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Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis

Institut für Genetik, Universität zu Köln, Weyertal 121, D-50931 Köln, Germany

View larger version (55K): [in a new window]   Fig. 1. Alignment of Engrailed homeodomains and Engrailed-specific domains. (A) The homeodomains are aligned to the Cs-EN-1 homeodomain. (B) Sequences of the Engrailed-specific domains are aligned to the consensus as defined by Duboule (Duboule, 1994). Engrailed-specific domains EH-1, EH-2, EH-3 and EH-5 are boxed. EH-4 corresponds to the homeodomain (HD), dashes indicate identical amino acids, dots indicate gaps. The spacing between the different domains are given in number of amino acids. Cs, Cupiennius salei (Chelicerata, AJ007437 and AJ315944); Al, Archegozetes longisetosus (Chelicerata, AF071404); Tc, Tribolium castaneum (Insecta, S73225); Dm, Drosophila melanogaster (Insecta, P02836 and P05527); Bm, Bombyx mori (Insecta, P27609 and P27610); Ag, Anopheles gambiae (Insecta, O02491); Af, Artemia franciscana (Crustacea, Q05640); Sc, Saculina carcina (Crustacea, AAC63993 and AAG40576); Ak, Acanthokara kaputenis (Onychophora, CAA71745); Ht, Helobdella triserialis (Annelida, P23397); Np, Nautilus pompilius (Mollusca, U23431 and U21857); Io, Ilyanassa obsoleta (Mollusca, S65924); Sm, Schistosoma mansoni (Platyhelminthes, S33640); Ce, Caenorhabditis elegans (Nematoda, P34326); Tg, Tripneustes gratilla (Echinodermata, P09532); He, Heliocidaris erythrogramma (Echinodermata, U58775); Amph, Branchiostoma floridae (Chordata, Cephalochordata, U82487); Mm, Mus musculus (Chordata, Vertebrata, P09065 and P09066).

View larger version (24K): [in a new window]   Fig. 2. Phylogram of Wingless/WNT sequences of mouse (Mm), Drosophila melanogaster (Dm) and Cupiennius salei (Cs). Phylogenetic analysis was done using PUZZLE (Strimmer and von Haeseler, 1996), as implemented in PAUP 4.0b6 (Swofford, 2001). Accession Numbers are as follows. Mm: Wnt1 (P04426), Wnt2 (P21552), Wnt3 (AAB38109), Wnt3a (P27467), Wnt4 (P22724), Wnt5a (AAA40567), Wnt5b (P22726), Wnt6 (AAA40569), Wnt7a (AAA40570), Wnt7b (AAA40571), Wnt8a (Q64527), Wnt8b (NP035850), Wnt10a (P70701), Wnt16 (AAD49352), Wnt13/2b (AAC25397). Dm: Wg (P09615), DWnt2 (S24559), DWnt3/5 (P28466), DWnt4 (P40589). Cs: Wg (AJ315945), Wnt5-1 (AJ315946).

View larger version (94K): [in a new window]   Fig. 3. Expression of Cs-en-1 in embryos of the spider Cupiennius salei. (A) Young germ band stage, five clear stripes of Cs-en-1 expression visible [Pp and legs (L)] and a weak stripe for the Cheliceres. No opisthosomal segments formed yet. (B-D) Lateral, anterior and posterior views, respectively, of the same embryo. Three opisthosomal segments are formed, marked by three stripes of Cs-en-1. Arrowheads in C indicate the ocular spots of Cs-en-1 expression. (E,F) Different views of the same embryo. Five opisthosomal segments have formed. Limb buds straddle the anterior border of Cs-en-1 expression. (G,H) Different views of the same embryo. Up to eleven Cs-en-1 stripes are visible in the opisthosoma. The posterior part of the appendages expresses Cs-en-1. In addition, note that the abdominal limb buds on opisthosomal segment 2-5 straddle the anterior border of Cs-en-1 expression. The expression of Cs-en-1 is weaker dorsal to these opisthosomal limb buds, compared with its expression in the limb buds themselves and in the neuroectoderm ventral to them. The furrow that forms is the result of the inversion that leads to longitudinal splitting of the germ band and to dorsal closure. The two halves of the germband are connected in the head region and at the most posterior end. This is even more evident in the embryos shown in J,L. (I-K) More advanced stage of development, Cs-en-1 expression anterior to the labrum. Twelve stripes of Cs-en-1 expression detectable in the opisthosoma, the 12th one only as two weak spots after DAPI staining (K, arrowhead). (L) Stage after dorsal closure. Arrowheads mark the ring of Cs-en-1 expression at the posterior end that probably represents the hind gut. Anterior is towards the left in all embryos. Ch, cheliceres; Pp, pedipalps; L1-L4, walking leg 1-4; Lb, labrum; Opisthosomal segments are indicated by 1-12.

