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
*Authors for correspondence [e-mail: shayashi{at}cdb.riken.go.jp (fly strains) and yshiga{at}ls.toyaku.ac.jp (Daphnia methods and sequences)]
Accepted 10 May 2002
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SUMMARY |
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Key words: HOM protein, Antennapedia, Distal-less, Evolution, Crustacean, Limb, Daphnia
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INTRODUCTION |
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Crustaceans bear several pairs of thoracic limbs in a variety of sizes as well as branching patterns that are grouped into two major types (Brusca and Brusca, 1990). Larger ones serve as legs for mainly locomotive functions. Another type of the thoracic limb, termed the maxilliped, is smaller and its branching is suppressed, resembling head appendages used for feeding functions. The number of maxillipeds varies from three pairs in Homarus to one in Mysidium and none in Artemia. Averof and Patel (Averof and Patel, 1997
) have reported that the anterior borders of Ubx/abdA expression varied in ten crustacean species examined, and that the changes correlate well with the borders of transition from maxillipeds to legs. The authors proposed that evolutional change in Ubx/abdA expression determines the number of segments bearing maxillipeds. However, what specifies the morphological characteristics of maxillipeds is not known to date. One prediction is that another homeotic gene expressed anterior to Ubx/abdA directly specifies the shape of the maxillipeds.
We have studied the expression and function of the homeotic gene Antennapedia of Daphnia magna. We first show that Daphnia Antennapedia (DapAntp) is regulated by a post-transcriptional mechanism that limits its protein expression to a subset of T1 leg primordium in a pattern complementary to the expression of Distal-less (DLL). Using assays in Drosophila, we show that DapAntp is capable of repressing Distal-less (Dll) expression and limb development. The protein region responsible for this repressive activity was mapped to the highly divergent N-terminal region of DapANTP, suggesting that functional alteration of homeotic proteins has played a significant role in the evolution of crustacean limb patterns.
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MATERIALS AND METHODS |
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Isolation of Daphnia Antp cDNA
Homeobox fragments were PCR amplified from the Daphnia magna cDNA library (Tokishita et al., 1997) using a set of primers 5'- CGCGGATTCAGACSCTGGAGCTGGAGAARGA-3' and 5'-TCCGGATCCCACTTCATGCGCCGRTTCTGRAACCA-3' that corresponds to highly conserved regions of ANTP-type genes. An ANTP-like fragment was used as a probe to screen the same library to identify full-length Antp cDNAs.
Antibody staining
Fixation and antibody staining of Daphnia embryos were basically carried out according to the protocol by Panganiban et al. (Panganiban et al., 1995) with modifications. Rabbit anti-DapANTP was raised against a recombinant peptide (residue 1 to 539) and affinity purified for immunostaining. Other antibodies used were anti-UBX/ABDA FP6.87 (Kelsh et al., 1994
), anti-DmANTP (4C3) (Glicksman and Brower, 1988
), anti-DLL (Panganiban et al., 1995
) and anti-TSH (Andrew et al., 1994
). For DapANTP/DLL double labeling, anti-DapANTP was biotinylated and affinity-purified for detection with an ABC elite kit (Vector Lab) and a TSA direct kit (New England Nuclear). Protocols for Drosophila are described in Sullivan et al. (Sullivan et al., 2000
).
Construction of mutant Antp genes
Mutant Antp genes were constructed by a series of PCR based site-directed mutagenesis, and after confirmation by DNA sequencing, cloned into pUAST (Brand and Perrimon, 1993) for germline transformation. Detailed protocols are available upon request.
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RESULTS AND DISCUSSION |
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A post-transcriptional mechanism limits Daphnia ANTP expression to Mx2 and anterior L1
To address the mechanism for regulating segmental differences of thoracic limbs, we examined homeotic gene expression in the trunk. We cloned cDNAs encoding Daphnia Antennapedia (DapAntp) and found that the encoded protein is highly homologous to Drosophila Antennapedia (DmAntp) in the region spanning the YPWM motif and the homeodomain, but the remaining protein-coding region was highly divergent (Fig. 2A,B). DapAntp mRNA was expressed broadly from the second maxilla (Mx2) to the post-thoracic segments (Fig. 3A), which was similar to the pattern reported for other crustaceans (Abzhanov and Kaufman, 2000; Averof and Akam, 1995
). By contrast, DapANTP protein was detectable only in Mx2 and the anterior region of the first thoracic segment (T1a), including L1 (Fig. 3B), suggesting that DapANTP expression is regulated post-transcriptionally.
