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.

DNA sequence analysis dictates new interpretation of phylogenic trees. Taxa that were once thought to represent successive grades of complexity at the base of the metazoan tree are being displaced to much higher positions inside the tree. This leaves no evolutionary ''intermediates'' and forces us to rethink the genesis of bilaterian complexity.A deep reorganization of the metazoan phylogenetic tree is presently taking place as a result of the input of molecular data. Far from being an exercise confined to a small circle of aficionados, the changing views on the pattern of animal interrelationships has profound consequences for understanding the underlying processes of animal diversification. As has repeatedly been stressed, we shall never be able to reason on the evolution of development and the way it has shaped animal diversity unless we have a reliable history of the path taken by this diversification. Here, we highlight the salient recent results based on genetic data, especially the displacement of taxa long thought to represent successive grades of complexity at the base of the metazoan tree, to much higher positions inside the tree. This leaves us with no evolutionary ''intermediates'' and forces us to rethink the genesis of bilaterian complexity. The reappraisal of animal evolution rests on several congruent approaches ranging from primary gene sequence analysis to qualitative molecular signatures within appropriate genes. Each of them, however, has its methodological difficulties; we shall, therefore, also try to briefly pinpoint the issues of contention and discuss the strength of the present view.Preliminary Comments on the Reliability of Phylogenetic Trees. To an outsider, the field of phylogenetic reconstruction may appear to be full of controversies and uncertainties. There have been acrimonious debates over the best methodology to use for reconstruction (phenetics vs. cladistics) and over the relative merits of morphological vs. molecular data. Worst of all, contradictory trees have kept pouring in, often with insufficient critical assessment. Obviously, we cannot review the whole field here, but we wish to emphasize three points to justify our reasoned optimism and confidence in the recent molecular phylogenies.

A posttranslational modification was detected in the carboxyl-terminal region of axonemal tubulin from Paramecium. Tubulin carboxyl-terminal peptides were isolated and analyzed by Edman degradation sequencing, mass spectrometry, and amino acid analysis. All of the peptides, derived from both alpha and beta tubulin subunits, were modified by polyglycylation, containing up to 34 glycyl units covalently bound to the gamma carboxyl group of glutamyl residues. This modification, present in one of the most stable microtubular systems, may influence microtubule stability or axoneme function, or both.

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.

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Abstract. The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sareoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation.This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg 2÷. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 t~M) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defmed the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions . Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000-19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000-250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt 40,000 . These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles ; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pl 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins . It will also be useful in future studies of trichocysts and mating reactions. J. CELL BIOLOGY