complex role in phology of the mandibular appendage. Thus, it is possible the cephalic appendages that dac has an additional function in the mandible, that of specifying mandible identity—in analogy to the selector In the Drosophila antenna, Dll is involved in the forma- gene function of Dll in the Drosophila antenna. It will be tion of the PD axis and the development of the larval interesting to test this hypothesis in an arthropod that is antennal sensory organ (Cohen and Ju¬rgens, 1989a). It is amenable to functional testing (e.g., Tribolium). Expression also a selector gene for antennal identity. Partial loss of Dll of Gm-dac in the maxilla is at first distributed in the entire function leads to transformation of antennal tissue into leg primordium of the stipes and the tissue contributing to the tissue (Sunkel and Whittle, 1987; Cohen and Ju¬rgens, intermaxillary plate, but later is restricted to cell clusters in 1989b; Dong et al., 2000). In the antenna of Glomeris, the primordia of the maxillary sensory organs. Thus, Gm- Gm-Dll is expressed in the entire distal part including the dac is apparently involved in sensory organ development in primordia of the sensory cones. This hints to a role in distal the maxilla. The role of the earlier ubiquitous expression in development, but makes it impossible to discriminate be- the stipes is unclear. Neither in the mandible nor in the tween a function in PD axis formation and a possible role in maxilla, is expression of Gm-dac found in a pattern that sensory organ development. In addition, a role of Gm-Dll in would suggest its involvement in instructing medial fates on specifying antennal identity cannot be substantiated without the PD axis of these appendages. In summary, Gm-dac functional tests, which are not possible to date. In the expression implicates this gene in at least two distinct pro- maxilla, however, expression of Gm-Dll relates exclusively cesses in the cephalic appendages: PD axis formation in the to the primordia of maxillary sensory organs. Therefore, a antenna and sensory organ development in antenna and role of Gm-Dll in establishing the PD axis of the maxilla of mouthparts. In addition, Gm-dac may have a third role, that Glomeris is unlikely. Also in the mandible, expression of of specifying mandible identity in Glomeris and other man- Gm-Dll does not suggest a role of this gene in PD axis dibulate arthropods. formation. Rather, like in the maxilla, Gm-Dll expression appears to correlate with mandibular sensory organs. These data suggest that Gm-Dll in the cephalic appendages has at Phylogenetic implications: serial homologies among least two distinct functions, that of instructing distal fates on mouthparts in insects, crustaceans and myriapods the PD axis in the antenna and that of guiding the development of sensory organs in the mouthparts. A function of Dll In order to reconstruct the evolution of the gene expres- in sensory organ development has also been proposed for sion pattern, the expression of Dll homologues has been other arthropods, including chelicerates, crustaceans, and studied in a great number of different arthropod species. The apterygote insects (Mittmann and Scholtz, 2001; Williams present study is the first detailed account for Dll expression et al., 2002), and our results support this notion. in a myriapod and the first report on expression of dac, hth, In the antenna, Gm-dac has two distinct expression do- and exd in a myriapod. The data on Dll presented here show mains. First, there is a ring-like expression at a medial PD that, in contrast to the antenna and trunk legs, in the two level. At early stages, expression forms a