Abstract: Chelicerates represent a basal arthropod group, which makes them an excellent system for the study of evolutionary processes in arthropods. To enable functional studies in chelicerates, we developed a double-stranded RNA-interference (RNAi) protocol for spiders while studying the function of the Distal-less gene. We isolated the Distal-less gene from the spider Cupiennius salei. Cs-Dll gene expression is first seen in cells of the prosomal segments before the outgrowth of the appendages. After the appendages have formed, Cs-Dll is expressed in the distal portion of the prosomal appendages, and in addition, in the labrum, in two pairs of opisthosmal (abdominal) limb buds, in the head region, and at the posterior-most end of the spider embryo. In embryos, in which Dll was silenced by RNAi, the distal part of the prosomal appendages was missing and the labrum was completely absent. Thus, Dll also plays a crucial role in labrum formation. However, the complete lack of labrum in RNAi embryos may point to a different nature of the labrum from the segmental appendages. Our data show that the expression of Dll in the appendages is conserved among arthropods, and furthermore that the role of Dll is evolutionarily conserved in the formation of segmental appendages in arthropods.

Abstract: Pigment patterns of Danio fishes are a tractable system for assessing the developmental genetic bases for the evolution of adult form in vertebrates. These pigment patterns include multiple horizontal melanophore stripes in the zebrafish D. rerio, a complete absence of stripes in D. albolineatus, a few broad stripes in D. kerri, and a combination of stripes and spots in D. nigrofasciatus. Here we assess the genetics of pigment pattern development and evolution using interspecific hybrids. We first reconstruct the phylogenetic relationships of these species by analyzing mitochondrial 12S and 16S rDNA sequences. We find a clade comprising several small species of danio, and within this clade a sister taxon relationship between D. rerio and D. nigrofasciatus. We also find that the large bodied D. dangila is more closely related to the clade of small danios than other large bodied species. As a first step in evaluating the genetics of pigment pattern diversification in the group, we then examine the phenotypes of interspecific hybrids. Adult pigment patterns of hybrids between D. rerio and other danios are in many respects more similar to D. rerio than the heterospecific danio, demonstrating that alleles of pigment pattern genes in other species typically are recessive to D. rerio alleles. Furthermore, hybrids between two additional striped species (D. kerri, D. nigrofasciatus) and D. albolineatus suggest that striped patterns are dominant or semi-dominant over an absence of stripes. Together, these analyses support a model in which pigment pattern differences between D. rerio and other species result from gain-of-function alleles in D. rerio, or loss-of-function alleles in other danios. Finally, because several D. rerio pigment pattern mutants resemble heterospecific danios, we use interspecific complementation tests to assess potential roles for these loci in pigment pattern diversification. Crosses between other danios and most D. rerio pigment pattern mutants develop stripes, similar to control hybrids with wild-type D. rerio. These complementation phenotypes allow us to exclude most of these loci as having major effect roles in generating pigment pattern differences between species. In contrast, hybrids between fms mutant D. rerio and D. albolineatus fail to develop stripes, similar to D. albolineatus. This non-complementation phenotype identifies changes in fms, or the pathway in which it acts, as candidates for contributing to the evolutionary loss of stripes in D. albolineatus.

Abstract: Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.

Abstract: Over the past few years, molecular studies of phylogeny have challenged the traditional view of evolutionary relationships among protostomian animal phyla. Based on analysis of 18S ribosomal RNA gene sequences, it has been suggested that some traditional groups, like the articulata and the pseudocoelomata, should be completely abandoned and that instead the protostomians should be split into two major clades: the Ecdysozoa and the Lophotrochozoa. However, this new molecular phylogeny still awaits confirmation by independent methods. In this study, we present a cytological feature that supports the new classification. The carbohydrate epitope that is recognised by antisera against the plant glycoprotein horseradish peroxidase (HRP) is known to be selectively expressed by membrane proteins on the surface of neural tissue in insects. We found that the major ecdysozoan phyla show neural expression of HRP immunoreactivity, which is completely absent in the nervous tissue of lophotrochozoans, deuterostomians, and cnidarians. This suggests that the presence of anti-HRP-reactive glycoproteins in neural tissue is an ecdysozoan autapomorphy.

