J. David Lambert

Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages

During development, different cell fates are generated by cell–cell interactions or by the asymmetric distribution of patterning molecules. Asymmetric inheritance is known to occur either through directed transport along actin microfilaments into one daughter cell or through capture of determinants by a region of the cortex inherited by one daughter. Here we report a third mechanism of asymmetric inheritance in a mollusc embryo. Different messenger RNAs associate with centrosomes in different cells and are subsequently distributed asymmetrically during division. The segregated mRNAs are diffusely distributed in the cytoplasm and then localize, in a microtubule-dependent manner, to the pericentriolar matrix. During division, they dissociate from the core mitotic centrosome and move by means of actin filaments to the presumptive animal daughter cell cortex. In experimental cells with two interphase centrosomes, mRNAs accumulate on the correct centrosome, indicating that differences between centrosomes control mRNA targeting. Blocking the accumulation of mRNAs on the centrosome shows that this event is required for subsequent cortical localization. These events produce a complex pattern of mRNA localization, in which different messages distinguish groups of cells with the same birth order rank and similar developmental potentials.

Widespread RNA segregation in a spiralian embryo

SUMMARY Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3–4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four‐cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24‐cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.

Nanos Is Required in Somatic Blast Cell Lineages in the Posterior of a Mollusk Embryo

During animal development, blast cell lineages are generated by repeated divisions of a mother cell into a series of daughter cells, often with a specific series of distinct fates. Nanos is a translational regulator that is involved in germline development in diverse animals and also involved in somatic patterning in insects. Recently, Nanos was found to be required for maintenance of stem cell divisions in the Drosophila germline. We have found that in the mollusk Ilyanassa, Nanos messenger RNA and protein are specifically localized in the mesendodermal blast cell lineage derived from the strongly conserved 4d cell. Nanos activity is required for differentiation of multiple tissues that are derived from the 4d cell, showing that IoNanos is required for somatic development in this embryo. At the cellular level, we show that IoNanos activity is required for the highly stereotyped cleavage pattern of the 4d lineage, the proliferative capacity of the blast cells, and the marked asymmetry of the blast cell divisions. These results suggest that IoNanos is involved in regulating blast cell behaviors in the 4d lineage.

Developmental Patterns in Spiralian Embryos

At least five animal phyla exhibit spiralian development, which is characterized by striking similarities in the geometry of the early cleavage pattern and the fate map of the blastula, along with similarities in larval morphology. Recent advances in reconstructing the phylogeny of spiralians and their relatives suggest that the common ancestor of a large clade of protostome phyla known as the Lophotrochozoa had spiralian development. In this minireview, I describe characteristics of spiralian development and some recent insights into its mechanisms and evolution.

Localization of Vasa mRNA during early cleavage of the snail Ilyanassa.

Members of the Vasa family of helicases are specifically localized to germ line lineages in embryos of many animal groups and, in some cases, have been shown to be required for germ line formation. Despite considerable attention to the embryology of gastropod molluscs, the germ line has not been identified in the early cleavage stages of these embryos. We have cloned a Vasa ortholog in the snail Ilyanassa and examined the distribution of IoVasa mRNA during early cleavage. Initially, the transcript is present in all cells and non-specifically localized to centrosomes in a subset of cells. The IoVasa mRNA becomes progressively more enriched in the dorsal quadrant of the embryo, and then becomes restricted to particular cells in the 4d lineage. At the 64-cell stage, IoVasa mRNA is detected in 4dL11, 4dL12, 4dR11, and 4dR12. Following another round of division in the 4d lineage, the mRNA is restricted to two cells: 4dL121 and 4dR121. By the 108-cell stage, IoVasa mRNA is no longer detectable. Because the germ line is thought to arise from the 4d lineage in spiralians, these data are consistent with the hypothesis that the Ilyanassa germ line is marked by inheritance of IoVasa and derived from the cells 4dL121 and 4dR121. Alternatively, IoVasa may be required in somatic lineages where it is expressed, and the germ line may be specified later in development.