single ring, but gnathal appendages, mandible and maxilla, no Gm-Dll ex- later this ring divides into two separate rings linked by pression can be found that can be confidently correlated residual expression. A similar separation of the dac expres- with PD axis formation. This implies that the Glomeris sion domain during PD axis formation has been shown to mouthparts lack elements serially homologous to the distal occur in the thoracic legs of the cricket Gryllus bimaculatus Gm-Dll-expressing parts of the antenna and legs. Thus, both (Inoue et al., 2002), suggesting that the dynamic expression mandible and maxilla are gnathobasic in nature and lack in Glomeris may play a similar role in PD axis formation. distal parts (telopodite, palp). A gnathobasic mandible tra- Second, Gm-dac shows prominent expression in the sensory ditionally is considered as a synapomorphy of crustaceans, cone primordia starting at late stage 3, clearly arguing that insects, and myriapods, uniting them into a monophyletic it is involved in the development of the antennal sensory Mandibulata (Snodgrass, 1938; see review by Koch, 2001). cones. Because of the complex role of Gm-dac in the an- A gnathobasic maxilla, however, is found only in myriapods tenna, a tentative assessment of its role in the remaining [apart from Glomeris (diplopod) also the maxilla of chilo- head appendages is difficult to make without functional pods, symphylans, and pauropods apparently lacks a telopo- tests. In the mandible, the expression in small clusters of dite judging from external morphology] and adults of sev- cells in the inner lobe probably correlates with the formation eral crustacean species. The maxilla in insects and most of sensory organs of this appendage. The very strong ex- crustaceans is not gnathobasic and has a palp (telopodite). pression in the outer lobe, which will give rise to the tooth, This demonstrates that the gnathobasic condition of an ap- is similar to mandibular dac expression in the crustacean pendage can evolve independently by convergence and thus 110 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 questions the homology of the mandibular gnathobasy in References crustaceans, insects, and myriapods. Indeed, recent molecular phylogenies corroborate two monophyletic groups Abu-Shaar, M., Mann, R.S., 1998. Generation of multiple antagonistic comprising chelicerates and myriapods on the one hand and domains along the proximodistal axis during Drosophila leg development. Development 125, 3821Ð3830. crustaceans and insects on the other hand (Hwang et al., Abu-Shaar, M., Ryoo, H.D., Mann, R.S., 1999. Control of the nuclear 2001; Friedrich and Tautz, 2001). This would argue for an localization of Extradenticle by competing nuclear import and export independent origin of the gnathobasic mandible in myri- signals. Genes Dev. 13, 935Ð945. apods and the so-called Tetraconata (insects and crusta- Abzhanov, A., Kaufman, T.C., 2000. Homologs of Drosophila appendage ceans; Dohle, 2001). However, the results with Gm-dac genes in the patterning of arthropod limbs. Dev. Biol. 227, 673Ð689. Aspo¬ck, G., Bu¬rglin, T.R., 2001. The Caenorhabditis elegans Distal-less presented here support the homology of the gnathobasy in ortholog ceh-43 is required for development of the anterior hypoder- the mandibles of crustaceans, insects, and myriapods. It has mis. Dev. Dyn. 222, 403Ð409. been shown previously that expression of dac is very strong Azpiazu, N., Morata, G., 2000. Function of homothorax in the wing in the mandibles of crustaceans and insects (Abzhanov and imaginal disc of Drosophila. Development 127, 2685Ð2693. Beermann, A., Jay, D.G., Beeman, R.W., Hulskamp, M., Tautz, D., Jur- Kaufman, 