Abstract: The homeobox gene Distal-less (Dll) is well known for its participation in the development of arthropod limbs and their derivatives. Dll activity has been described for all groups of arthropods, but also for molluscs, echinoderms and vertebrates. Generally, Dll participates in the establishment of the proximo-distal-axis and differentiation along this axis. During our investigation of the expression pattern in the silverfish Lepisma saccharina and the horseshoe crab Limulus polyphemus, we found several expressions in late stages which cannot be explained with the "normal" limb-specific function. The antenna, cerci and terminal filament of the silverfish show a striped expression; single cells on the labrum, mandibles, maxillary palps and anal valves are also strongly stained by the Dll antibody. In addition to cell groups in the developing ganglia of the CNS, in the coxal endites and several nerve cells in femur and the trochanter of the prosomal limbs, the whole prosomal shield of Limulus polyphemus is surrounded by Dll-positive cell clusters. Furthermore, the lateral processes of the opisthosoma and the edges of the opisthosomal appendages are Dll positive. To get an indication of the cell fate of these regions, we examined hatched larvae and juvenile stages of both species with the SEM. We found a striking correlation of these Dll-positive areas and different sense organs, especially mechanoreceptors. Since many sense organs in arthropods are situated on the limbs, interpretation of the Dll expression in limbs is problematical. This has critical implications for comparative analysis of Dll expression patterns between arthropods and for the claim of homology between limb-like structures. Furthermore, we discuss the possibility of convergent appendage evolution in various bilaterian groups based on the improvement of spatial sensory resolution.

Abstract: With accumulating evidence for the appendicular nature of the labrum, the question of its actual segmental origin remains. Two existing insect head segmentation models, the linear and S-models, are reviewed, and a new model introduced. The L-/Bent-Y model proposes that the labrum is a fusion of the appendage endites of the intercalary segment and that the stomodeum is tightly integrated into this segment. This model appears to explain a wider variety of insect head segmentation phenomena. Embryological, histological, neurological and molecular evidence supporting the new model is reviewed.

Abstract: We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

Abstract: We have cloned a Hox-like gene, cnox-2Am, from a staghorn coral, Acropora millepora, an anthozoan cnidarian, and characterised its embryonic and larval expression. cnox-2Am and its orthologs in other cnidarians and Trichoplax most closely resemble the Gsx and, to a lesser extent, Hox 3/4 proteins. Developmental northern blots and in situ hybridisation are consistent in showing that cnox-2Am message appears in the planula larva shortly after the oral/aboral axis is formed following gastrulation. Expression is localised in scattered ectodermal cells with a restricted distribution along the oral/aboral body axis. They are most abundant along the sides of the cylindrical larva, rare in the oral region and absent from the aboral region. These cells, which on morphological grounds we believe to be neurons, are of two types; one tri-or multipolar near the basement membrane and a second extending projections in both directions from a mid-ectodermal nucleus. Anti-RFamide staining reveals neurons with a similar morphology to the cnox-2Am-expressing cells. However, RFamide-expressing neurons are more abundant, especially at the aboral end of the planula, where there is no cnox-2Am expression. The pattern of expression of cnox-2Am resembles that of Gsx orthologs in Drosophila and vertebrates in being expressed in a spatially restricted portion of the nervous system.

Abstract: A number of genes that are involved in somitogenesis in vertebrates are cyclically expressed in the presomitic mesoderm. These include homologues of the Drosophila genes fringe and hairy. We have analysed here two genes that belong to these classes in the zebrafish, namely the apparent orthologues of lunatic fringe (l-fng) and of c-hairy1 (called her9). However, unlike the respective mouse and chicken genes, they are not expressed cyclically in the presomitic mesoderm. Instead, both genes are mainly expressed in the central nervous system. her9 is predominantly expressed in the fore- and midbrain, and transiently in the hindbrain. Thus, the previously identified and only very distantly related her1 gene of zebrafish has more similarities to the expression of the c-hairy1 gene than its apparent orthologue her9, indicating that sequence similarity and similarity of function are not necessarily linked in this case. l-fng expression is found in alternating pre-rhombomeres, comparable to the equivalent mouse gene expression and in the anterior compartments of the mature somites, which was also shown for the chicken l-fng gene. The latter expression indicates that it might be involved in boundary definition and cell fate decision processes, rather than in pre-patterning of the somites. Interestingly, a similar role has previously been inferred for the grasshopper homologue of l-fng. This suggests that the function of l-fng in boundary definition of the somites might be ancestral, while its recruitment to the pre-patterning process of the somites might be a derived feature in higher vertebrates.

