Dev Genes Evol (2006) 216: 443–449 DOI 10.1007/s00427-006-0088-1
ORIGINA L ARTI CLE
Élodie Géant . Emmanuèle Mouchel-Vielh . Jean-Pierre Coutanceau . Catherine Ozouf-Costaz . Jean S. Deutsch
Are Cirripedia hopeful monsters? Cytogenetic approach and evidence for a Hox gene cluster in the cirripede crustacean Sacculina carcini Received: 24 October 2005 / Accepted: 1 May 2006 / Published online: 14 June 2006 # Springer-Verlag 2006
Abstract The “hopeful monster” has haunted evolutionary thinking since Richard Goldschmidt coined the phrase in 1933. The phrase is directly related to genetic mechanisms in development and evolution. Cirripedes are peculiar crustaceans in that they all lack abdomens as adults. In a previous study aimed at describing the repertoire of Hox genes of the Cirripedia, we failed to isolate the abdominalA gene in three species representative of all three cirripede orders. To address the question of whether the cirripede ancestor could have been a “hopeful monster” arising from a rearrangement of the Hox complex, we have performed a cytogenetic analysis of the Hox complex of the cirripede Sacculina carcini. We present here molecular and cytogenetic evidence for the grouping of the Hox genes on a single chromosome. This is the first direct evidence reported for the grouping of Hox genes on the same chromosome in a non-insect arthropod species. Keywords Hox complex . Cytogenetics . FISH . Evolution of body plans
Communicated by guest editors Jean Deutsch and Gerhard Scholtz É. Géant . J. S. Deutsch (*) Développement et Évolution, UMR 7622, CNRS et Université P. et M. Curie, case 24, 9 quai St-Bernard, 75252 Paris cedex 05, France e-mail:
[email protected] Tel.: +33-1-44272576 Fax: +33-1-46459671 E. Mouchel-Vielh Chromatine et Développement, UMR 7622, CNRS et Université P. et M., Curie, Paris, France J.-P. Coutanceau . C. Ozouf-Costaz Service de Systématique Moléculaire, IFR 101, Département Systématique et Évolution, Muséum National d’Histoire Naturelle, Paris, France
Introduction: the hopeful monster and the cirripedes The “hopeful monster” has haunted evolutionary thinking since Richard Goldschmidt coined the phrase in 1933. The phrase is directly related to genetic mechanisms in development and evolution. The provocative ideas of Richard Goldschmidt were rejected in his time, and still are nowadays (e.g. Akam 1998), on two grounds: First he claimed that the Synthetic Theory was not able to account for the origin of species, but only for differentiation (genetic structuring) within a species. Second, he rejected Morgan’s concept of the particulate gene. He also put emphasis on homeotic mutations and on the importance of chromosomal arrangement for gene function. As for the first point, we now have good examples of speciation that fit with the “Synthetic Theory”, as they can be drawn by accumulation of small mutations and change in allelic frequencies. But this does not imply rejection of other possible mechanisms being instrumental in the speciation process. Gradualism in evolution has been challenged in the past few decades (e.g. Gould and Eldredge 1977). On the second point, the view of the chromosome where indivisible genes are arranged as a collar of pearls is now abandoned, but not in favour of the “systemic mutation” proposed by Richard Goldschmidt (1940). On another side, the genetic analysis of homeotic mutants in Drosophila, their grouping in gene complexes (Lewis 1978), the discovery of the homeobox (Scott and Weiner 1984; McGinnis et al. 1984) and the discovery of the conservation of the structure and function of the Hox genes were milestones in the biology of the second half of the 20th century, and were at the origin of developmental genetics in model organisms and of comparative developmental genetics, i.e. so-called “evo–devo”. The Hox complexes would have been the honey of Richard Goldschmidt (who died in 1958). To give examples of what he meant by “hopeful monsters”, Goldschmidt wrote: “What appear to-day as a monster will be to-morrow the origin of a line of special adaptations. The