2000; Prpic et al., 2001). Similarly, strong ex- gens, G., 2001. The Short antennae gene of Tribolium is required for pression of Gm-dac is seen in the mandible of Glomeris. limb development and encodes the orthologue of the Drosophila Distal- The role of the strong dac expression in the development of less protein. Development 128, 287Ð297. the mandible is unclear, but it seems possible to correlate it Berthelsen, J., Kilstrup-Nielsen, C., Blasi, F., Mavilio, F., Zappavigna, V., with the specific tooth-like morphology of this appendage 1999. The subcellular localization of PBXI and EXD proteins depends on nuclear import and export signals and is modulated by association (see discussion above). Thus, a common genetic mechanism with PREP1 and HTH. Genes Dev. 13, 946Ð953. appears to underlie the gnathobasic nature of the mandibles Bessa, J., Gebelein, B., Pichaud, F., Casares, F., Mann, R.S., 2002. Com- in myriapods, crustaceans, and insects. In contrast to this, no binatorial control of Drosophila eye development by eyeless, homo- comparable dac expression has been found in the first lo- thorax, and teashirt. Genes Dev. 16, 2415Ð2427. Campbell, G., Tomlinson, A., 1998. The roles of the homeobox genes comotory leg (L1) in chelicerates (Abzhanov and Kaufman, aristaless and Distal-less in the patterning the legs and wings of 2000; unpublished observations). The L1 segment in cheli- Drosophila. Development 125, 4483Ð4493. cerates corresponds to the mandibular segment in the other Casares, F., Mann, R.S., 2000. A dual role for homothorax in inhibiting arthropods (Damen et al., 1998; Telford and Thomas, 1998; wing blade development and specifying proximal wing identities in Mittmann and Scholtz, 2003). Drosophila. Development 127, 1499Ð1508. Caubit, X., Thangarajah, R., Theil, T., Wirth, J., Nothwang, H.G., Ru¬ther, In summary, the results presented here support earlier U., Krauss, S., 1999. Mouse Dac, a novel nuclear factor with homology reports that the mandible in myriapods is indeed gnatho- to Drosophila Dachshund shows a dynamic expression in the neural basic (Popadic et al., 1998; Scholtz et al., 1998). Moreover, crest, the eye, the neocortex, and the limb bud. Dev. Dyn. 214, 66Ð80. our results indicate that also the maxilla is gnathobasic, Chen, R., Amoui, M., Zhang, Z., Mardon, G., 1997. Dachshund and eyes absent proteins form a complex and function synergistically to induce which is different from insects and most crustaceans and ectopic eye development in Drosophila. Cell 91, 893Ð903. also from chelicerates which do not possess a single gna- Cohen, B., Simcox, A.A., Cohen, S.M., 1993. Allocation of the thoracic thobasic appendage. Strong expression of dac in the man- imaginal primordia in the Drosophila embryo. Development 117, 597Ð dible in crustaceans, insects, and myriapods suggests that 608. the mandibular morphology is produced by a homologous Cohen, B., Wimmer, E.A., Cohen, S.M., 1991. Early development of leg and wing primordia in the Drosophila embryo. Mech. Dev. 33, 229Ð mechanism and supports the homology of the mandible in 240. all mandibulate arthropods. Although a homologous man- Cohen, S.M., 1990. Specification of limb development in the Drosophila dible in crustaceans, insects, and myriapods would support embryo by positional cues from segmentation. Nature 343, 173Ð177. the monophyly of the Mandibulata, it has to be pointed out Cohen, S.M., Bro¬nner, G., Ku¬ttner, F., Ju¬rgens, G., Ja¬ckle, H., 1989. Distal-less encodes a homeodomain protein required for limb develop- that it is also compatible with recent molecular phylogenies ment in Drosophila. Nature 338, 