The cephalopod arm crown: appendage formation and differentiation in the Hawaiian bobtail squid .

Background: Cephalopods are a highly derived class of molluscs that adapted their body plan to a more active and predatory lifestyle. One intriguing adaptation is the modification of the ventral foot to form a bilaterally symmetric arm crown, which constitutes a true morphological novelty in evolution. In addition, this structure shows many diversifications within the class of cephalopods and therefore offers an interesting opportunity to study the molecular underpinnings of the emergence of phenotypic novelties and their diversification.

Culture of Hawaiian bobtail squid (Euprymna scolopes) embryos and observation of normal development.

The ability to rear Hawaiian bobtail squid (Euprymna scolopes) embryos under controlled environmental conditions is a basic and necessary tool for developmental studies. It negates the need to know when fertilization occurred, allows correlation of the phase of development with the time of development (thereby facilitating collection of embryos at specific stages), and allows comparisons between cephalopod species. Embryonic development in E.

Whole-mount in situ hybridization of Hawaiian bobtail squid (Euprymna scolopes) embryos with DIG-labeled riboprobes: II. Embryo preparation, hybridization, washes, and immunohistochemistry.

Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry.

Whole-mount in situ hybridization of Hawaiian bobtail squid (Euprymna scolopes) embryos with DIG-labeled riboprobes: I. DNA template preparation and in vitro transcription of riboprobes.

Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of a DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess, unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry.

Confocal immunocytochemistry of embryonic and juvenile Hawaiian bobtail squid (Euprymna scolopes) tissues.

The Hawaiian bobtail squid Euprymna scolopes is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. This protocol describes the preparation of whole juvenile squids by whole-mount immunocytochemistry for visualization by confocal microscopy.

Preparation of genomic DNA from Hawaiian bobtail squid (Euprymna scolopes) tissue by cesium chloride gradient centrifugation.

This procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic methods and commercial kits for the preparation of genomic DNA based on proteolytic digestion of chromatin components, followed by selective binding of nucleic acids to ion-exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also, molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain conditions.

The embryonic development of the Hawaiian bobtail squid (Euprymna scolopes).

A staging series based on easily distinguishable morphological features is a basic and necessary tool for developmental studies. It provides a consistent reference for comparisons between independent studies, negates the need to know when fertilization occurred, allows correlation of the phase of development with the time of development (to facilitate collection of embryos at specific stages), and allows comparisons between species. Given the growing interest in Hawaiian bobtail squid (Euprymna scolopes) as a contemporary cephalopod developmental system, this article provides a detailed survey of E.

The Hawaiian bobtail squid (Euprymna scolopes): a model to study the molecular basis of eukaryote-prokaryote mutualism and the development and evolution of morphological novelties in cephalopods.

The Hawaiian bobtail squid, Euprymna scolopes, is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. E. scolopes is studied in three principal contexts: (1) as a model of cephalopod development; (2) as a model of animal-bacterial symbioses; and (3) as a system for studying adaptations of tissues that interact with light.

Mutational analysis of the eyeless gene and phenotypic rescue reveal that an intact Eyeless protein is necessary for normal eye and brain development in Drosophila.

Pax6 genes encode evolutionarily highly conserved transcription factors that are required for eye and brain development. Despite the characterization of mutations in Pax6 homologs in a range of organisms, and despite functional studies, it remains unclear what the relative importance is of the various parts of the Pax6 protein. To address this, we have studied the Drosophila Pax6 homolog eyeless.

Developmental expression of apterous/Lhx2/9 in the sepiolid squid Euprymna scolopes supports an ancestral role in neural development.

The transcription factors Apterous/Lhx2/9 play many pivotal roles in the development of protostomes and deuterostomes, most notably limb patterning, eye morphogenesis, and brain development. Full-length apterous/lhx2/9 homologs have been isolated from several invertebrate species, but hitherto not from a lophotrochozoan. Here, we report the isolation, characterization, and spatio-temporal expression of apterous in the sepiolid squid Euprymna scolopes.

Immunization with hybrid recombinant Mycobacterium tuberculosis H37Rv proteins increases the TH1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria.

The aim of this research was to identify subunit immunogens that can generate enhanced CD8 T cell and TH1 responses against Mycobacterium tuberculosis. A genomic comparison of the M. tuberculosis H(37)R(V) and M.

Global phylogeography of the scalloped hammerhead shark (Sphyrna lewini).

Large marine fishes typically have little population genetic structure. The exceptions are associated with sedentary behaviour, disjunct distributions, or reproductive philopatry. Scalloped hammerhead sharks (Sphyrna lewini) incorporate the contrasting traits of oceanic habitat (usually associated with high dispersal) and possible fidelity to nursery grounds (for reproductive females).