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dachshund and the bulldog are monsters. But the first reptile with rudimentary legs or fish species with bulldog-heads were also monsters” (Goldschmidt 1933, p 544). “A Manx cat with a hereditary concrescence of the tail vertebrae, or a comparable mouse or rat mutant, is just a monster. But a mutant of Archaeopteryx producing the same monstrosity was a
Fig. 1 “Hopeful monsters”. a-b Goldschmidt’s example: the ancestor of modern birds. “A mutant of Archaeopteryx with a hereditary concrescence of the tail vertebrae was a monster (Goldschmidt 1940, p 390). a Skeleton of Archeopteryx; b skeleton of Columba, from Strickberger 1990 (Fig. 17–27, p 359). c-d Monstrous reduction of the abdomen in Cirripedia vs Ascothoracida. The Ascothoracida are the sister group of the Cirripedia. A conspicuous synapomorphy of Cirripedia is their drastic reduction of the abdomen. c Sacculina carcini, Rhizocephala Cirripedia Thecostraca, cypris larva. Adapted from Mourlan et al. 1985, Fig. 1; t1–t6: thoracic segments bearing cirri; f furca, i.e. the appendage of the telson. Note that there is no segment appearing in between the thorax and the telson. d Dendrogaster astericola, Ascothoracida Thecostraca, adult. Adapted from Calman 1909 (Fig. 78, p 128). a abdomen
hopeful monster because the resulting fanlike arrangement of the tail feathers was a great improvement in the mechanics of flight” (Goldschmidt 1940, p 390) (Fig. 1a,b). It is noteworthy that, in these examples, the monstrosity comes from the reduction of some organ. To all naturalists, this sounds familiar. Darwin (1859) devoted almost half a chapter of The Origin of Species (p 428 et ss.) to “Rudimentary, atrophied or aborted organs”. He discussed in two places (p 177 and p 428) the known case of beetle species with rudimentary or absent wings, and how this could be favoured by natural selection. It is clear that a pterygote insect without wings is a monster. Now we found that cirripedes strikingly resemble birds in this respect: they have lost a posterior part of the body that was present in their ancestor’s body plan (Fig. 1c,d). Naturalists have been interested in cirripedes since antiquity because of their strange morphology and habits. Indeed, the most common cirripedes, i.e. ordinary barnacles, possess two characters rarely found together: they live in a calcareous shell, like bivalve molluscs, and they bear articulated biramous appendages, like crustaceans. They develop, through nauplius larvae, a feature that permitted their classification as bona fide crustaceans. As an additional puzzling character, they have no complete abdomen (Darwin 1851, 1854; Anderson 1994). During the last decade, we have undertaken the revisiting of the cirripedes’ body plan using molecular tools in an “evo– devo” approach (see Deutsch et al. 2004 for review). We have shown that tiny vestigial abdominal segments are still present in nauplius larvae of the rhizocephalan cirripede Sacculina carcini (Gibert et al. 2000; Blin et al. 2003). We have performed an extensive search for Hox genes and could not find any abdominal-A (abdA) gene in three cirripede species representative of the three orders of Cirripedia, whereas we found it in Ulophysema oeresundense, representative of the Ascothoracida, Cirripedia’s sister-taxon (Mouchel-Vielh et al. 1998). Using an antibody that recognises both Ultrabithorax (Ubx) and AbdA proteins, we found no evidence for any abdA expression during larval development of Sacculina (Blin et al. 2003). We thus concluded that the Hox gene abdA must have been profoundly derived or lost during the evolution of the cirripede stem lineage. As Richard Goldschmidt put emphasis on chromosome rearrangements as examples of possible “systemic mutations” involving several genes at once while keeping a valid “rate of reaction” (Goldschmidt 1940), we sought to look into whether the Hox complex of cirripedes is rearranged. We here report a cytogenetic analysis of the Hox genes by fluorescent in situ hybridisation (FISH) on metaphase chromosomes of S. carcini. All Hox probes assayed hybridised on the same chromosome. This is the first evidence for Hox genes being clustered on the same chromosome in a non-insect arthropod.