Abstract: In the discussion on arthropod phylogeny, the structural evolution of compound eyes and optic ganglia in Crustacea and Insecta is an important topic. On the one hand, many morphological features as well as developmental aspects of the visual system in Insecta and Crustacea correspond in so much detail that eye design in these two groups is likely to have a common euarthropodan ancestor. On the other hand, however, some authors advocate a convergent evolution of the crustacean and insect visual system founding their arguments on differences in the arrangement of the visual neuropils and the fibre connections between Malacostraca and Entomostraca (the "entomostracan enigma"). Therefore, information about cellular aspects of visual system formation in entomostracan Crustacea is likely to enliven this debate, but is not yet available. To fill this gap, we examined the proliferation of neuronal stem cells in the developing visual system of the tadpole shrimp Triops longicaudatus (LeConte, 1846) (Entomostraca, Branchiopoda, Phyllopoda, Calmanostraca, Notostraca) by in vivo incorporation of the proliferation marker bromodeoxyuridine and subsequent immunohistochemical detection. Our results indicate that in the developing visual system of T. longicaudatus, three band-shaped zones containing neuronal stem cells are present corresponding to the proliferation zones found in Malacostraca. We therefore conclude that the ontogenetic mechanisms of visual-system formation are evolutionarily conserved (homologous) in Branchiopoda, Malacostraca, and Insecta.

Abstract: The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.

Abstract: Brachyury is a key transcription factor whose homologs have been identified in many animal species. Different Brachyury expression patterns have been observed amongst echinoderms. We have isolated PlBra, the Brachyury ortholog from the sea urchin Paracentrotus lividus and analyzed its expression during development. PlBra is first expressed at the end of cleavage in a ring of cells at the border between the presumptive endoderm and mesoderm territories. At later stages, PlBra is expressed around the blastopore and in the stomodaeum area as in most basal deuterostomes.

Abstract: The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulans nudC3 mutant, demonstrating evolutionary conservation of function. A C. elegans nud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.

Abstract: The HB9 homeobox gene has been clonedfrom several vertebrates and is implicated in motor neurondifferentiation. In the chick, a related gene,MNR2, actsupstream ofHB9 in this process. Here we report anamphioxus homologue of these genes and show that itdiverged before the gene duplication yieldingHB9 andMNR2. AmphiMnx RNA is detected in two irregularpunctate stripes along the developing neural tube, com-parable to the distribution of ‘dorsal compartment’ motorneurons, and also in dorsal endoderm and posteriormesoderm. We propose a new homeobox class, Mnx, toinclude AmphiMnx, HB9, MNR2 and theirDrosophilaand echinoderm orthologues; we suggest that vertebrateHB9 is renamed Mnx1 and MNR2 be renamed Mnx2.Keywords Homeobox · Motor neuron · Amphioxus ·Gene duplication Introduction The HB9 homeobox gene (also known asHLXB9) wasfirst isolated in humans (Deguchi and Kehrl 1991) andsubsequently in other vertebrates (Saha et al. 1997;Tanabe et al. 1998; Harrison et al. 1999).HB9 is marginallymore closely related to Hox, ParaHox, En, Emx, and NKclass homeobox genes than it is to the Prd, Prd-like ormore divergent homeobox classes (Burglin 1994). Itsevolutionary origin is unclear, however. One early sug-gestion was thatHB9 is related to, or derived from, theDrosophila homeotic geneproboscipedia (Burglin 1994;Harrison et al. 1994). This now seems unlikely asHB9maps to 7q36 (nearEn2 and Gbx1) and not within thehuman Hox clusters (Pollard and Holland 2000). An alter-native possibility is thatHB9 is a member of a distinctclass of homeobox genes, analogous to other definedclasses such as Emx, Msx, Dlx, Mox, etc. Consistent withthis suggestion, a second homeobox gene with highsequence identity toHB9 over the homeobox was reportedfrom chick, and namedMNR2 (Tanabe et al. 1998). Inter-estingly, MNR2 and HB9 are both expressed during motorneuron differentiation, and form part of a cascade of genesregulating this process (Tanabe et al. 1998; Arber et al.1999). HB9 is also expressed in the developing gut, brain,testis and lymphoid lineage (Harrison et al. 1994, 1999;Saha et al. 1997) and mutations inHB9 are implicated inthe human congenital condition sacral agenesis (Lynch etal. 1995; Ross et al. 1998). To investigate the relatednessbetween HB9 and MNR2, and the evolution of their devel-opmental roles, we have examined a homologous gene ina cephalochordate (amphioxus), the group of animalsthought to be the sister group of the vertebrates.

