André Adoutte

Hox genes in brachiopods and priapulids and protostome evolution

Understanding the early evolution of animal body plans requires knowledge both of metazoan phylogeny and of the genetic and developmental changes involved in the emergence of particular forms. Recent 18S ribosomal RNA phylogenies suggest a three-branched tree for the Bilateria comprising the deuterostomes and two great protostome clades, the lophotrochozoans and ecdysozoans. Here, we show that the complement of Hox genes in critical protostome phyla reflects these phylogenetic relationships and reveals the early evolution of developmental regulatory potential in bilaterians. We have identified Hox genes that are shared by subsets of protostome phyla. These include a diverged pair of posterior (Abdominal-B -like) genes in both a brachiopod and a polychaete annelid, which supports the lophotrochozoan assemblage, and a distinct posterior Hox gene shared by a priapulid, a nematode and the arthropods, which supports the ecdysozoan clade. The ancestors of each of these two major protostome lineages had a minimum of eight to ten Hox genes. The major period of Hox gene expansion and diversification thus occurred before the radiation of each of the three great bilaterian clades.

The Segmented Urbilateria: A Testable Scenario

Abstract The idea that the last common ancestor of bilaterian animals (Urbilateria) was segmented has been raised recently on evidence coming from comparative molecular embryology. Leaving aside the complex debate on the value of genetic evidence, the morphological and developmental evidence in favor of a segmented Urbilateria are discussed in the light of the emerging molecular phylogeny of metazoans. Applying a cladistic character optimization procedure to the question of segmentation is vastly complicated by the problem of defining without ambiguity what segmentation is and to what taxa this definition applies. An ancestral segmentation might have undergone many complex derivations in each different phylum, thus rendering the cladistics approaches problematic. Taking the most general definitions of coelom and segmentation however, some remarkably similar patterns are found across the bilaterian tree in the way segments are formed by the posterior addition of mesodermal segments or somites. Postulating that these striking similarities in mesodermal patterns are ancestral, a scenario for the diversification of bilaterians from a metameric ancestor is presented. Several types of evolutionary mechanisms (specialization, tagmosis, progenesis) operating on a segmented ancestral body plan would explain the rapid emergence of body plans during the Cambrian. We finally propose to test this hypothesis by comparing genes involved in mesodermal segmentation.

The major lines of metazoan evolution: Summary of traditional evidence and lessons from ribosomal RNA sequence analysis

Contrary to a widespread belief among biologists, and to diagrams still found in many elementary biology or zoology textbooks, the general pattern of the phylogeny of Metazoa (the multicellular animals) is far from being settled. While there is a strong body of essentially congruent morphological, paleontological and molecular data concerning the branching pattern within some phyla or classes, most notably the vertebrates, the problem of the overall relationships of the invertebrate phyla is much more open (see Fig. 1 for a summary of conflicting schemes). These 32–36 phyla, however, account by far for most of the biological diversity of animals. They include some huge groups such as Arthopoda or Mollusca, to which many of the favorite experimental organisms discussed at this meeting belong as well as many additional groups also containing experimentally important species (e.g. Nematoda: Caenorhabditis; Echinodermata: the various sea urchins, both extensively used models in developmental biology; Annelida and Platyhelminthes, classical organisms for the study of embryology and regeneration, etc.). As illustrated in detail in books recently devoted to the subject (Barnes, 1987; Brusca and Brusca, 1990; Willmer, 1990), summarizing and updating over 200 years of comparative anatomy and embryology, defining a phylum is usually straightforward, but it is the establishment of the evolutionary relationships linking the different phyla that is difficult to achieve.

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post‐translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post‐translational epitope (non‐reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N‐terminal domain, one in the β C‐terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post‐translational modifications. Among these, three antibodies are directed against an N‐terminal acetylated epitope of α‐tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post‐translational modifications, located in the C‐terminal domain of both α‐ and β‐tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.

Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa

SummaryIn spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised againstParamecium andTetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.

Development of surface pattern during division in Paramecium: III. Study of stomatogenesis in the wild type using antitubulin antibodies and confocal microscopy

Summary Stomatogenesis, during the vegetative division of Paramecium, is a complex morphogenetic process involving massive proliferation of basal bodies and their progressive patterning to generate a new oral apparatus in the immediate vicinity of the old one. This new oral apparatus will be inherited by the posterior daughter cell while the old one remains in the anterior daughter cell. We have carried out a detailed description of the whole process, using new antibodies and confocal microscopy, allowing visualization of all basal bodies and a number of other cytoskeletal elements, thus completing our previous description of morphogenesis. The following inferences could be made: 1) The new oral structures are exclusively formed from the anarchic field by a two step wave of basal body duplication. These two steps strictly parallel, slightly in advance, those we have previously detailed at the level of the “somatic” cortex, suggesting *) that a common set of biochemical cascades is involved, **) that the OA may be triggering these cascades. 2) The initial proliferation of basal bodies occurs in the immediate vicinity of the parental “paroral kinety” which itself remains invariant and appears to dictate the progressive patterning of the oral anlage (as can be inferred from the staining intensity, distance and alignment of the basal bodies of the anarchic field with respect to the paroral kinety). This suggests that the concept of cytotaxis or structural guidance can be extended to the genesis of an elaborate set of basal bodies. 3) The number of basal bodies within the anarchic field is determined during this formation of the new oral apparatus. Thus, the size and possibly part of the pattern of the new OA is determined in one generation, with storage of morphogenetic potentialities used in the next generation. In this process, a slight cortical rotation around the cell axis is effected at each division. 4) The whole morphogenetic assemblage up to the level of the whole cell displays an overall left-right asymmetry which is progressively built up upon the basic asymmetry of the basal body.

Purification, in vitro reassembly, and preliminary sequence analysis of epiplasmins, the major constituent of the membrane skeleton of Paramecium.

The epiplasmic layer, a continuous rigid granulo-fibrillar sheet directly subtending the surface membranes of Paramecium, is one of the outermost of the various cytoskeletal networks that compose it cortex. We have previously shown that the epiplasm consists of a set of 30 to 50 protein bands on SDS-PAGE in the range 50 to 33 kDa, the epiplasmins. We report a purification procedure for the set of epiplasmic proteins, a description of their physicochemical and reassembly properties, and a preliminary characterization of their sequence. The conditions for solubilization of the epiplasm and for in vitro reassembly of its purified constituents ar described. Reassembly of the entire set of proteins and of some (but not all) subsets are shown to yield filamentous aggregates. Microsequences of two purified bands of epiplasmins reveal a striking amino acid sequence consisting of heptad repeats of only three main amino acids, P, V, and Q. These repeats were confirmed by DNA sequencing of polymerase chain reaction products. The motif is QPVQ-h, in which h is a hydrophobic residue. This may constitute the core of the epiplasmin sequence and, in view of the tendency of such a sequence to form a coiled-coil, may account for the remarkable self-aggregation properties of epiplasmins.

The surface pattern of Paramecium tetraurelia in interphase: an electron microscopic study of basal body variability, connections with associated ribbons and their epiplasmic environment

Summary In Paramecium tetraurelia, thousands of basal bodies with their three associated rootlets are anchored into the submembranous skeleton, the epiplasm, which is itself segmented into cortical units. In this study, we redescribe more precisely (after Pitelka [31], Hufnagel [19] and Allen [1]) the architecture of these structures after detergent treatment and tannic acid contrast reinforcement. Although this technique leads to loss of membranes and of some labile cytoskeletal elements, it allows to better underline how the three rootlets are linked together and to the basal body. The postciliary microtubules and ciliary rootlets (Kd fibers) are bound together by a “fingered node”, and the transverse microtubules to the previous fibers by dense links. The anterior basal body of the pairs is linked to the ciliary rootlet by a newly identified architecture in this species, the “bone-like node” [1]. A specific ciliary domain can be defined in the epiplasm of each cortical unit: epiplasmic rings encircling the basal bodies are closely apposed on the thicker part of the epiplasm and can include some apertures along the basal bodies towards the cilia. The thickness of the epiplasm seems to vary according to the presumptive elongation direction of the cortical units. Each basal body associated rootlet is specifically linked to the epiplasm itself. Because of their unexpected set of linkage organization, these rootlets may ensure the ciliary cohesiveness in the interphase cell as well as the transmission of the ciliary polarity during division as it was previously discussed [19]. We also show for the first time that there is a variability of the basal body architecture at the cellular level: its height, and the number of cartwheel elements change depending on its location on the cortex and its morphogenetical history; the longest basal bodies with 5 cartwheel elements are found in territories which result from hyperduplication and become invariant, i.e. do not undergo duplication during the next divisions, as is the case along the sutures and in the left oral and cortical areas [22]. These results are discussed in terms of morphogenetical behavior.