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Materials and methods Biological material Carcinus maenas (green crabs) infested by the rhizocephalan parasite S. carcini were collected on the seashore near the Marine Biology Station at Roscoff (Brittany, France). The external part of the parasite, so-called “externa”, is nothing but a sack containing the ovaries filled with thousands of developing embryos. Usually, a crab is infested by a single Sacculina individual. Colchicin treatment The parasites were injected with 0.5 to 1 ml, depending on size, of a colchicin solution (20 ng/μl) through the opening of the externa. Crabs were then kept at 4°C for 20 h. Cell and chromosome preparation The externa was removed from the crab and opened in saline buffer. Ovaries were dissected to isolate embryos in hypotonic buffer (tri-sodium citrate 0.9 g/l). Ovarian tubules were smashed and filtered on a nylon screen (50 μm mesh) upon a tube. After filtration, the volume was completed to 15 ml with hypotonic buffer. After 30 min, at room temperature with gentle agitation, the hypotonised cell suspension was pre-fixed with two drops of Carnoy fixative (methanol to acetic acid ratio of 3:1) and centrifuged at 1,500 turns (t)/min at 4°C for 10 min. The pellet was gently re-suspended in ice-cold Carnoy fixative up to 15 ml. The pellet was left for 7 min at room temperature then centrifuged at 2,000 t/min at 4°C for 7 min. This step was repeated twice. Slides were prepared under standard procedures after controlling mitotic index and cell dispersion and metaphase plate quality on a Giemsa-stained chromosome preparation. Series of airdried slide preparations were kept at 4°C (a few days) or at −20°C (several weeks).
MEGA) kits. Labelled probes were purified using Quick Spin columns, then precipitated with ethanol in the presence of sodium acetate. For each lambda phage, two probes were prepared, one digoxygenin- and the other biotin-labelled. A probe stock solution was prepared with labelled DNA dissolved at a concentration of 50 ng/μl in hybridisation buffer [50% formamide; 2× saline-sodium citrate (SSC); 10% dextran sulphate, pH 7]. Fluorescence in situ hybridisation Chromosome preparations were thawed just before use. Slide preparations were denaturated for 10 s in (50% formamide, 2× SSC, pH 7), then dehydrated by successive baths in cold ethanol (70, 80 and 100%), 2 min each, and left drying at room temperature for a few minutes. For hybridisation with a single probe, a working solution was used by diluting the stock solution in hybridisation buffer to 15–20 ng/μl. For double probing, the hybridisation mixture contained each probe at 20 ng/μl. Probes were denaturated by heating at 74°C for 5 min, then cooled in crushed ice. Of this mixture, 15 μl was laid on each slide, and then covered with a plastic cover slip (22×22 mm). Slides were allowed to hybridise for 3 to 4 days at 37°C in a wet chamber. Coverslips were then removed and the slides were washed with 2× SSC at 74°C for 5 min, then transferred to phosphate buffer-detergent (PBD, Q-Biogene) for 1 to 2 min at room temperature. Detection was performed with either 40 μl of flourescein isothiocyanate– avidine (for biotin probes) or rhodamine–anti-dig (for digoxygenin probes) from Roche, according to the supplier’s protocol. Briefly, reagents were laid on each slide and incubated under coverslip (24×40 mm) at 37°C for 5 min in wet chamber. The cover slides were then
Hox probes A genomic DNA library of S. carcini has been constructed in λ phage FIX-II (Stratagene) (Gibert et al. 2000). Phages containing the Hox genes Sex combs reduced (Scr), fushi tarazu (ftz) and Antennapedia (Antp) have been previously described (Mouchel-Vielh et al. 2002). Using as probes cDNAs of the Hox genes labial (lab), Ubx and AbdominalB (AbdB) (GenBank accession numbers DQ231558, AF393442 and AF481736, respectively), phages containing the corresponding genes were isolated. The size of genomic DNA inserts lies between 15 and 20 kb. Phage DNA was isolated using the Lambda System kit from Qiagen. Fluorescent probes were prepared by random priming using either BioPrime DNA Labelling System (GIBCO-BRL) or Prime-a-Gene Labelling System (PRO-
