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. Specifically, we have generated new eyeless alleles, each with single missense mutations in one of the four domains of the protein. We show that these alleles result in abnormal eye and brain development while maintaining the OK107 eyeless GAL4 activity from which they were derived. We performed in vivo functional rescue experiments by expressing in an eyeless-specific pattern Eyeless proteins in which either the paired domain, the homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain phenotypes was only observed when full-length Eyeless was expressed, while all deletion constructs failed to rescue. These data, along with the phenotypes observed in the four newly characterized eyeless alleles, demonstrate the requirement for an intact Eyeless protein for normal Drosophila eye and brain development. They also suggest that some endogenous functions may be obscured in ectopic expression experiments.

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. The isolated composite cDNA encodes a hypothetical protein of 448 amino acid residues with a typical LIM-homeodomain (LIM-HD) structure and the greatest overall sequence similarity to vertebrate Lhx2/9 proteins. The Euprymna scolopes apterous (Es-ap) expression patterns provided no indication of a role in the early dorso/ventral patterning or growth of the arm crown that showed expression only in two ventral cords running in parallel inside the arms and tentacles and at the base of the suckers, a region rich in nerve endings and chemosensory neurons. The Es-ap hybridization signal was also conspicuous in the eyes, olfactory organs, optic lobes, and in several lobes of the supraesophageal mass, among these the olfactory and vertical lobes, and paravertical bodies. The observed expression patterns suggest gene involvement in eye morphogenesis and neural wiring of sensory structures, including those for olfaction and vision.

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. E. scolopes embryos can be obtained continually and can be reared in the laboratory over an entire generation. The embryos and protective chorions are optically clear, facilitating in situ developmental observations, and can be manipulated experimentally. Many molecular protocols have been developed for studying E. scolopes development. This species is best known, however, for its symbiosis with the luminous marine bacterium Vibrio fischeri and has been used to study determinants of symbiont specificity, the influence of symbiosis on development of the squid light organ, and the mechanisms by which a stable association is achieved. Both partners can be grown independently under laboratory conditions, a feature that offers the unusual opportunity to manipulate the symbiosis experimentally. Molecular and genetic tools have been developed for V. fischeri, and a large expressed sequence tag (EST) database is available for the host symbiotic tissues. Additionally, comparisons between light organ form and function to those of the eye can be made. Both types of tissue interact with light, but have divergent embryonic development. As such, they offer an opportunity to study the molecular basis for the evolution of morphological novelties.

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. scolopes embryogenesis from cleavage through hatching under controlled environmental conditions, including detailed descriptions of externally visible morphological features that are easily distinguished in either live or freshly fixed embryos under a dissecting microscope. Photomicrographs are also provided to aid in the accurate and rapid staging of E. scolopes embryos.

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. Although nucleic acids prepared this way can serve as a template for polymerase chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these contaminants. The method described here yields high-molecular-weight DNA that can be readily restricted for Southern hybridization. The procedure uses brain tissue under the assumption that its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and avoids potential sources of enzymatic contaminants and parasites from the intestinal sac. However, the method can be applied to other tissue sources and works well with other species. The purification of DNA by gradient centrifugation is an established method based on the specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA, which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved and expensive and produces large amounts of ethidium bromide waste.

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.

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. This protocol outlines the techniques for preparing RNA probes for whole-mount in situ hybridization in Hawaiian bobtail squid (Euprymna scolopes) embryos from linearized plasmid DNA or polymerase chain reaction (PCR) products.

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. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody.

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. scolopes is robust over a range of temperatures, is relatively rapid (approximately 21 d), and proceeds normally under laboratory conditions at ambient temperature (27 degrees C-29 degrees C). Here we present methods for maintaining E. scolopes embryos in culture from cleavage through hatching, as well as observing and recording live or freshly fixed embryos under a dissecting microscope.

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). To evaluate the expectations of these contrasting behaviours, we examined the global genetic structure of S. lewini based on collections (n = 271 individuals) from 20 nursery areas. A 548-bp fragment of mitochondrial DNA control region revealed 22 polymorphic sites, 24 haplotypes, and three lineages distinguished by 2.56-3.77% sequence divergence. Coalescence analyses based on a provisional molecular clock indicate an origin in the Indo-West Pacific with late Pleistocene radiations into the central Pacific (Hawaii) and eastern Pacific (Central America), as well as recent interchange between oceans via southern Africa. Population subdivisions are strong (overall Phi(ST) = 0.749, P < 0.0001 and among oceans Phi(ST) = 0.598, P < 0.0098). Genetic discontinuity within oceans (Phi(ST) = 0.519, P < 0.0001) is primarily associated with oceanic barriers (migration across oceans M approximately 0), with much less structure along continental margins (M > 10). We conclude that nursery populations linked by continuous coastline have high connectivity, but that oceanic dispersal by females is rare. Although we cannot rule out philopatry to natal nurseries, oceanic barriers appear to have a much stronger influence on the genetic architecture of this species and may indicate a mechanism for recent evolutionary radiations in the genus Sphyrna.

The adaptor protein X11Lalpha/Dmint1 interacts with the PDZ-binding domain of the cell recognition protein Rst in Drosophila.

The Drosophila cell adhesion molecule Rst plays key roles during the development of the embryonic musculature, spacing of ommatidia in the compound eye and of sensory organs on the antenna, as well as in the neuronal wiring of the optic lobe. In rst(CT) mutants lacking the cytoplasmic domain of the Rst protein, cell sorting and apoptosis in the eye are affected, suggesting a requirement of this domain for Rst function. To identify potential interacting proteins, yeast two-hybrid screens were performed using the cytoplasmic domains of Rst and its paralogue Kirre as baits. Among several putative interactors, two paralogous Drosophila PDZ motif proteins related to X11/Mint were identified. X11/Mint family members in C. elegans (LIN-10) and vertebrates are believed to function as adaptor proteins and to regulate the assembly of multi-subunit complexes at the synapse, thereby linking the vesicle cycle to cell adhesion. Using genetic, cell biological, and biochemical approaches, we show that the interaction of Rst with X11Lalpha is of biological significance. The proteins interact, for example, in the context of cell sorting in the pupal retina.

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. We found that in flies these proteins are mainly expressed in axons at the root of fiber tracts, in places where strong fasciculation is required, e.g. at the neck of the peduncle of the mushroom bodies and in the optic chiasms. Despite their evolutionary conservation which implies fundamental and important functions, a P-element-induced null mutant (KG00210) of reggie1/flotillin2 (reggie1/flo2) in D. melanogaster shows no apparent phenotypic defects. This was even more surprising as we show that in this reggie1/flo2 null mutant the paralogous Reggie2/Flo1 protein is unstable and degraded, while the transcript is still present. The requirement of Reggie1/Flo2 for Reggie2/Flo1 stabilization is confirmed by misexpression experiments. Reggie2/Flo1 can only be misexpressed when Reggie1/Flo2 is provided as well. Conversely, Reggie1/Flo2 immunoreactivity can be detected, when its transgene is misexpressed alone. Using appropriate Gal4 driver lines, misexpression of Reggie1/Flo2 alone or together with Reggie2/Flo1 in the eye imaginal disc results in a specific and severe mislocalization of cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) (while DE-Cadherin is unaffected) and in differentiation defects pointing to impaired signaling. In the wing imaginal disc, global overexpression of Reggie/Flotillin proteins leads to a significant extension of the Wingless signal and severely disrupts normal wing development. Our data support the notion that Reggie/Flotillin proteins are implicated in signaling processes at cellular contact sites.

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.

