RESUMEN

Many marine animals depend upon a larval phase of their life cycle to locate suitable habitat, and larvae use light detection to influence swimming behaviour and dispersal. Light detection is mediated by the opsin genes, which encode light-sensitive transmembrane proteins. Previous studies suggest that r-opsins in the eyes mediate locomotory behaviour in marine protostomes, but few have provided direct evidence through gene mutagenesis. Larvae of the marine annelid Capitella teleta have simple eyespots and are positively phototactic, although the molecular components that mediate this behaviour are unknown. Here, we characterize the spatio-temporal expression of the rhabdomeric opsin genes in C. teleta and show that a single rhabdomeric opsin gene, Ct-r-opsin1, is expressed in the larval photoreceptor cells. To investigate its function, Ct-r-opsin1 was disrupted using CRISPR/CAS9 mutagenesis. Polymerase chain reaction amplification and DNA sequencing demonstrated efficient editing of the Ct-r-opsin1 locus. In addition, the pattern of Ct-r-opsin1 expression in photoreceptor cells was altered. Notably, there was a significant decrease in larval phototaxis, although the eyespot photoreceptor cell and associated pigment cell formed normally and persisted in Ct-r-opsin1-mutant animals. The loss of phototaxis owing to mutations in Ct-r-opsin1 is similar to that observed when the entire photoreceptor and pigment cell are deleted, demonstrating that a single r-opsin gene is sufficient to mediate phototaxis in C. teleta. These results establish the feasibility of gene editing in animals like C. teleta, and extend previous work on the development, evolution and function of the C. teleta visual system . Our study represents one example of disruption of animal behaviour by gene editing through CRISPR/CAS9 mutagenesis, and has broad implications for performing genome editing studies in a wide variety of other understudied animals.

Asunto(s)

Anélidos/genética , Organismos Acuáticos/genética , Sistemas CRISPR-Cas , Opsinas/genética , Fototaxis , Animales , Anélidos/fisiología , Organismos Acuáticos/fisiología , Edición Génica , Larva/genética , Larva/fisiología , Mutagénesis Sitio-Dirigida , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología

RESUMEN

We are interested in understanding whether the annelids and arthropods shared a common segmented ancestor and have approached this question by characterizing the expression pattern of the segment polarity gene engrailed (en) in a basal annelid, the polychaete Chaetopterus. We have isolated an en gene, Ch-en, from a Chaetopterus cDNA library. Genomic Southern blotting suggests that this is the only en class gene in this animal. The predicted protein sequence of the 1.2-kb cDNA clone contains all five domains characteristic of en proteins in other taxa, including the en class homeobox. Whole-mount in situ hybridization reveals that Ch-en is expressed throughout larval life in a complex spatial and temporal pattern. The Ch-en transcript is initially detected in a small number of neurons associated with the apical organ and in the posterior portion of the prototrochophore. At later stages, Ch-en is expressed in distinct patterns in the three segmented body regions (A, B, and C) of Chaetopterus. In all segments, Ch-en is expressed in a small set of segmentally iterated cells in the CNS. In the A region, Ch-en is also expressed in a small group of mesodermal cells at the base of the chaetal sacs. In the B region, Ch-en is initially expressed broadly in the mesoderm that then resolves into one band/segment coincident with morphological segmentation. The mesodermal expression in the B region is located in the anterior region of each segment, as defined by the position of ganglia in the ventral nerve cord, and is involved in the morphogenesis of segment-specific feeding structures late in larval life. We observe banded mesodermal and ectodermal staining in an anterior-posterior sequence in the C region. We do not observe a segment polarity pattern of expression of Ch-en in the ectoderm, as is observed in arthropods.

Asunto(s)

Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Anélidos , Secuencia de Bases , Southern Blotting , Tipificación del Cuerpo , Clonación Molecular , ADN Complementario/metabolismo , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Hibridación in Situ , Larva/fisiología , Mesodermo/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular

RESUMEN

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.