View larger version (109K): [in a new window]   Fig. 4. Expression of Cs-en-2 in embryos of the spider Cupiennius salei. (A-C) Different views of the same embryo (embryo is slightly younger than the one in Fig. 3B). Two stripes and a posterior cap of Cs-en-2 expression are visible in the opisthosoma. The stripes in the more anterior segments are doublet stripes (most prominent for the Ch and Pp, A). Arrowheads in A indicate the ocular spots. Expression of Cs-en-2 seems to diminish in the ventral midline, in contrast to Cs-en-1 expression (compare with Fig. 2A-F). (D,E) Slightly older stage; four opisthosomal segments are present. Limb buds straddle the anterior border of each double stripe. Arrowheads in D indicate the ocular spots of Cs-en-2 expression that became small stripes (F). (F,G) germ band stage at onset of inversion (comparable stage as in Fig. 3G). Strong expression of Cs-en-2 in the stomodeum (st). Strong dorsal Cs-en-2 expression and expression in two paired segmental spots in the neuro-ectoderm. (H) Detail of a DAPI counter-stained embryo. Expression of Cs-en-2 has an anterior border in the appendage at the same position as Cs-en-1, but, by contrast, it does not cover the complete posterior part of the appendage. (I) Dorsal view of an embryo after dorsal closure (comparable stage to that in Fig. 3K). Arrowheads indicate the ring-like expression that probably represents the onset of the hind gut. Anterior is towards the left in all embryos. Ch, cheliceres; Pp, pedipalps; L1-L4, walking leg 1-4; St, stomodeum; Opisthosomal segments are indicated by 1-12.

View larger version (86K): [in a new window]   Fig. 5. Details of Cs-en-1 expression point to a parasegmental organization of the spider embryo. (A) The opisthosomal Cs-en-1 stripes have a sharp anterior boundary (arrowhead). The opisthosomal stripes of Cs-en-1 expression are shown in an embryo in which three opisthosomal stripes are present. (B) Limb buds of the L1-L3 segments that straddle the boundary of Cs-en-1 expression (arrowhead). (C) Anterior border of Cs-en-1 expression correlates with a morphological visible groove (arrowheads). The opisthosomal stripes 1-4 in an embryo are shown in which seven opisthosomal stripes are present. (D) Longitudinal section of a Cs-en-1 stained embryo. The grooves are visible at the domains of en expression (arrowhead). (E) DAPI stained embryo (no in situ hybridization). Grooves are clearly visible. Anterior is towards the left in all embryos. L1-L4, walking leg 1-4; Opisthosomal segments are indicated by 1-12.

View larger version (71K): [in a new window]   Fig. 6. Expression of Cs-wg in embryos of the spider Cupiennius salei. (A,B) Different views of the same embryo, showing the head region and the posterior region, respectively. Cs-wg is expressed in the ventral-anterior portion of the appendages. In B, Cs-wg is clearly expressed just anterior to the grooves that delimit the parasegments. (C) Slightly older embryo. Cs-wg expression dorsally to the opisthosomal limb buds. (D-G) Different views of one embryo, showing Cs-wg expression dorsal to the prosomal appendages (D), in the anterior labrum (arrowhead), the ventral-anterior region of the appendages (arrowheads) (E) and the opisthosoma (F). (G) A detail of the expression in a stripe dorsally (white circle) and a spot ventral (star) to the opisthosomal limb buds. Expression is also seen posterior to the opisthosomal limb buds of O2, O4 and O5 (arrowheads). In O2 these represent the lamellae of the book lung. (H) Posterior view of an embryo at the inversion stage. Cs-wg is still expressed at the very posterior end of the embryo (arrowhead). Anterior is towards the left in all embryos. Ch, cheliceres; Pp, pedipalps; L1-L4, walking leg 1-4; Lb, labrum; Opisthosomal segments are indicated by 1-12.