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Daphnia ANTP specifies thoracic identities in the Drosophila head
The proposed role of DapANTP in modifying DLL expression and limb morphologies may have arisen through changes in transcriptional enhancers of its target genes such as Dll. An alternative, but non-exclusive possibility, is that a change occurred in the protein-coding region of DapANTP to alter its target specificity. To test the latter possibility, we compared the activities of DapANTP and DmANTP by using assays in Drosophila, where DmANTP is compatible with limb development. Misexpression of DapANTP in eye-antennal discs caused transformation of the antennae to the leg and of the dorsal head to the thorax similar to those caused by DmANTP (Schneuwly et al., 1987) (Fig. 4A-C). When misexpressed in embryonic heads, it induced ectopic expression of the trunk-specific gene teashirt (tsh) (Fasano et al., 1991
) (Fig. 4D-F), demonstrating that DapANTP is a functional homolog of DmANTP.
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Daphnia ANTP regulates Dll enhancer through the region normally mediating repression by UBX and ABDA
To elucidate the mechanism by which DapANTP represses DLL, we examined the transcriptional enhancer of Dll that reproduces Dll expression in the limb primordium (Vachon et al., 1992) (Dll304; Fig. 5G). While ectopic DmANTP has no effect on Dll304, DapANTP strongly repressed Dll304 expression, leaving only a small number of Dll304-expressing cells remaining (Fig. 5H,I). It has been shown that Dll304 is induced at the ventral edge of limb primordia and those cells migrate in the dorsal direction (Goto and Hayashi, 1997
). Therefore, the remaining Dll304-positive cells in DapANTP expressing embryos may be the earliest born limb primordial cells that have yet to receive the repressive effect of DapANTP. The enhancer fragment used to construct Dll304 contains multiple binding sites for UBX and ABDA. Deletion of all but one of those sites in the construct Dll305 caused de-repression in abdominal segments (Vachon et al., 1992
) (Fig. 5J). DapANTP failed to repress Dll305 effectively (Fig. 5L), suggesting that DapANTP regulates Dll enhancer through the region normally mediating repression by UBX and ABDA. It should be noted that in Drosophila embryos, the expression domain of DmANTP covers those of DLL (Casares and Mann, 1998
) (Fig. 5H). Therefore, DmANTP does not repress DLL in this stage of development.
N-terminal region of Daphnia ANTP contributes to the major functional differences
To map the region responsible for the functional differences in the two proteins, we constructed chimeric proteins and repeated all analyses. We divided ANTP proteins into three regions: the diverged N-terminal region (N), highly conserved YPWM motif and homeodomain (HD) and C-terminal tails (C, Fig. 2, Table 1). All constructs retained the activities to transform the head to the thorax, and to induce TSH expression in the head. Replacement of NDm with NDap conferred the Daphnia-specific activity (compare constructs 1 and 3 in Table 1), and a reciprocal replacement of NDap with NDm greatly compromised DapANTP activity (constructs 2, 6). Those results suggest that the majority of Daphnia-specific activity resides in the N-terminal region of DapANTP (NDap). Analyses also revealed a negative effect of CDm on Daphnia-specific activity when combined with the rest of DapANTP (construct 5). In CDm, there are two casein kinase II (CKII) phosphorylation consensus sites that are conserved in several insect species, but are not present in DapANTP. These sites are required to modulate ANTP activity in Drosophila (Jaffe et al., 1997). Mutation of CDap to create CKII sites compromised DapANTP activity (construct 8), suggesting that CKII phosphorylation inhibits DapANTP activity. However, addition of CDap to DmANTP, or mutations of CDm to disrupt CKII sites failed to provide Daphnia-specific activity to DmANTP (constructs 4, 7). Taken together, the results suggest that NDap is a major determinant of Daphnia specific activity of ANTP to repress Dll, and CDm can interfere with this activity, possibly through phosphorylation of CKII sites. NDap is two times as long as NDm and does not contain blocks of obvious sequence homology, except for a short region at the N terminus. In two places, HDDap differs from HDDm: one is conservative F to Y substitution in the homeodomain and the other in the region connecting YPWM motif and homeodomain, the latter being affected by alternative use of splicing acceptor sites (Bermingham and Scott, 1988
). The significance of these differences remains to be determined.
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We have shown here that diversification of the ANTP protein outside the homeodomain contributed to its functional variation in modifying limb patterns. The region responsible for Daphnia-specific activity was mapped to the N terminal region of ANTP that is highly diverged. Two recently works on Ubx proteins (Galant and Carroll, 2002; Ronshaugen et al., 2002
) reported that functional alteration of homeotic proteins played a significant role in restricting the number of insect limbs. This work demonstrates that an evolutional change in Antennapedia protein has contributed to a micro-evolutionary event that has produced the difference in the shape of T1 leg and T2-4 legs of Daphnia. Taken together, homeotic proteins have undergone a number of alterations in regions outside the homeodomain to change their target specificity and the way they control limb development. More importantly, Daphnia-specific ANTP activity and the pattern of its expression account for segment-specific limb morphology of Daphnia, suggesting that protein-coding regions of Hox genes serve as rich substrates for evolutional alterations that have generated segmental diversities of the crustacean limb.
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ACKNOWLEDGMENTS |
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