Ctenophore relationships and their placement as the sister group to all other animals.

Ctenophora, comprising approximately 200 described species, is an important lineage for understanding metazoan evolution and is of great ecological and economic importance. Ctenophore diversity includes species with unique colloblasts used for prey capture, smooth and striated muscles, benthic and pelagic lifestyles, and locomotion with ciliated paddles or muscular propulsion. However, the ancestral states of traits are debated and relationships among many lineages are unresolved. Here, using 27 newly sequenced ctenophore transcriptomes, publicly available data and methods to control systematic error, we establish the placement of Ctenophora as the sister group to all other animals and refine the phylogenetic relationships within ctenophores. Molecular clock analyses suggest modern ctenophore diversity originated approximately 350 million years ago ± 88 million years, conflicting with previous hypotheses, which suggest it originated approximately 65 million years ago. We recover Euplokamis dunlapae-a species with striated muscles-as the sister lineage to other sampled ctenophores. Ancestral state reconstruction shows that the most recent common ancestor of extant ctenophores was pelagic, possessed tentacles, was bioluminescent and did not have separate sexes. Our results imply at least two transitions from a pelagic to benthic lifestyle within Ctenophora, suggesting that such transitions were more common in animal diversification than previously thought.

Author Correction: Ctenophore relationships and their placement as the sister group to all other animals.

In the version of this Article originally published the location of Punta Arenas was incorrect and should have read 'Chile' in Figures 3-5 and in the Supplementary Information. This has been corrected in all versions of the Article.

The ctenophore genome and the evolutionary origins of neural systems.

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

Single-cell semiconductor sequencing.

RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field. It is especially critical for developmental, aging, and cancer biology as well as neuroscience where the enormous heterogeneity of cells present a significant methodological and conceptual challenge. Here we present two methods that allow for fast and cost-efficient transcriptome sequencing from ultra-small amounts of tissue or even from individual cells using semiconductor sequencing technology (Ion Torrent, Life Technologies). The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts' directionality. The second, a template-switch protocol, is designed for small mammalian neurons. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The efficiency of these protocols has been validated with single hippocampal neurons and various invertebrate tissues including individually identified neurons within a simpler memory-forming circuit of Aplysia californica and early (1-, 2-, 4-, 8-cells) embryonic and developmental stages from basal metazoans.

Genetic manipulation of AML1-ETO-induced expansion of hematopoietic precursors in a Drosophila model.

Among mutations in human Runx1/AML1 transcription factors, the t(8;21)(q22;q22) genomic translocation that creates an AML1-ETO fusion protein is implicated in etiology of the acute myeloid leukemia. To identify genes and components associated with this oncogene we used Drosophila as a genetic model. Expression of AML1-ETO caused an expansion of hematopoietic precursors in Drosophila, which expressed high levels of reactive oxygen species (ROS). Mutations in functional domains of the fusion protein suppress the proliferative phenotype. In a genetic screen, we found that inactivation of EcRB1 or activation of Foxo and superoxide dismutase-2 (SOD2) suppress the AML1-ETO-induced phenotype by reducing ROS expression in the precursor cells. Our studies indicate that ROS is a signaling factor promoting maintenance of normal as well as the aberrant myeloid precursors and suggests the importance of antioxidant enzymes and their regulators as targets for further study in the context of leukemia.

The 2M6 antigen is a Müller cell-specific intracellular membrane-associated protein of the sarcolemmal-membrane-associated protein family and is also TopAP.

PURPOSE: The differentiation marker 2M6 has been used to identify Müller cells within the developing chick retina for several years, although the molecular identity of 2M6 was not known. This study was aimed at determining the identity of the protein antigen recognized by the 2M6 monoclonal antibody. METHODS: Affinity chromatography and subsequent mass spectrometry were used to determine the molecular identity of the 2M6 antigen. Immunohistochemistry of monolayer preparations and paraffin-embedded sections of chick retina were performed to localize expression of the 2M6 antigen within cells of the chick retina. RESULTS: Mass spectrometry analyses revealed that the 2M6 antigen is identical (with 95% probability) to the protein known as Top(AP), which is a member of the sarcolemmal membrane-associated protein family of proteins. The 2M6 polypeptide is expressed by Müller glial cells as well as boundary cells within the chick retina. Expression localizes to intracellular membrane structures within those cells. CONCLUSIONS: Members of the sarcolemmal membrane-associated protein family of proteins have been implicated in structural and functional roles related to the cytoskeleton and Ca(+2) release from internal stores. It is thought that 2M6 plays a similar role in Müller cells of the vertebrate retina.

Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Carbonic anhydrase XIV identified as the membrane CA in mouse retina: strong expression in Müller cells and the RPE.