View larger version (64K): [in a new window]   Fig. 7. Expression of Cs-Wnt5-1 in embryos of the spider Cupiennius salei. (A,B) Different views of the same embryo. Expression of Cs-Wnt5-1 is visible in segmentally iterated stripes. Furthermore, expression is seen in a ring in the appendages, four spots in the head (arrowheads) and the labrum. (C,D) Slightly older embryos with six and seven opisthosomal segments formed, respectively. The Cs-Wnt5-1 stripes are located ventrally and do not completely extend dorsally. The arrowhead marks a weak expression domain at the posterior end of the germband. (E) Slightly older embryo. The stripes of Cs-Wnt5-1 expression are ventral to the opisthosomal appendages; there is an incomplete ring of expression in these opisthosomal appendage anlagen. Expression becomes visible in segmental dorsal spots that almost form a continuous stripe. (F,G) An even older embryo. The dorsal spots are clearly visible now. The region dorsal to the appendage anlagen is free of Cs-Wnt5-1 expression. (H) Expression of Cs-wg and Cs-Wnt5-1 in one embryo. Together the two genes form one stripe of expression covering the complete width of the embryo, just interrupted by the opisthosomal limb anlagen. Anterior is towards the left in all embryos. Ch, cheliceres; Pp, pedipalps; L1-L4, walking leg 1-4; Lb, labrum; Opisthosomal segments are indicated by 1-12.

View larger version (104K): [in a new window]   Fig. 8. Expression of Cs-ci in embryos of the spider Cupiennius salei. (A) Segmentally iterated expression of Cs-ci in opisthosoma of young germ band stage embryo. (B) Slightly older embryo, new stripes form posteriorly. (C,D) Expression of Cs-ci is in the anterior part of the opisthosomal limb anlagen (C) and appendages (D), showing that Cs-ci is expressed in the engrailed negative cells. Anterior is towards the left in all embryos. Pp, pedipalps; L1-L2, walking leg 1-2; Opisthosomal segments are indicated by 1-12.

View larger version (33K): [in a new window]   Fig. 9. Engrailed expression in the spider marks probable fundamental boundaries. The upper part shows the situation in Drosophila. The stripes of en expression mark the anterior parasegment boundaries, whereas wg marks the posterior parasegment boundaries. The parasegments 1-14 are dependent on the action of the pair-rule genes. The anterior expression border of the posterior Hox genes obeys a parasegmental boundary (purple), in contrast to the anterior one that obeys a segmental boundary (orange). Lower part shows the situation in the spider Cupiennius. The insect and chelicerate segments are homologized as proposed by Telford and Thomas (Telford and Thomas, 1998) and Damen et al. (Damen et al., 1998). Note that the Cs-Ubx1 gene probably obeys a segmental boundary. For Abd-B, the domain of strong expression is used. Data are taken from Kaufman et al. (Kaufman et al., 1990), Schmidt-Ott and Technau (Schmidt-Ott and Technau, 1992), Jürgens and Hartenstein (Jürgens and Hartenstein, 1993), Damen et al. (Damen et al., 1998), Damen and Tautz (Damen and Tautz, 1998; Damen and Tautz, 1999) and M. Schoppmeier and W. G. M. D. (unpublished). Ant, antennal segment; Int, intercalary segment; La, labial segment; Ma, mandibular segment; Mx, maxilar segment; Oc, ocular segment/acron; ps, parasegment; T1-T3, thoracic segment 1-3; A1-A8, abdominal segment 1-8; Tel, telson; Ch, cheliceres; Pp, pedipalps; L1-L4, walking leg 1-4; O1-O12, opisthosomal segments 1-12; ftz, fushi tarazu; eve, even-skipped.