Fig. 2 Sketches of the S. carcini Hox complex. a The putative ancestral arthropod Hox complex. From various sources, we can derive that the Hox genes’ repertoire of the ancestral arthropod comprised 10 Hox genes, clustered in a precise order in a single complex. The ancestral genes are here given the names of their orthologous Drosophila genes. b The S. carcini Hox complex, as determined from our previous and present works, under the hypothesis of maximal similarity with the ancestral arthropod complex. White squares: non-identified genes; stars: accumulation of mutations including repetitive sequences. c Sacculina carcini Hox complex, under the hypothesis of inversion of a part of the complex due to recombination within repetitive sequences. This rearrangement may lead to partial or complete loss of the abdA gene. This hypothesis is equally consistent with our data
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Results and discussion Molecular walk on the chromosome
Fig. 3 Sacculina carcini karyotype. The karyotype was established from 4′,6-diamidino-2-phenylindole staining of metaphase plates. Chromosomes are classified by decreasing size and not according to their shape because, for some pairs, centromere position could not be clearly established due to the pairs’ very small size. Scale bar: 5 μm. The smallest chromosome pair hybridised with Hox probes (bottom right)
removed and slides were rinsed three times 2 min with PBD. After drying, slides were mounted with 100 μg/ml 4′,6-diamidino-2-phenylindole counterstaining in anti-fade (Vectashield from Vector). Slides were analysed with a karyotyping–FISH imaging workstation (Applied Imaging, GENUS software) equipped with a cooled digital camera monochrome camera and a Zeiss Axiophot fluorescence microscope. Negative control A control was made using the vector phage DNA without insert as probe. This control DNA was treated the same way as experimental phage DNAs containing Hox genes’ inserts, similarly labelled with biotin and digoxygenin and hybridised on the same preparations as the experimental probes. No signal was detected on chromosomes (see below).
Fig. 4 FISH with ftz and Antp on Sacculina chromosomes. aftz probe labelled with biotin (green). b Antp probe labelled with digoxygenin (red). c Merge
A molecular walk (Spierer et al. 1983) using a genomic library probed with cDNA clones corresponding to Hox genes previously enabled us to build a contig and a molecular map showing that the Hox genes Scr, ftz and Antp are physically linked in the S. carcini genome (Mouchel-Vielh et al. 2002). As we failed to find the Hox gene abdA in our extensive search for Hox genes in three cirripede species (Mouchel-Vielh et al. 1998), we sought to establish a similar contig between Ubx and AbdB, assuming the location of the abdA gene in between (Fig. 2a). However, walking from both the 3′ and 5′ sides of the AbdB gene, we encountered repetitive sequences that prevented the selection of adjacent genomic clones. This was quite disappointing because it prevented us from building a contig including Ubx and AbdB. On the other side, finding repetitive sequences near Hox genes is very unexpected if the Hox complex were integral. Indeed, Hox complexes of mouse and human are devoid of repetitive sequences, although repetitive sequences represent more than 90% of mammalian DNA. Similarly, no repetitive sequence was detected by hybridisation using any of the phage DNA comprising the Scr–Antp contig. Hence, this could be taken as evidence for an unusual structure in the Ubx–AbdB region of the Hox complex. Sacculina carcini karyotype By examining metaphase plates, we were able to determine the karyotype of S. carcini. It is composed of 28 chromosome pairs (Fig. 3). Previous determinations (Austin 1987; Lécher et al. 1995) gave a range from 14 to 28 for chromosome pair numbers in rhizocephalan cirripedes, whereas this number is much more constant (2n=32) among various thoracican species (Austin 1987). For S. carcini, two different isolates yielded two different numbers (2n=42 and 2n=48) (Lécher et al. 1995). Our own determination gives a third number. Sacculina, like all other rhizocephalans, has separate sexes (Hoeg 1995). However, these variations in chromosome number do not reflect sex differentiation, contrary to what was once assumed (Yanagimachi 1961).