Abstract: Previously, the only anuran amphibian known to regenerate the lens of the eye was Xenopus laevis. This occurs during larval stages through transdifferentiation of the outer cornea epithelium under control of factors presumably secreted by the neural retina. This study demonstrates that a distantly related species, X. tropicalis, is also able to regenerate lenses through this process. A transgenic line of X. tropicalis was used to examine the process of cornea-lens transdifferentiation in which green fluorescent protein (GFP) is expressed in differentiated lens cells under the control of the Xenopus γ1-crystallin promoter element. Unlike X. laevis, the process of cornea-lens transdifferentiation typically occurs at a very low frequency in X. tropicalis due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening. The inner cornea endothelium serves as a key physical barrier that normally prevents retinal signals from reaching the outer cornea epithelium. If this barrier is circumvented by implanting outer cornea epithelium of transgenic tadpoles directly into the vitreous chamber of non-transgenic X. tropicalis larval eyes, a higher percentage of cases formed lenses expressing GFP. Lenses were also formed if these tissues were implanted into X. laevis larval eyes, suggesting the same or similar inducing factors are present in both species. When pericorneal ectoderm and posteriolateral flank ectoderm were implanted into the vitreous chamber, only in rare cases did pericorneal ectoderm form lens cells. Thus, unlike the case in X. laevis, competence to respond to the inducing factors is tightly restricted to the cornea epithelium in X. tropicalis. As controls, all these tissues were implanted into the space located between the inner and outer corneas. None of these implants, including outer cornea epithelium, exhibited GFP expression. Thus, the essential inductive factors are normally contained within the vitreous chamber. One explanation why this type of lens regeneration is not seen in some other anurans could be due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening once the original lens is removed. These findings are discussed in terms of the evolution of this developmental process within the anurans.

Abstract: Many insects possess abdominal prolegs, raising the question of whether these prolegs are homologous or convergent structures. One way to address this issue is to compare mechanisms controlling the development of prolegs in different insects. Segmental morphologies along the insect body are controlled by the regulatory activities of the Hox proteins, and one well-studied regulatory target is the Distal-less (Dll) gene, which is required for the development of distal limb structures in arthropods. In Drosophila abdominal segments, Dll transcription is prevented by Hox proteins of the Bithorax Complex (BX-C). In lepidopteran abdominal segments, circular holes lacking BX-C protein expression allow Dll to be expressed and prolegs to develop. For comparison, we examined protein expression patterns in two species of sawfly from the hymenopteran suborder Symphyta; these insects develop prolegs on all abdominal segments. Interestingly, sawfly prolegs did not express Dll protein at any time, and expressed BX-C proteins throughout development. These results suggest that sawfly prolegs lack distal elements that are present in lepidopteran prolegs. Consistent with this interpretation, the proximal determinant extradenticle (exd) was present in cell nuclei all of the way to the tip of the sawfly proleg, whereas it was not detectable in the nuclei of cells near the tip of the lepidopteran proleg. Our results support the hypothesis that larval prolegs have evolved independently in the Lepidoptera and Hymenoptera.

Abstract: Members of the Smad family of TGFβ signal transducers are important regulators of proliferation and cell fate during axis formation, organogenesis, and tumorigenesis. A canonical TGFβ/Smad signaling pathway is conserved in nematodes, insects, and vertebrates. However, its evolutionary origin before the divergence of protostomes and deuterostomes is unclear. Here, we present the cloning and expression of a highly conserved orthologue of receptor-activated Smads in Hydra, which represents clear evidence of the presence of TGFβ signaling in the ancient phylum Cnidaria. HySmad1 is expressed rather uniformly during asexual reproduction and regeneration, and is transcriptionally upregulated during oocyte development. This suggests that multiple functions of TGFβ/Smad signaling might be conserved between diploblastic and triploblastic metazoans.