Loss- and gain-of-function analysis of the lipid raft proteins Reggie/Flotillin in Drosophila: they are posttranslationally regulated, and misexpression interferes with wing and eye development.

Reggie/Flotillin proteins are upregulated after optic nerve dissection and evolutionary highly conserved components of lipid rafts. Whereas many biochemical and cell culture studies suggest an involvement in the assembly of multiprotein complexes at cell contact sites, not much is known about their biological in vivo functions. We therefore set out to study the expression pattern and the effects of loss- and gain-of-function in the Drosophila melanogaster model system.

Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest gene in Drosophila melanogaster.

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, we previously identified several cis-regulatory modules that mediate specific expression of the roughest gene in Drosophila developmental processes.

Cephalopod Hox genes and the origin of morphological novelties.

Cephalopods are a diverse group of highly derived molluscs, including nautiluses, squids, octopuses and cuttlefish. Evolution of the cephalopod body plan from a monoplacophoran-like ancestor entailed the origin of several key morphological innovations contributing to their impressive evolutionary success. Recruitment of regulatory genes, or even pre-existing regulatory networks, may be a common genetic mechanism for generating new structures.

Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster.

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad.

Interspecies comparison of a gene pair with partially redundant function: the rst and kirre genes in D. virilis and D. melanogaster.

The D. melanogaster rst and kirre genes encode two highly related immunoglobulin-like cell adhesion molecules that function redundantly during embryonic muscle development. The two genes appear to be derived from a common ancestor by gene duplication.

Pax6 in the sepiolid squid Euprymna scolopes: evidence for a role in eye, sensory organ and brain development.

The cloning of a Pax6 orthologue from the sepiolid squid Euprymna scolopes and its developmental expression pattern are described. The data are consistent with the presence of a single gene encoding a protein with highly conserved DNA-binding paired and homeodomains. A detailed expression analysis by in situ hybridization and immunodetection revealed Pax6 mRNA and protein with predominantly nuclear localization in the developing eye, olfactory organ, brain lobes (optic lobe, olfactory lobe, peduncle lobe, superior frontal lobe and dorsal basal lobe), arms and mantle, suggestive of a role in eye, brain, and sensory organ development.

HOX genes in the sepiolid squid Euprymna scolopes: implications for the evolution of complex body plans.

Molluscs display a rich diversity of body plans ranging from the wormlike appearance of aplacophorans to the complex body plan of the cephalopods with highly developed sensory organs, a complex central nervous system, and cognitive abilities unrivaled among the invertebrates. The aim of the current study is to define molecular parameters relevant to the developmental evolution of cephalopods by using the sepiolid squid Euprymna scolopes as a model system. Using PCR-based approaches, we identified one anterior, one paralog group 3, five central, and two posterior group Hox genes.

rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila.

The polynucleate myotubes of vertebrates and invertebrates form by fusion of myoblasts. We report the involvement of the Drosophila melanogaster Roughest (Rst) protein as a new membrane-spanning component in this process. Rst is strongly expressed in mesodermal tissues during embryogenesis, but rst null mutants display only subtle embryonic phenotypes.

Novel proteins interacting with the leucine-rich repeat domain of human flightless-I identified by the yeast two-hybrid system.

The flightless-I gene encodes a member of the gelsolin-like family of actin-binding proteins linked to a leucine-rich repeat (LRR) domain. It is required for cellularization during early embryogenesis and normal development of the indirect flight muscles in Drosophila melanogaster. Although the association between actin and the gelsolin-like domain of the human Flightless-I homologue (FLI) has been established, its biological role is unknown.

Data transferability from model organisms to human beings: insights from the functional genomics of the flightless region of Drosophila.

At what biological levels are data from single-celled organisms akin to a Rosetta stone for multicellular ones? To examine this question, we characterized a saturation-mutagenized 67-kb region of the Drosophila genome by gene deletions, transgenic rescues, phenotypic dissections, genomic and cDNA sequencing, bio-informatic analysis, reverse transcription-PCR studies, and evolutionary comparisons. Data analysis using cDNA/genomic DNA alignments and bio-informatic algorithms revealed 12 different predicted proteins, most of which are absent from bacterial databases, half of which are absent from Saccharomyces cerevisiae, and nearly all of which have relatives in Caenorhabditis elegans and Homo sapiens. Gene order is not evolutionarily conserved; the closest relatives of these genes are scattered throughout the yeast, nematode, and human genomes.

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally.

Molecular and mutational analysis of a gelsolin-family member encoded by the flightless I gene of Drosophila melanogaster.

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene.