Nmf9 Encodes a Highly Conserved Protein Important to Neurological Function in Mice and Flies.

Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.

A DTI-based template-free cortical connectome study of brain maturation.

Improved understanding of how the human brain is "wired" on a macroscale may now be possible due to the emerging field of MRI connectomics. However, mapping the rapidly developing infant brain networks poses challenges. In this study, we applied an automated template-free "baby connectome" framework using diffusion MRI to non-invasively map the structural brain networks in subjects of different ages, including premature neonates, term-born neonates, six-month-old infants, and adults. We observed increasing brain network integration and decreasing segregation with age in term-born subjects. We also explored how the equal area nodes can be grouped into modules without any prior anatomical information--an important step toward a fully network-driven registration and analysis of brain connectivity.

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.

Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled.

The relationship between egg polarity and the adult body plan is well understood in many bilaterians. However, the evolutionary origins of embryonic polarity are not known. Insight into the evolution of polarity will come from understanding the ontogeny of polarity in non-bilaterian forms, such as cnidarians. We examined how the axial properties of the starlet sea anemone, Nematostella vectensis (Anthozoa, Cnidaria), are established during embryogenesis. Egg-cutting experiments and sperm localization show that Nematostella eggs are only fertilized at the animal pole. Vital marking experiments demonstrate that the egg animal pole corresponds to the sites of first cleavage and gastrulation, and the oral pole of the adult. Embryo separation experiments demonstrate an asymmetric segregation of developmental potential along the animal-vegetal axis prior to the 8-cell stage. We demonstrate that Dishevelled (Dsh) plays an important role in mediating this asymmetric segregation of developmental fate. Although NvDsh mRNA is ubiquitously expressed during embryogenesis, the protein is associated with the female pronucleus at the animal pole in the unfertilized egg, becomes associated with the unipolar first cleavage furrow, and remains enriched in animal pole blastomeres. Our results suggest that at least one mechanism for Dsh enrichment at the animal pole is through its degradation at the vegetal pole. Functional studies reveal that NvDsh is required for specifying embryonic polarity and endoderm by stabilizing beta-catenin in the canonical Wnt signaling pathway. The localization of Dsh to the animal pole in Nematostella and two other anthozoan cnidarians (scleractinian corals) provides a possible explanation for how the site of gastrulation has changed in bilaterian evolution while other axial components of development have remained the same and demonstrates that modifications of the Wnt signaling pathway have been used to pattern a wide variety of metazoan embryos.

A WNT of things to come: evolution of Wnt signaling and polarity in cnidarians.

The conserved family of Wnt signaling molecules mediates various developmental processes including governing cell fate, proliferation, and polarity. The diverse developmental functions of the Wnt genes in bilaterians have obscured the evolutionary origin of this important signaling pathway. Recent work in the Cnidaria has shown the diversity of Wnt genes, and regulatory components of Wnt signaling, evolved early in metazoan evolution, prior to the divergence of cnidarians and bilaterians. Evidence from Hydra and the sea anemone, Nematostella, demonstrates a role for Wnt signaling in axis formation and patterning, as well as gastrulation and germ-layer specification. In this review, we examine what is currently known about Wnt signaling in cnidarians, and discuss what this group of "simple" animals may reveal about the evolution of Wnt signaling and polarity.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

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. Hox genes encode a family of transcriptional regulatory proteins with a highly conserved role in axial patterning in bilaterians; however, examples highlighting the importance of Hox gene recruitment for new developmental functions are also known. Here we examined developmental expression patterns for eight out of nine Hox genes in the Hawaiian bobtail squid Euprymna scolopes, by whole-mount in situ hybridization. Our data show that Hox orthologues have been recruited multiple times and in many ways in the origin of new cephalopod structures. The manner in which these genes have been co-opted during cephalopod evolution provides insight to the nature of the molecular mechanisms driving morphological change in the Lophotrochozoa, a clade exhibiting the greatest diversity of body plans in the Metazoa.

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.