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. Gene duplications have been proposed to be of major evolutionary significance since duplicated redundant sequences can accumulate mutations without detrimental effects for the organism and leave the duplicated genes free to assume novel functions. To address the issue of conservation of the duplicated sequences and their putative redundancy, as well as to identify putative functional divergence of the paralogs during drosophilid evolution, we performed an interspecies comparison of the rst and kirre genes from D. virilis and D. melanogaster. The D. virilis genome contains orthologues of both rst and kirre and hence the duplication took place before the split of the two lineages and has subsequently been conserved. However, whilst the Rst orthologues show a high degree of sequence similarity, this similarity is lower in Kirre orthologues. Especially the intracellular domains of D. virilis and D. melanogaster Kirre sequences are highly divergent: the D. virilis kirre gene lacks the 3'-most exon present in D. melanogaster, which contains motifs conserved between kirre and rst in D. melanogaster. Hence, while each of the two genes is highly conserved at the level of its exon-intron organization, the selection forces acting on the rst and kirre coding sequences are different. These findings are discussed in the light of general evolutionary mechanisms.

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. The deduced homeodomain sequences of the E. scolopes Hox cluster genes are most similar to known annelid, brachiopod, and nemertean Hox gene homeodomain sequences. Our results are consistent with the presence of a single Hox gene cluster in cephalopods. Our data also corroborate the proposed existence of a differentiated Hox gene cluster in the last common ancestor of Bilaterians. Furthermore, our phylogenetic analysis and in particular the identification of Post-1 and Post-2 homologs support the Lophotrochozoan clade.

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. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.

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. Evidence is presented that this is due to functional redundancy between Rst and its paralogue Kirre. Both are highly related single-pass transmembrane proteins with five extracellular immunoglobulin domains and three conserved motifs in the intracellular domain. The expression patterns of kirre and rst overlap during embryonic development in muscle founder cells. Simultaneous deletion of both genes causes an almost complete failure of fusion between muscle founder cells and fusion-competent myoblasts. This defect can be rescued by one copy of either gene. Moreover, Rst, like Kirre is a myoblast attractant.

Drosophila Pin1 prolyl isomerase Dodo is a MAP kinase signal responder during oogenesis.

The mammalian cis-trans prolyl isomerase Pin1 and its yeast orthologue Ess1/Ptf1 have been implicated in cell cycle control but a correlation between biochemical and physiological functions has not been established conclusively. Pin1 targets the proline residue carboxy-terminal to the phosphorylated threonine or serine residue, which constitutes part of the phosphorylated mitogen-activated protein kinase (MAPK) site PXpT/SP. Here we show that the Drosophila Pin1 homologue, the Dodo protein, is involved in dorsoventral patterning of the follicular epithelium in the egg chamber. Its function is to facilitate the degradation of transcription factor CF2, which requires, a priori, activated epidermal growth factor receptor-MAPK signalling.

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. The human FLI gene is mapped within the Smith-Magenis microdeletion region of chromosome 17. We report the identification of two related genes, LRRFIP1 and LRRFIP2, encoding proteins that interact with the LRR domain of human FLI using the yeast two-hybrid system. LRRFIP1 exhibits sequence identity with the TRIP RNA-binding protein and GCF-2 transcriptional repressor, which are also related to the murine FLAP-1 gene. LRRFIP2 is a novel gene that shares sequence homology with LRRFIP1 and FLAP-1. LRRFIP1 and LRRFIP2 both express alternative splice variants in heart and skeletal muscle tissue. A coiled-coil domain, conserved within each encoded protein, serves as a potential interaction motif for FLI LRR. The occurrence of multiple proteins able to interact with FLI within the same tissue suggests that they may compete for the same binding site. Sequencing and PCR-directed genomic analysis indicate that LRRFIP1 and LRRFIP2 are related genes that arose from gene duplication.

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. Most gene expression is pleiotropic, and deletion studies reveal that a morphological phenotype is seldom observed when these genes are removed from the genome. These data pinpoint some general bottlenecks in functional genomics, and they reveal the acute emerging difficulties with data transferability above the levels of genes and proteins, especially with complex human phenotypes. At these higher levels the Rosetta stone analogy has almost no applicability. However, newer transgenic technologies in Drosophila and Mus, combined with coherency pattern analyses of gene networks, and synthetic neural modeling, offer insights into organismal function. We conclude that industrially scaled robogenomics in model organisms will have great impact if it can be realistically linked to epigenetic analyses of human variation and to phenotypic analyses of human diseases in different genetic backgrounds.