Abstract: Brachyury is a T-box-containing transcription factor involved in mesoderm formation during vertebrate gastrulation. To analyse whether the regulation of gastru- lation varies as much as the timing of gastrulation does with respect to implantation, we isolated a bovine brachyury cDNA fragment. The amino acid sequence shows high similarity to mouse and human Brachyury and clear differences to other T-box genes. Whole-mount in situ hybridisation reveals a normal expression pattern except for a transiently reduced expression in the anteri- or part of the primitive streak. According to these results, gastrulation in mammals is implemented and regulated irrespective of implantation.

Abstract: The totipotent stem cells called neoblasts seem to be concerned with the remarkable regeneration ability of planarians. However, the pharynx is able to regenerate after the amputation of its distal part, in spite of a lack of neoblasts in the pharynx. The process of regeneration has been referred to as morphallaxis, based on conventional histochemical observations. We examined it again immuno-histochemically using anti-Dugesia japonica proliferating cell nuclear antigen (DjPCNA) antibody for neoblasts and anti-D. japonica myosin heavy chain-A (DjMHC-A) antibody for pharynx muscle fibers. This immuno-histochemical study, together with observations of the regeneration process of planarians irradiated with X-rays in particular regions, revealed that after the amputation, neoblasts from outside the pharynx entered that organ, moved through the mesenchyme of the pharynx to the wounded area, and differentiated into the cells that had been lost there. We show here that the regeneration after amputation of the distal part of the pharynx is an 'epimorphic' process.

Abstract: Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. We examined the expression patterns of four eye specification genes, eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac) throughout all time points of embryogenesis and show that only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. We also compare the expression of Ey with that of a putative antennal specifying gene Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field.

Abstract: Bicoid directs anterior development in Drosophila embryos by activating different genes along the anterior-posterior axis. However, its activity is down-regulated at the anterior tip of the embryo, in a process known as retraction. Retraction is under the control of the terminal polarity system, and results in localized repression of Bicoid target genes. Here, we describe a Drosophila homolog of human SAP18, a member of the Sin3A/Rpd3 histone deacetylase complex. dSAP18 interacts with Bicoid in yeast and in vitro, and is expressed early in development coincident with Bicoid. In tissue culture cells, dSAP18 inhibits the ability of Bicoid to activate reporter genes. These results suggest a model in which dSAP18 interacts with Bicoid to silence expression of Bicoid target genes in the anterior tip of the embryo.

Abstract: Molecular and morphological evidence (expression patterns of pair-rule genes and segmental position of the genital openings and other segmental markers) suggest that the segmental units of the arthropod body are specified, in early ontogeny, by three spatially and/or temporally distinct mechanisms and do not appear in a strict antero-posterior sequence. A first anterior set of indivisible segments (naupliar segments, possibly three in all arthropods) is followed by a set of more caudal (post-naupliar) primary units (eosegments, possibly ten in all arthropods) which then undergo a process of secondary segmentation, thus giving rise to a higher number of definitive segments (merosegments). The number of merosegments deriving from each eosegment is characteristic of the different arthropod clades and is mostly stable at the level of the traditional arthropodan classes or subclasses. All their segmentation patterns, however, including those found in the segmental organisation of highly segmented forms (such as centipedes and millipedes, notostracan, lipostracan and anostracan crustaceans, and trilobites) are reducible to the basic groundplan with three naupliar and ten postnaupliar segments. These basic units of arthropod segmentation may also have an equivalent in other Ecdysozoa, despite the lack of any segmentation (nematodes) or, at least, of an overt segmentation (kinorhynchs).

Abstract: Although embryonic development in ascidians has been studied for over a century, the signals involved in coordinating post-larval development and metamorphosis are just beginning to be investigated. In this paper, we demonstrate that transcription is necessary for both the acquisition of metamorphic competence and the completion of the initial events of metamorphosis in Boltenia villosa. Transcripts expressed during metamorphic competence were isolated by a suppressive PCR subtraction of Boltenia villosa larval cDNAs. One of these transcripts is homologous to cornichon. Cornichon has a crucial but undefined role in epidermal growth factor (EGF) signaling during Drosophila embryogenesis. In situ hybridization demonstrates that Boltenia cornichon (Cnib) is expressed in the anterior papillary region of larvae as they gain competence. Our hypothesis is that Cnib acts to potentiate EGF signaling, thereby allowing Boltenia larvae to respond to cues for metamorphosis. Further research into the role of Cnib in urochordate metamorphosis may provide insight into the function of cornichon in other organisms. A better molecular understanding of urochordate metamorphosis will also provide a foundation for exploring the role of metamorphosis in chordate evolution.