432Ð434. (Hwang et al., 2001; Friedrich and Tautz, 2001) on the Cohen, S.M., Ju¬rgens, G., 1989a. Proximal-distal pattern formation in assumption that a true mandible already existed in the last Drosophila: cell autonomous requirement for Distal-less gene activity common ancestor of all extant arthropod classes and has in limb development. EMBO J. 8, 2045Ð2055. been lost secondarily in the chelicerates. Cohen, S.M., Ju¬rgens, G., 1989b. Proximal-distal pattern formation in Drosophila: graded requirement for Distal-less gene activity during limb development. Roux’s Arch. Dev. Biol. 198, 157Ð169. Damen, W.G., Hausdorf, M., Seyfarth, E.A., Tautz, D., 1998. A conserved mode of head segmentation in arthropods revealed by the expression Acknowledgments pattern of Hox genes in a spider. Proc. Natl. Acad. Sci. USA 95, 10665Ð10670. We thank Wim Damen and Ralf Janssen for their help in Davis, R.J., Shen, W., Heanue, T.A., Mardon, G., 1999. Mouse Dach,a establishing Glomeris as an experimental animal, and Wim homologue of Drosophila dachshund, is expressed in the developing retina, brain and limbs. Dev. Genes Evol. 209, 526Ð536. Damen for critical reading of the manuscript. This work has Dohle, W., 1964. Die Embryonalentwicklung von Glomeris marginata been funded by the Deutsche Forschungsgesellschaft (Grant (Villers) in Vergleich zur Entwicklung anderer Diplopoden. Zool. Jb. TA 99/19-1). Anat. 81, 241Ð310. N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112 111

Dohle, W., 2001. Are the insects terrestrial crustaceans? A discussion of interacts with extradenticle in the patterning of the embryonic PNS. some new facts and arguments and the proposal of the proper name Development 125, 1037Ð1048. “Tetraconata” for the monophyletic unit Crustacea plus Hexapoda. Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W.J., Furukubo- Ann. Soc. Entomol. Fr. (N.S.) 37, 85Ð103. Tokunaga, K., 2000. Genetic control of development of the mushroom Dong, P.D.S., Chu, J., Panganiban, G., 2000. Coexpression of the ho- bodies, the associative learning centers in the Drosophila brain, by the meobox genes Distal-less and homothorax determines Drosophila an- eyeless, twin of eyeless, and dachshund genes. Proc. Natl. Acad. Sci. tennal identity. Development 127, 209Ð216. USA 97, 2140Ð2144. Dong, P.D.S., Chu, J., Panganiban, G., 2001. Proximodistal domain spec- Mardon, G., Solomon, N.M., Rubin, G.M., 1994. dachshund encodes a ification and interactions in developing Drosophila appendages. De- nuclear protein required for normal eye and leg development in Dro- velopment 128, 2365Ð2372. sophila. Development 120, 3473Ð3486. Dove, H., Stollewerk, A., 2003. Comparative analysis of neurogenesis in Martini, S.R., Roman, G., Meuser, S., Mardon, G., Davis, R.L., 2000. The the myriapod Glomeris marginata (Diplopoda) suggests more similar- retinal determination gene, dachshund, is required for mushroom body ities to chelicerates than to insects. Development 130, 2161Ð2171. cell differentiation. Development 127, 2663Ð2672. Friedrich, M., Tautz, D., 2001. Arthropod rDNA phylogeny revisited: a Mittmann, B., Scholtz, G., 2001. Distal-less expression in embryos of consistency analysis using Monte Carlo simulation. Ann. Soc. Ento- Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina mol. Fr. (N.S.) 37, 21Ð40. (Insecta, Zygentoma) suggests a role in the development of mechano- Gonzalez-Crespo, S., Morata, G., 1996. Genetic evidence for the subdivi- receptors, chemoreceptors, and the CNS. Dev. Genes Evol. 211, 232Ð sion of the arthropod limb into coxopodite and telopodite. Development 243. 