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Evolving role of Antennapedia protein in arthropod limb patterning

Yasuhiro Shiga1,*, Ryusuke Yasumoto1, Hideo Yamagata1 and Shigeo Hayashi2,3,*

1 School of Life Science, Tokyo University of Pharmacy and Life Science 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
2 National Institute of Genetics and Graduate University for Advanced Studies, 1111 Yata, Mishima, Shizuoka-ken 411-8540 Japan
3 Morphogenetic Signaling Group, Riken Center for Developmental Biology, 2-2-3, Minatojima-Nakamachi, Chuo-ku, Kobe, Hyogo, 650-0047 Japan



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  		Fig. 1. Limb development in Daphnia. (A) First to fifth trunk limbs of Daphnia and their relationship to DLL expression. Epipod (Ep), exopod (Ex), endopod (En) and gnathobase (Gn) or filter comb (Fc) are arranged in a dorsal-to-ventral orientation. Endites of second to fourth leg bear prominent comb-like structures (Gn and Fc) that correspond to strong expression of DLL. No such branches or DLL expression are present in T1 or T5 (asterisks). (B,D,F) Daphnia embryos stained with nuclear marker YOYO-1. (C,E,G) DLL expression. Ventral view, anterior is upwards. (B,C) At 18 hours (Obreshkove and Fraser, 1940Go), with visible furrows separating thoracic segments, DLL expression begins in a dorsal cluster of cells in the first thoracic leg primordia, and in dorsal and ventral clusters in the second and the third primordia. Ventral DLL-expressing clusters in T2 and dorsal clusters in T3 are out of focus in this image. (D,E) At 21 hours, the DLL-expressing domain is expanded and clear nuclear localized signals are detected in future internal branches of developing thoracic legs and in first antenna, labrum, first maxilla, but not in mandibles and second maxilla. (F,G) Later stage embryos (24-27 hours) show strong DLL signals in limb branches, which show characteristic segment-specific morphologies. An1, first antennae; An2, second antennae; Lb, labrum; Mn, mandible; Mx1, first maxillae; Mx2, second maxillae; T1-T5, first to fifth thoracic segment; L1-L5, first to fifth thoracic limb. Scale bars: 100 µm.

 


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  		Fig. 2. Structures of ANTP. (A) An alignment of amino acid sequences around YPWM motifs and homeodomains (blocked) of DapANTP (DDBJ Accession Number, AB069680) and DmANTP (cDNA G1100) (Laughon et al., 1986Go). Identical amino acid residues are indicated by asterisks and conserved ones by dots. Borders of N, HD and C regions are indicated. Amino acid sequences matching the consensus CKII phosphorylation sites are underlined and diagnostic amino acid residues are indicated in bold. (B) Structures of native and mutant ANTP constructs used in this study. Amino acid sequences derived from DapANTP are shown in pink and those from DmANTP in blue. Black bars, blocks and the open box indicate the YPWM motifs, homeodomains and the homology found in several insects, respectively. Asterisks indicate putative CKII sites in the CDm.

 


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  		Fig. 3. Expression of ANTP and UBX/ABDA, and their relationship to DLL. (A) Antp mRNA was expressed in Mx2 and more posterior segments. (B-E) Confocal images of embryos double labeled with DLL (purple in B,C,D,D',E,E') and ANTP (green in B,D,D'') or UBX/ABDA (green in C,E,E'') (green). (B,D) ANTP expression was limited to Mx2 and anterior of first thoracic segment (T1a). Most of the DLL expression in T1 did not overlap with ANTP. Note that prominent expression of ANTP in ventral T1 corresponds to reduced expression of DLL (arrowheads in D). The dorsal cells expressing DLL are located in a different cell layer expressing ANTP but appear white in some cells because of the projection of multiple confocal sections. (C,E) UBX/ABDA expression was detected in dorsal part of T1 and in posterior segments. In the ventral region, the anterior staining border is at the posterior edge of the first thoracic segment, abutting the posterior border of ANTP. As reported in other crustaceans, millipedes and insects (Panganiban et al., 1995Go), UBX/ABDA are co-expressed with DLL in a large number of leg cells that appear white in the merged image. Scale bar: 100 µm.

 


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  		Fig. 4. DapANTP specifies thoracic identities in the Drosophila head. (A-C) Transformation of adult heads into thorax by ectopic expression of ANTP. Dorsal head views of (A) wild-type y w strain, (B) dppGal4; UAS-DmAntp and (C) dppGal4; UAS-DapAntp flies. Transformation of antennae to legs characterized by bracted bristles, dorsal thoracic cuticle in place of dorsal head and compound eyes were observed in B,C. (D-F) Induction of an ANTP target gene Teashirt (TSH). (D) y w, (E) DllGal4; UAS-DmAntp, (F) DllGal4; UAS-DapAntp embryos. Ectopic head expression of TSH was observed in E,F (arrowheads).

 


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  		Fig. 5. Repression of Drosophila limb development by DapANTP. Genotypes (B,E,H,K) AntpGal4; UAS-DmAntp; (C,F,I,L) AntpGal4; UAS-DapAntp. (A-C) Larval cuticle phenotypes. (B) Mild type II phenotype (see Table 1 legend), (C) Severe type V phenotype. Positions of Keilin’s organ, T1 and A1 denticle belts are indicated by arrowhead, cross and asterisk, respectively. (D-F) DLL expression (arrowheads). DapANTP eliminated DLL in T2 and T3 (asterisks in F). (G-L) DapANTP represses Dll through a DNA element that is a target for Drosophila UBX and ABDA. (G-I) Expression pattern of Dll-304 (purple). It reproduces DLL expression in the thorax (G) and is repressed by DapANTP (I, asterisks). (J-L) Expression of Dll-305 was not affected by either DmANTP (K) or DapANTP (L). In H and K, DmANTP was stained green to indicate the domain of ANTP expression that overlaps with T2 and T3. Leg primordia are indicated by arrowheads.

 

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