Abstract: We report the full-length coding sequence and the expression pattern during neurulation and early organogenesis of ScOtx5, a novel member of the Otx gene family in the dogfish Scyliorhinus canicula. Phylogenetic analyses confirm that ScOtx5 is closely related to the Xenopus XlOtx5/5b genes, and also to the Crx genes characterized in mammals and zebrafish. This supports the hypothesis that these genes define a third gnathostome Otx orthology class. During neurulation, ScOtx5 transcripts are detected in the foregut diverticulum and the anterior neuroectoderm. At the onset of organogenesis, ScOtx5 is transcribed over a broad domain spanning the whole prosencephalon and mesencephalon, albeit with a much lower signal intensity than its paralogues Otx1 and Otx2. At later stages, four major expression sites are observed: the developing eye and epiphysis, the olfactory placodes and a broad epidermal domain in the dorsal part of the head. In the embryonic eye, the signal is first detected in the presumptive pigmented retina and slightly later in the adjacent outer layer of the neural retina, fated to photoreceptors. The comparison of this expression pattern with those of osteichthyan Otx genes suggests that a role in the specification of photoreceptors may correspond to a functional specialization of Otx5 and Crx genes, fixed early in the gnathostome lineage, prior to the splitting of chondrichthyans and osteichthyans. In contrast, the roles played by ScOtx5 in the retinal pigmented epithelium or in the olfactory placodes may be fulfilled by different combinations of paralogous genes in other gnathostome taxa.

Abstract: In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.

Abstract: We have conducted an evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes resulted from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences showed there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2–5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the Caenorhabditis elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal of the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function.

Abstract: The Drosophila genome contains a single orthologue of mammalian puromycin-sensitive aminopeptidases, dPsa. Even though dPsa was expressed in many tissues during development, animals lacking dPsa activity were viable. Ubiquitous overexpression of dPsa during embryonic or larval development resulted in lethality and overexpression in isolated tissues during development resulted in localized lesions. These results suggest that even though dPsa function was not essential for viability, dPsa expression must be tightly regulated for normal development. By screening the Drosophila genome we found 43 predicted aminopeptidases and generated a phylogenetic tree of aminopeptidases related to dPsa by sequence. We discuss possible functions of dPsa and the idea that other Drosophila aminopeptidases might perform redundant functions with dPsa for regulating protein turnover.

Abstract: During the final step of Drosophila vitelline membrane formation, the structural proteins composing this layer become cross-linked by covalent bonds. In the present report, we analyzed the vitelline membrane cross-linking in mutants having defects either in this layer or in the chorionic layers. In the fs(1)Nasrat and fs(1)polehole mutant alleles conferring defects in vitelline membrane formation, disruption of vitelline membrane cross-linking was observed, indicating the involvement of these two genes in the process. On the contrary, in the fs(1)Nasrat and fs(1)polehole alleles showing defects only at the termini of the embryo the vitelline membrane is properly formed, confirming a multifunctional activity of their gene products. Altered vitelline membrane cross-linking was also detected in a mutant of the chorion protein gene Cp36 and in the chorion amplification mutant fs(1)K1214, suggesting a role of the structural components of chorion layers in the process of vitelline membrane hardening.

Abstract: The mammalian genome contains over 30 genes whose expression is dependent upon their parent-of-origin. Of these imprinted genes the majority are involved in regulating the rate of fetal growth. In this report we show that in the deer mouse Peromyscus the placental lactogen-1-variant (pPl1-v) gene is paternally expressed throughout fetal development, whereas the linked and closely related pPl1 gene is expressed in a biallelic manner. Neither the more distantly related pPl2A gene, nor the Mus Pl1 gene displays any preferential expression of the paternal allele, suggesting that the acquisition of imprinting of pPl1-v is a relatively recent event in evolution. Although pPl1 expression is temporally mis-regulated in the dysplastic placentae of hybrids between two Peromyscus species, its over-expression cannot account for the aberrant phenotypes of these placentae. We argue that the species-specific imprinting of pPl1-v, encoding a growth factor that regulates nutrient transfer from mothers to their offspring, is consistent with the parent-offspring conflict model that has been proposed to explain the evolution of genomic imprinting.