122, 3921Ð3928. Mittmann, B., Scholtz, G., 2003. Development of the nervous system in the Goodman, C.S., Doe, C.Q., 1993. Embryonic development of the Dro- “head”of Limulus polyphemus (Chelicerata: Xiphosura): morphological sophila central nervous system, in: Bate, M., Martinez-Arias, A. (Eds.), evidence for a correspondence between the segments of the chelicerae The Development of Drosophila melanogaster, Cold Spring Harbor and of the (first) antennae of Mandibulata. Dev. Genes Evol. 213, 9Ð17. Laboratory Press, Cold Spring Harbor, NY, pp 1131Ð1206. Nagao, T., Endo, K., Kawauchi, H., Walldorf, U., Furukubo-Tokunaga, K., Gorfinkiel, N., Morata, G., Guerrero, I., 1997. The homeobox gene Distal- 2000. Patterning defects in the primary axonal scaffolds caused by the less induces ventral appendage development in Drosophila. Genes mutations of the extradenticle and homothorax genes in the embryonic Dev. 11, 2259Ð2271. Drosophila brain. Dev. Genes Evol. 210, 289Ð299. Goto, S., Hayashi, S., 1997. Specification of the embryonic limb primor- Noveen, A., Daniel, A., Hartenstein, V., 2000. Early development of the dium by graded activity of decapentaplegic. Development 124, 125Ð Drosophila mushroom body: the roles of eyeless and dachshund. De- 132. velopment 127, 3475Ð3488. Hammond, K.L., Hanson, I.M., Brown, A.G., Lettice, L.A., Hill, R.E., Pai, C.Y., Kuo, T.S., Jaw, T.J., Kurant, E., Chen, C.T., Bessarab, D.A., 1998. Mammalian and Drosophila dachshund genes are related to the Salzberg, A., Sun, Y.H., 1998. The Homothorax homeoprotein acti- Ski proto-oncogene and are expressed in eye and limb. Mech. Dev. 74, vates the nuclear localization of another homeoprotein, Extradenticle, 121Ð131. and suppresses eye development in Drosophila. Genes Dev. 12, 435Ð Henderson, K.D., Andrew, D.J., 2000. Regulation and function of Scr, exd, 446. and hth in the Drosophila salivary gland. Dev. Biol. 217, 362Ð274. Panganiban, G., 2000. Distal-less function during Drosophila appendage Hwang, U.W., Friedrich, M., Tautz, D., Park, C.J., Kim, W., 2001. Mito- and sense organ development. Dev. Dyn. 218, 554Ð562. chondrial protein phylogeny joins myriapods with chelicerates. Nature Panganiban, G., Sebring, A., Nagy, L., Carroll, S.B., 1995. The develop- 413, 154Ð157. ment of crustacean limbs and the evolution of arthropods. Science 270, Inoue, Y., Mito, T., Miyawaki, K., Matsushima, K., Shinmyo, Y., Heanue, 1363Ð1366. T.A., Mardon, G., Ohuchi, H., Noji, S., 2002. Correlation of expression Panganiban, G., Rubenstein, J.L.R., 2002. Developmental functions of the patterns of homothorax, dachshund, and Distal-less with the proximo- Distal-less/Dlx homeobox genes. Development 129, 4371Ð4386. distal segmentation of the cricket leg bud. Mech. Dev. 113, 141Ð148. Pichaud, F., Casares, F., 2000. homothorax and iroquois-C genes are Jaw, T.J., You, L.R., Knoepfler, P.S., Yao, L.C., Pai, C.Y., Tang, C.Y., required for the establishment of territories within the developing eye Chang, L.P., Berthelsen, J., Blasi, F., Kamps, M.P., Sun, Y.H., 2000. disc. Mech. Dev. 96, 15Ð25. Direct interaction of two homeoproteins, Homothorax and Extraden- Popadic, A., Panganiban, G., Rusch, D., Shear, W.A., Kaufman, T.C., ticle, is essential for EXD nuclear localization and function. Mech. 1998. Molecular evidence for the gnathobasic derivation of arthropod Dev. 91, 279Ð291. mandibles and for the appendicular origin of the labrum and other Jhaveri, D., Sen, A., Rodrigues, V., 2000. Mechanisms underlying olfac- structures. Dev. Genes Evol. 208, 142Ð150. tory neuronal connectivity in Drosophila-the atonal lineage organizes Prpic, N.M., Wigand, B., Damen, W.G.M., Klingler, M., 2001. Expression the periphery while sensory neurons and glia pattern the olfactory lobe. of