The presence of carbonic anhydrase (CA) activity in the neural retina has been known for several decades. CA-II, a soluble cytoplasmic isoform expressed by Müller cells and a subset of amacrine cells, was thought to be the sole source of CA activity in the neural retina. However, CA-II deficient mice retain CA activity in the neural retina, which implies that another isoform must be present in that tissue. Recently CA-XIV, an integral membrane protein, was cloned and characterized. We, therefore, sought to determine whether CA-XIV is expressed in the neural retina, and hence is responsible for the CA activity observed in CA-II null animals. Immunohistochemical analyses of histological sections from CA-II null, CA-XIV null, and control mice were performed to localize the CA-XIV isoform, as well as other known retinal markers. Immunoblotting and real-time RT-PCR analyses were also performed to test for CA-XIV expression in retina and other mouse tissues. We determined herein that CA-XIV, a approximately 45kDa membrane protein, is expressed in retina, as it is in kidney. In the retina, CA-XIV is expressed on the plasma membrane of Müller cells. CA-XIV is also found on both the apical and basal membranes of the retinal pigmented epithelium. The data presented here indicate that like CA-II, CA-XIV is highly expressed in the neural retina and, like CA-II, more specifically by the Müller cells. The cellular compartmentalization of the two isoforms in the Müller cell-one cytoplasmic and the other on the plasma membrane-suggest that the two enzymes have specific and unique functions. Future studies will be necessary to assign functions to CA-II and CA-XIV in the mouse neural retina.

Developmental analyses of 5A11/Basigin, 5A11/Basigin-2 and their putative binding partner MCT1 in the mouse eye.

Recent reports by this laboratory and others have demonstrated an association between 5A11/Basigin, a member of the immunoglobulin gene superfamily, and monocarboxylate transporter-1 (MCT1), a lactose transporter. Indeed, it was determined in the 5A11/Basigin null mouse retina that MCT1 does not properly integrate into the cell membranes of Müller cells (MCs) or the retinal-pigmented epithelium, where the two are colocalized. The purpose of this study was to elucidate the association of 5A11/Basigin and MCT1 in the developing mouse retina. Immunocytochemical localization and real-time RT-PCR were used to evaluate the expression and localization of 5A11/Basigin and MCT1 at embryonic days 12, 15, and 18, as well as post-natal days 1, 7, 14, and 21. Expression of both proteins progressed from a more generalized distribution throughout the undifferentiated neural retina to specific staining of retina-pigmented epithilia, the MCs, photoreceptor cells and the ciliary apparatus. Although these two membrane glycoproteins were often colocalized, distinct differences in the location and magnitude of their expression over time was observed. These findings suggest that although 5A11/Basigin and MCT1 can associate within the cell membrane, their expression is not always associated and colocalized.

Retina-specific expression of 5A11/Basigin-2, a member of the immunoglobulin gene superfamily.

PURPOSE: 5A11/Basigin has recently been identified as a critical glycoprotein for full maturity and function of the mouse retina. However, the biological function of 5A11/Basigin has yet to be determined. Previous reports indicate the presence of multiple 5A11/Basigin polypeptides within the retina. Therefore, in an effort to determine the function of 5A11/Basigin, the molecular diversity of its expression was evaluated. METHODS: Northern blot and immunoblot techniques were used to evaluate the number of forms of 5A11/Basigin in the mouse retina. cDNA cloning, using a mouse retina library or RT-PCR from rat, chicken, zebrafish, and human retina, was performed to determine the sequence of 5A11/Basigin transcripts. A peptide was generated, based on the deduced amino acid sequence, for subsequent antibody production. Localization of 5A11/Basigin expression was evaluated by immunoblot, immunohistochemistry, and real-time RT-PCR. RESULTS: Two 5A11/Basigin transcripts of approximately 1.5 kb and approximately 1.8 kb, which correspond to glycosylated proteins of approximately 45 and approximately 55 kDa, respectively, were identified in mouse retina. The shorter form was previously cloned. However, the longer form, a splice variant of mouse 5A11/Basigin, is a member of the immunoglobulin gene superfamily and has been named 5A11/Basigin-2. Homologous transcripts were also cloned from rat, chicken, zebrafish, and human retina. 5A11/Basigin-2 expression was limited to the retina, specifically to photoreceptor cells, where it appeared to be most concentrated in the inner segments. CONCLUSIONS: The specific and limited expression of 5A11/Basigin-2 explicitly within photoreceptor cells implies that this glycoprotein plays a fundamental role within the retina. However, its role remains to be determined.

Inactivation of the Basigin gene impairs normal retinal development and maturation.

5A11/Basigin is an immunoglobulin-like glycoprotein expressed on the surface of Müller cells, the apical and basal surfaces of the retinal pigmented epithelium, and photoreceptor cell bodies and their inner segments. Disruption of the 5A11/Basigin gene in the mouse results in photoreceptor degeneration and a corresponding decrease in electroretinogram amplitudes in mature mice. The purpose of this study was to examine the electrophysiology of the 5A11/Basigin null mouse retina at earlier ages than previously examined. Although the architecture of the 5A11/Basigin null mouse retina appears normal, the ERG amplitudes are severely depressed at eye opening, indicating failure in retinal maturation.