447 Fig. 5 Negative control: FISH with phage probes. a Lambda phage vector labelled with digoxygenin. b Lambda phage vector labelled with biotin. Background staining outside nuclei ensures that probes were actually present on the slides
Sacculina is a parasite, so its dispersal as an adult is limited by its host. Dispersal is performed by larvae, but may be limited (Reisser and Forward 1991), thus enabling genetic divergence between distant isolates. This variation in karyotype may reveal a hidden process of speciation. Indeed, the number of morphological characters that could permit the distinguishing of different isolates as different species or sub-species is low or null. In addition, the actual number we observed in our isolate of S. carcini (2n=56), compared to that reported for another species, Sacculina eriphiae (2n=28) (Lécher et al. 1995), leads us to postulate a diploidisation event during the evolution of this genus. Whatever the case, this putative duplication event did not lead to duplication of the Hox cluster (see below). The shape and small size of the chromosomes we observed (about 1 to 4 μm) are consistent with previous reports on rhizocephalan chromosomes (Lécher et al. 1995). Chromosomal localisation of Sacculina Hox genes Among the eight Hox genes previously identified in S. carcini (Mouchel-Vielh et al. 1998), five were assayed in the present study for localisation by FISH on chromosomes, namely, lab, ftz, Antp, Ubx and AbdB. Hybridisations with a single probe and with two probes together were performed, each time with two fluorescent dyes, biotin and digoxygenin. For double hybridisations, both combinations were assayed. Thus, for each couple of genes, six experiments were performed: gene A-biotin, gene A-dig, gene B-biotin, gene B-dig, gene A-biotin and gene B-dig, gene A-dig and gene B-biotin. Several metaphase plates
Fig. 6 FISH with Ubx and lab on Sacculina chromosomes. a lab probe labelled with biotin (green). b Ubx probe labelled with digoxygenin (red). c Merge
were observed per slide. In all cases, results were consistent. As we knew from the molecular walk on the chromosome that the Scr, ftz and Antp genes were linked on the same contig (Mouchel-Vielh et al. 2002), we chose the ftz and Antp probes to perform the first hybridisations as a positive control. Results confirmed our previous finding: ftz and Antp are located on the same chromosome (Fig. 4). More precisely, these Hox genes are located on the smallest chromosome (Fig. 3). Both signals are superimposed, giving a yellow colour (Fig. 4c). As our negative controls using a phage probe with no insert never showed fluorescence spots on chromosomes (Fig. 5), fluorescence could not be due to unspecific sequences being part of the phage vector. From the molecular map, the two probes do not overlap, and the distance between them can be estimated to be about 33 kb. Overlapping fluorescence signals is expected because FISH on chromosomes cannot resolve genes that are located less than 1 Mb apart (C. Ozouf-Costaz and J.-P. Coutanceau, personal observations). Going further, we showed that Antp and Ubx are similarly linked (not shown). To have a more extended view on the Hox complex, we performed hybridisations using Ubx and lab, the latter being, supposedly, the most 3′ gene in the complex (Fig. 2a). Again, it showed that the two Hox genes are located on the same chromosome (Fig. 6). From the molecular walk in the Scr to Antp region (Mouchel-Vielh et al. 2002), the distance between two Hox genes can be estimated as 50 kb at most. This gives an estimated distance of 350 to 400 kb between Ubx and lab. This is again consistent with overlapping signals (Fig. 6c).