Asunto(s)

Tipificación del Cuerpo , Linaje de la Célula , Sanguijuelas/embriología , Animales , Evolución Biológica , Drosophila/embriología , Epidermis/embriología , Rayos Láser , Sanguijuelas/citología , Sistema Nervioso/embriología , Especificidad de la Especie , Células Madre

RESUMEN

We have investigated whether the development of segmental repeats is autonomous in the embryo of the leech Helobdella robusta. The segmental tissues of the germinal band arise from progeny of five stem cells called teloblasts. Asymmetric divisions of the teloblasts form chains of segment founder cells (called primary blast cells) that divide in a stereotypical manner to produce differentiated descendants. Using two distinct techniques, we have looked for potential interactions between neighboring blast cell clones along the anterior-posterior axis. In one technique, we prevented the birth of primary blast cells by injection of DNase I into the teloblast, thereby depriving the last blast cell produced before the ablation of its normal posterior neighbors. We also ablated single blast cells with a laser microbeam, which allowed us to assess potential signals acting on either more anterior or more posterior primary blast cell clones. Our results suggest that interactions along the anterior-posterior axis between neighboring primary blast cell clones are not required for development of normal segmental organization within the blast cell clone. We also examined the possibility that blast cells receive redundant signals from both anterior and posterior neighboring clones and that either is sufficient for normal development. Using double blast cell laser ablations to isolate a primary blast cell clone by removal of both its anterior and its posterior neighbor, we found that the isolated clone still develops normally. These results reveal that the fundamental segmental repeat in the leech embryo, the primary blast cell clone, can develop normally in the apparent absence of signals from adjacent repeats along the anterior-posterior axis.

Asunto(s)

Tipificación del Cuerpo , Sanguijuelas/embriología , Animales , Desarrollo Embrionario , Sanguijuelas/anatomía & histología

RESUMEN

Annelids, unlike their vertebrate or fruit fly cousins, are a bilaterian taxon often overlooked when addressing the question of body plan evolution. However, recent data suggest that annelids offer unique insights on the early evolution of spiral cleavage, anteroposterior axis formation, body axis segmentation, and head versus trunk distinction.

Asunto(s)

Anélidos/anatomía & histología , Anélidos/embriología , Evolución Biológica , Tipificación del Cuerpo/genética , Genes Homeobox , Animales , Anélidos/genética , Embrión no Mamífero

RESUMEN

REGA-1 is a glycoprotein localized to sheath cell processes in the developing CNS when NBs are producing progeny and neurons are maturing and extending processes. It is also present on a subset of muscles and on the lumenal surface of the ectoderm in the embryonic appendages when pioneer neurons are growing into the CNS. REGA-1 is associated with the extracellular side of the cell membrane by a glycosyl-phosphatidylinositol linkage. We have identified a cDNA clone encoding REGA-1 using a sequence from purified protein. Sequence analysis defines REGA-1 as a novel member of the immunoglobulin superfamily containing three immunoglobulin domains and one fibronectin type III repeat. Each Ig domain has distinct sequence characteristics that suggest discrete functions. REGA-1 is similar to other Ig superfamily members involved in cell adhesion events and neurite outgrowth.

Asunto(s)

Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Glicosilfosfatidilinositoles/metabolismo , Saltamontes/embriología , Inmunoglobulinas/química , Proteínas de Insectos , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/química , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía de Afinidad , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , ADN Complementario , Fibronectinas/química , Inmunoglobulinas/biosíntesis , Inmunohistoquímica , Glicoproteínas de Membrana/análisis , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Mapeo Restrictivo , Homología de Secuencia de Aminoácido

RESUMEN

To identify molecules important for pathfinding by growing axons, monoclonal antibodies (mAb) have been generated against embryonic grasshopper tissue. One mAb, 2B2, shows labeling exclusively in the nervous system. It recognizes a surface epitope on neuronal growth cones, filopodia and axons in the central nervous system (CNS). Initially, the antigen is expressed on all processes of the CNS; after 70% of embryonic development, localization of the 2B2 mAb is restricted to a small subset of axon tracts within the ganglia. Immunoprecipitation from embryonic membrane extracts with the 2B2 mAb reveals a unique band of 160 x 10(3) Mr. Functional studies with the 2B2 mAb demonstrate that the antigen is important in growth cone-axon interactions during process outgrowth. Growth cones that extend along axonal substrata are either blocked in growth or grow along an aberrant pathway when embryos are cultured in the presence of the 2B2 mAb. However, pioneer neurons that extend processes on non-neuronal substrata grow normally.