ATP synthase subunit c/III/9 gene sequences as a tool for interkingdom and metaphytes molecular phylogenies.

SummaryThe 38 sequences of the ATPase c/III/9 gene determined in bacteria, fungi, mammals, and higher plants have been used to construct phylogenetic trees by distance matrix and parsimony methods (checked by bootstrapping); alignments have been performed on the deduced amino-acid sequences and then transferred back to the nucleotide sequences. Three lineages stand out: (1) eubacteria (except cyanobacteria and a purple bacteria), (2) chloroplasts, together with cyanobacteria, and (3) mitochondria together with nuclei and α purple bacteria. The clear monophyly of the mitochondrial/nuclear lineage, taken all together, strongly suggests that the nuclear copies of the gene now residing in the eukaryotic nucleus originate from a mitochondrial transfer. Within this lineage, metaphytes emerge late and as a cohesive group, after fungi (as a dispersed group) and metazoa, yielding an order that markedly differs from that obtained through typical RNA nuclear molecules. The possible biphyletic origin of mitochondria based on mitochondrial rRNA sequences is not evidenced by these sequences. Internal branches within both the chloroplastic and the mitochondrial lineages are consistent with botanical evolutionary schemes based on morphological characters. In spite of its relatively small size, the ATPase c/III/9 gene therefore displays remarkable properties as a phylogenetic index and adds a new tool for molecular evolutionary reconstructions, especially within the metaphytes.

Nuclear and cortical regulation in doublets of Paramecium: I. Interactions between macronucleus and contractile vacuoles and their implication on the frequency of autogamy

Summary The macronucleus of ciliates is a huge “bag” of DNA whose mode of segregation during cell division is still not understood (no chromosome condensation, no spindle, no centrosomes or functional equivalents). We unexpectedly obtained some new hints on this process through the analysis of doublets of Paramecium . We had previously noted [32] that autogamy occurred at a seemingly higher frequency in regulating lines. In the present work, we studied this process in P. tetraurelia and in P. undecaurelia which has a long, more convenient, interautogamous interval. We confirmed a highly significant decrease in duration of the interautogamous interval in both species and tried to determine its causes. The process of regulation which concerns the return of doublet state to the singlet one was previously described in part [24. 61, 35]. We observed its course through successive vegetative and sexual cycles. We first described precisely the contractile vacuole (CV) systems, and their number, extension and relations with the nuclear division events, in normal cells and in tam mutant cells of P. tetraurelia which are known to show abnormal nuclear division [6, 58]. We deduced that cortical and nuclear events are correlated during division by means of the contractile vacuole system (immunocytology and TEM observations). In regulating doublets, the enlarged cell volume leads to an increase in the number of CVs on two meridians M1, M2 and to the changes of their positioning and extension. CVM2 disappears and the number of CVs decreases when the angle between both oral apparatuses decreases from 180° towards 90°. Our results strongly suggest that abnormal number, size and position of macronuclei in doublets are related to number, spreading and positioning of the CVs. During division of doublets, the macronuclei appear to be “quartered” between both dorsal surfaces bearing the CVMs, and this leads to gross asymmetries in the distribution of the macronuclear DNA to the two daughter doublets [8]. Abnormal numbers of micronuclei and macronuclear anlagen are observed along with the evolution of the regulation process and, in turn, appear to lead to numerous anomalies at autogamy, a process which also requires correct intracellular positioning of meiotic and postmeiotic nuclei. From such observations, we deduced that contractile vacuoles, through their microtubular rootlets, exert a major function in ensuring proper macronuclear segregation during amitosis, such as the role played by “atractophores” in other protists during extranuclear pleuromitosis [57].