Germ cells play a unique role in gamete production, heredity and evolution. Therefore, to understand the mechanisms that specify germ cells is a central challenge in developmental and evolutionary biology. Data from model organisms show that germ cells can be specified either by maternally inherited determinants (preformation) or by inductive signals (epigenesis). Here we review existing data on 28 metazoan phyla, which indicate that although preformation is seen in most model organisms, it is actually the less prevalent mode of germ cell specification, and that epigenetic germ cell specification may be ancestral to the Metazoa.

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.

Most of our knowledge about the mechanisms of segmentation in arthropods comes from work on Drosophila melanogaster. In recent years it has become clear that this mechanism is far from universal, and different arthropod groups have distinct modes of segmentation that operate through divergent genetic mechanisms. We review recent data from a range of arthropods, identifying which features of the D. melanogaster segmentation cascade are present in the different groups, and discuss the evolutionary implications of their conserved and divergent aspects. A model is emerging, although slowly, for the way that arthropod segmentation mechanisms have evolved.

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of (7,8), their sequences were determined by the dideoxy chain-termination method (7), and overlaps were determined by computer methods (9). TMV proved to be a poor template for the synthesis of cDNA longer than 2,000 nucleotides, and we found that the efficiency of the second-strand reaction was highly sequence dependent. These problems were circumvented by using a series of synthetic oligodeoxynucleotides to prime synthesis at either random or specific sites along the molecule. Efficient cloning of molecules that were rendered double-stranded was achieved by cleaving the synthetic DNA with restriction endonucleases. The use of several restriction enzymes ensured that overlapping sequences were cloned.Priming with Synthetic Oligonucleotides. Mixtures of fourto seven-residue oligonucleotides, synthesized by phosphodiester chemistry (10), were used as nonspecific primers on TMV RNA or on cDNA. Double-stranded cDNA to most of TMV genome could be synthesized by using these primer "cocktails." To direct the synthesis of double-stranded cDNA to the termini and other poorly sampled regions of TMV RNA, oligonucleotide primers of 13 to 17 residues were synthesized by the solid-phase phosphotriester method (11 1,813-1,829 and 3,159-3,172) were synthesized to obtain clones to the 5' sides of regions of TMV RNA well represented by cDNA clones from previous "shotgun" experiments. cDNA priming was with 10-to 100-fold molar excess of oligonucleotide over TMV RNA template (usually 5 ,ug of TMV RNA in a 25-,u reaction mixture) and standard incubation conditions for reverse transcription were used (12): 42°C and 60 min, with a 30°C and 15-min preincubation for the short oligonucleotide cocktails.Second-Strand Synthesis, Cloning, and Assembly of the Sequence. cDNA freed from RNA by alkaline hydrolysis (100 mM NaOH, 1 mM EDTA for 15 min at 70°C) was used as a template for second-strand synthesis primed by "flip back" or added oligonucleotide primers. The standard reaction used Klenow DNA polymerase and incubation was in 10 mM Tris HCI, pH 7.4/ 10 mM MgCl2/10 mM dithiothreitol/100 mM NaCI/50 AM each deoxynucleoside triphosphate at 37°C for 30 min. In some experiments no attempt was made to purify the cDNA. Oligonucleotides generated in the first-strand reaction were used to prime second-strand synthesis on TMV cDNA by using the conditions of Wickens et al. (12) or the conditions described above after melting and annealing desalted products ofthe firststrand reaction (alkali treatment of the first-strand reaction product was shown by these approaches not to cause deamination of cytidine residues). These double-stranded cDNAs were digested by restriction endonu...

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

Crustaceans and insects share a common origin of segmentation, but the specialization of trunk segments appears to have arisen independently in insects and various crustacean subgroups. Such macroevolutionary changes in body architecture may be investigated by comparative studies of conserved genetic markers. The Hox genes are well suited for this purpose, as they determine positional identity along the body axis in a wide range of animals. Here we examine the expression of four Hox genes in the branchiopod crustacean Artemia franciscana, and compare this with Hox expression patterns from insects. In Artemia the three 'trunk' genes Antp, Ubx and abdA are expressed in largely overlapping domains in the uniform thoracic region, whereas in insects they specify distinct segment types within the thorax and abdomen. Our comparisons suggest a multistep process for the diversification of these Hox gene functions, involving early differences in tissue specificity and the later acquisition of a role in defining segmental differences within the trunk. We propose that the branchiopod thorax may be homologous to the entire pregenital (thoracic and abdominal) region of the insect trunk.

The bithorax complex in Drosophila melanogaster is a cluster of homeotic genes that specify developmental pathways for many of the body segments of the fly. The DNA of the bithorax complex has been isolated, and a region of 195,000 base pairs that covers the left half of the complex is described here. The lesions associated with many of the bithorax complex mutants have been identified, and most are due to DNA rearrangements. Most of the spontaneous mutants have insertions of a particular mobile element named "gypsy." This element affects the functions of sequences removed from the site of insertion. Mutant lesions for a given phenotypic class are distributed over large DNA distances of up to 73,000 base pairs

The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network.