Asunto(s)

Antígenos/fisiología , Axones/fisiología , Sistema Nervioso Central/embriología , Saltamontes/embriología , Animales , Anticuerpos Monoclonales , Axones/inmunología , Axones/ultraestructura , Sistema Nervioso Central/ultraestructura , Técnica del Anticuerpo Fluorescente , Saltamontes/ultraestructura , Inmunohistoquímica , Microscopía Inmunoelectrónica , Pruebas de Precipitina

RESUMEN

The product of the nuclear Saccharomyces cerevisiae gene PET54 is specifically required, along with at least two other nuclear gene products, for translation of the mitochondrial mRNA encoding subunit III of cytochrome c oxidase (coxIII). We have genetically mapped PET54 (to the right arm of chromosome VII, 4.8 cM centromere-distal to SUF15), and have biochemically characterized the gene and its product. We determined the nucleotide sequence of a 1.6-kb DNA fragment carrying PET54 and identified the PET54 reading frame by determining the sequence of an ochre mutant allele as well as frameshift and frameshift-revertant alleles of the gene. The wild-type PET54 gene encodes a slightly basic 293-amino acid protein. PET54 is expressed from two mRNAs, both with unusual features: a major transcript with an extremely short 5'-untranslated leader, and a minor transcript with a relatively long 5'-leader carrying three short open reading frames. Antiserum raised against a trpE-PET54 fusion protein was used to probe subcellular fractions. These experiments showed that the PET54 protein is specifically associated with mitochondria, suggesting that it is likely to act directly in coxIII translation.

Asunto(s)

Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/análisis , Datos de Secuencia Molecular , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/análisis

RESUMEN

We describe several yeast nuclear mutations that specifically block expression of the mitochondrial genes encoding cytochrome c oxidase subunits II (COXII) and III (COXIII). These recessive mutations define positive regulators of mitochondrial gene expression that act at the level of translation. Mutations in the nuclear gene PET111 completely block accumulation of COXII, but the COXII mRNA is present in mutant cells at a level approximately one-third of that of the wild type. Mitochondrial suppressors of pet111 mutations correspond to deletions in mtDNA that result in fusions between the coxII structural gene and other mitochondrial genes. The chimeric mRNAs encoded by these fusions are translated in pet111 mutants; this translation leads to accumulation of functional COXII. The PET111 protein probably acts directly on coxII translation, because it is located in mitochondria. Translation of the mitochondrially coded mRNA for COXIII requires the action of at least three nuclear genes, PET494, PET54 and a newly discovered gene, provisionally termed PET55. Both the PET494 and PET54 proteins are located in mitochondria and therefore probably act directly on the mitochondrial translation system. Mutations in all three genes are suppressed in strains that contain chimeric coxIII mRNAs with the 5'-untranslated leaders of other mitochondrial transcripts fused to the coxIII coding sequence. The products of all three nuclear genes may form a complex and carry out a single function.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Núcleo Celular/metabolismo , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Regulación de la Expresión Génica , Genes Fúngicos , Genes , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Sustancias Macromoleculares , Mitocondrias/enzimología , Mutación , ARN Mensajero/genética , Saccharomyces cerevisiae/enzimología , Transcripción Genética

RESUMEN

Mitochondrial translation of the oxi2 mRNA, encoding yeast cytochrome c oxidase subunit III (coxIII), has previously been shown to specifically require the mitochondrially located protein product of the nuclear gene PET494. We show here that this specific translational activation involves at least one other newly identified gene termed PET54. Mutations in PET54 cause an absence of the coxIII protein despite the presence of normal levels of its mRNA. pet494 mutations are known to be suppressible by mitochondrial gene rearrangements that replace the normal 5'-untranslated leader of the oxi2 mRNA with the leaders of other mitochondrial mRNAs. In this study we show that pet54, pet494 double mutants are suppressed by the same mitochondrial gene rearrangements, showing that the PET54 product is specifically required, in addition to the PET494 protein, for translation of the oxi2 mRNA. Since, as we show here, PET54 is not an activator of PET494 gene expression, our results suggest that the products of both of these genes may act together to stimulate coxIII translation.

Asunto(s)

Núcleo Celular/metabolismo , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Genes Fúngicos , Genes , Biosíntesis de Proteínas , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Sustancias Macromoleculares , Mitocondrias/enzimología , Saccharomyces cerevisiae/enzimología , beta-Galactosidasa/genética