448 Fig. 7 FISH with AbdB and Ubx, AbdB and lab. a Merge picture of AbdB probe labelled with digoxygenin (red) and Ubx probe labelled with biotin (green). b Merge picture of AbdB probe labelled with biotin (green) and lab probe labelled with digoxygenin (red)
As we were most interested in the region near the AbdB gene where the abdA gene could be located, we performed hybridisations using probes corresponding to the following Hox genes: Ubx and AbdB, and lab and AbdB. This showed that all three genes are located on the same chromosome (Fig. 7).
Conclusion Bringing together cytogenetic analysis and molecular walk on chromosomes, we showed that the six Hox genes lab, Scr, ftz, Antp, Ubx and AbdB are located on the same chromosome of S. carcini (Fig. 2b). This is the first experimental evidence for the grouping of Hox genes in a complex in a non-insect arthropod. It is very likely that the remaining two Hox genes previously identified, namely, proboscipedia and Deformed (Mouchel-Vielh et al. 1998), are situated between lab and Scr on the same chromosome. The unusual presence of repetitive sequences in the vicinity of the Ubx and AbdB genes could be indicative of a chromosomal rearrangement. Our results exclude any event leading to transposition to another chromosome of a part of the Hox complex, for instance, AbdB and, partially or totally, the abdA gene. However, a chromosomal rearrangement within the chromosome is still not excluded. An extreme version of a possible rearrangement is shown on Fig. 2c. Such rearrangements, with breaks of the Hox complex still maintaining all Hox genes on the same chromosome, have occurred several times during insect evolution. In Drosophila melanogaster and closely related species, the complex is broken between Antp and Ubx genes (Lewis et al. 2003), it is broken between Ubx and abdA genes in Drosophila virilis (von Allmen et al. 1996) and in Drosophila immigrans, whereas in Drosophila buzzatii and Drosophila repleta an additional break brings the lab gene in the vicinity of abdA (Negre et al. 2003). Outside flies, a split is observed between lab and the other Hox genes in the silk moth Bombyx mori (Yasukochi et al. 2004). If abdA were lost in the genome of the Cirripedia, it would have been possible to detect a break of the Hox complex between Ubx and AbdB. Unfortunately, such a break, if any, could not be detected in S. carcini because of the lack of resolution of the FISH technique and the small
size of the chromosome we studied. Thus, between deletion of abdA and accumulation of point mutations, both hypotheses stand unfalsified. There is still no evidence for any “systemic mutation”, as postulated by Richard Goldschmidt. This conclusion may seem disappointing to some people, and satisfactory to others. Acknowledgements The authors want to warmly thank Catherine Rigolot, who isolated and sequenced the Sacculina labial cDNA, and Danièle Chassoux, who gave advice and participated on the FISH on DNA fibres. J.S. Deutsch warmly thanks all those who have accompanied him during this cirripede scientific adventure. In the first place, his mentor in cirripede biology, Professor Yves Turquier, and then, many thanks to all the students and members of his team that collaborated on the cirripede project for 12 years or so. In addition, many thanks to Professor Jean Guerdoux for his constant support, to Professor André Toulmond for providing all the facilities of the Station Biologique de Roscoff and all members of the Station for their welcome, and to Professor Jens Hoeg for discussions on our common favourites, the cirripedes, and for his insightful comments on a previous version of this manuscript. Authors’ contributions E. Géant performed most of the experimental work (i.e. preparing probes and performing FISH on chromosomes and fibres) as part of her undergraduate lab training. E. Mouchel-Vielh constructed the S. carcini’s genomic DNA and cDNA libraries, isolated the Hox cDNAs, performed the genomic walk on the chromosomes and prepared the high-molecular weight DNA used as probe for FISH on DNA fibres, J.-P. Coutanceau and C. Ozouf-Costaz helped with FISH on chromosomes, J.-P. Coutanceau prepared and analysed the karyotype, C. Ozouf-Costaz gave precious advice in preparing chromosomes and hybridisation, J.S. Deutsch dissected the crabs and their cirripede parasites, launched the project and wrote the paper. Although this was a friendly, cooperative work, J.S. Deutsch is solely responsible for debatable ideas on the “hopeful monster”.
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