dachshund in wild-type and Distal-less mutant Tribolium corrobo- Dev. Biol. 226, 73Ð87. rates serial homologies in insect appendages. Dev. Genes Evol. 211, Klingler, M., Gergen, P., 1993. Regulation of runt transcription by Dro- 467Ð477. sophila segmentation genes. Mech. Dev. 43, 3Ð19. Rauskolb, C., 2001. The establishment of segmentation in the Drosophila Koch, M., 2001. Mandibular mechanisms and the evolution of hexapods. leg. Development 128, 4511Ð4521. Ann. Soc. Entomol. Fr. (N.S.) 37, 129Ð174. Rauskolb, C., Irvine, K.D., 1999. Notch-mediated segmentation and Kozmik, Z., Pfeffer, P., Kralova, J., Paces, J., Paces, V., Kalousova, A., growth control of the Drosophila leg. Dev. Biol. 210, 339Ð350. Cvekl, A., 1999. Molecular cloning and expression of the human and Rauskolb, C., Peifer, M., Wieschaus, E., 1993. extradenticle, a regulator of mouse homologues of the Drosophila dachshund gene. Dev. Genes homeotic gene activity, is a homolog of the homeobox-containing Evol. 209, 537Ð545. human proto-oncogene pbxl. Cell 74, 1101Ð1112. Kraus, O., 2001. “Myriapoda” and the ancestry of the Hexapoda. Ann. Soc. Rauskolb, C., Smith, K.M., Peifer, M., Wieschaus, E., 1995. extradenticle Entomol. Fr. (N.S.) 37, 105Ð127. determines segmental identities throughout Drosophila development. Kumar, J.P., Moses, K., 2001. Expression of evolutionarily conserved eye Development 121, 3663Ð3673. specification genes during Drosophila embryogenesis. Dev. Genes Rieckhof, G.E., Casares, F., Ryoo, H.D., Abu-Shaar, M., Mann, R.S., 1997. Evol. 211, 406Ð414. Nuclear translocation of Extradenticle requires homothorax, which Kurant, E., Pai, C.Y., Sharf, R., Halachmi, N., Sun, Y.H., Salzberg, A., encodes an Extradenticle-related homeodomain protein. Cell 91, 171Ð 1998. dorsotonals/homothorax, the Drosophila homologue of Meisl, 183. 112 N.-M. Prpic, D. Tautz / Developmental Biology 260 (2003) 97Ð112

Scholtz, G., Mittmann, B., Gerberding, M., 1998. The pattern of Distal-less Tautz, D., Pfeifle, C., 1989. A non-radioactive in situ hybridization method expression in the mouthparts of crustaceans, myriapods and insects: for the localization of specific RNAs in Drosophila embryos reveals new evidence for a gnathobasic mandible and the common origin of translational control of the segmentation gene hunchback. Chromo- Mandibulata. Int. J. Dev. Biol. 42, 801Ð810. soma 98, 81Ð85. Schoppmeier, M., Damen, W.G.M., 2001. Double-stranded RNA interfer- Telford, M.J., Thomas, R.H., 1998. Expression of homeobox genes shows ence in the spider Cupiennius salei: the role of Distal-less is evolu- chelicerate arthropods retain their deutocerebral segment. Proc. Natl. tionarily conserved in arthropod appendage formation. Dev. Genes Acad. Sci. USA 95, 10671Ð10675. Evol. 211, 76Ð82. Williams, T.A., 1998. Distalless expression in crustaceans and the pattern- Shen, W., Mardon, G., 1997. Ectopic eye development in Drosophila ing of branched limbs. Dev. Genes Evol. 207, 427Ð434. induced by directed dachshund expression. Development 124, 45Ð52. Williams, T.A., Nulsen, C., Nagy, L.M., 2002. A complex role for Snodgrass, R.E., 1938. Evolution of the Annelida, Onychophora and Ar- Distal-less in crustacean appendage development. Dev. Biol. 241, thropoda. Smithsonian Misc. Coll. 97, 1Ð159. 302Ð312. Sunkel, C.S., Whittle, J.R.S., 1987. Brista: a gene involved in the speci- Wu, J., Cohen, S.M., 1999. Proximodistal axis formation in the Drosophila fication and differentiation of distal cephalic and thoracic structures in leg: subdivision into proximal and distal domains by homothorax and Drosophila melanogaster. Roux’s Arch. Dev. Biol. 196, 124Ð132. Distal-less. Development 126, 109Ð117.