Characterizing the Embryonic Transcriptome of the Snail Ilyanassa

The snail Ilyanassa obsoleta is a useful model for a variety of investigations in the fields of developmental biology, cell biology, larval ecology, ecotoxicology, parasitology, and chemical ecology. To enhance such studies, we have carried out two cDNA sequencing projects to characterize the mRNA transcripts that are present during development of this embryo. These efforts have generated 480 megabases of new sequence, which have been assembled into transcript contigs and represent thousands of newly identified Ilyanassa genes. We identified the orthologs of 182 transcription factors in these data, focusing on families that are likely to be sequence-specific transcriptional regulators. To demonstrate the utility of identifying and examining such transcripts, we describe the expression pattern during organogenesis for IoOnecut, an Ilyanassa ortholog of the HNF6/onecut family of transcription factors.

Spiralian quartet developmental potential is regulated by specific localization elements that mediate asymmetric RNA segregation

Spiralian embryos are found in a large group of invertebrate phyla but are largely uncharacterized at a molecular level. These embryos are thought to be particularly reliant on autonomous cues for patterning, and thus represent potentially useful models for understanding asymmetric cell division. The series of asymmetric divisions that produce the micromere quartets are particularly important for patterning because they subdivide the animal-vegetal axis into tiers of cells with different developmental potentials. In the embryo of the snail Ilyanassa, the IoLR5 RNA is specifically segregated to the first quartet cells during the third cleavage. Here, we show that this RNA, and later the protein, are maintained in the 1q121 cells and their descendents throughout development. Some IoLR5-expressing cells become internalized and join the developing cerebral ganglia. Knockdown of IoLR5 protein results in loss of the larval eyes, which normally develop in association with these ganglia. Segregation of this RNA to the first quartet cells does not occur if centrosomal localization is bypassed. We show that the specific inheritance of the RNA by the first quartet cells is driven by a discrete RNA sequence in the 3′ UTR that is necessary and sufficient for localization and segregation, and that localization of another RNA to the first quartet is mediated by a similar element. These results demonstrate that micromere quartet identity, a hallmark of the ancient spiralian developmental program, is controlled in part by specific RNA localization motifs.

Patterning a spiralian embryo: a segregated RNA for a Tis11 ortholog is required in the 3a and 3b cells of the Ilyanassa embryo.

Spiralian embryogenesis is found in a number of animal phyla, but the molecular mechanisms that pattern these embryos remain poorly understood. A hallmark of spiralian development is the production of tiers of cells, called quartets, that share distinct developmental potentials. Many RNAs have been discovered that are segregated into particular quartets, raising the possibility that such RNAs could be involved in establishing quartet-specific developmental potentials. In the spiralian embryo of the mollusc Ilyanassa, the IoTis11 RNA is segregated into the second and third quartets, then decays in nearly all lineages except for the ventral-anterior cells of the third quartet, 3a and 3b. Previously published fate-mapping studies, extended here, show that 3a and 3b make bilaterally symmetrical contributions to the esophagus, head ectoderm, and larval musculature. Deletion of either 3a or 3b has only mild effects on development, but ablating both cells impairs development of the esophagus and several other organs. Knockdown of IoTis11 with a translation-blocking morpholino oligonucleotide causes a very similar set of phenotypes as ablation of 3a and 3b, showing that translation of this transcript is required for normal development of 3a and 3b. These results show that a segregated RNA is necessary for the cells that inherit it in a spiralian embryo. Given that RNAs are asymmetrically segregated in nearly all the early cleavages in this embryo, these results suggest that the embryo is extensively patterned by segregated factors. Our experiments also uncovered two previously unappreciated non-autonomous events during Ilyanassa development. First, we found that the embryo can regulate to develop normal esophagus after deletion of either 3a or 3b. Second, we found that the 3a or 3b lineages are required for normal development of the digestive glands, which arise from the fourth order macromeres.

Dpp/BMP2-4 Mediates Signaling from the D-Quadrant Organizer in a Spiralian Embryo.

In some animal groups, the secondary embryonic axis is patterned by a small group of cells, often called an organizer, that signals to other cells to establish the correct pattern of cell fates. The Dpp/BMP2-4 pathway plays a central role in secondary axis patterning in many animals [1-11], but it has not been examined during early axial patterning in spiralian embryogenesis. This is a deeply conserved mode of development found in mollusks, annelids, nemerteans, entoprocts, and some marine platyhelminth groups (reviewed in [12, 13]). In the spiralian embryo of the mollusk Ilyanassa, we find that the Dpp ortholog (IoDpp) is expressed most strongly on the dorsal side, in cells of the embryonic organizer and its neighbors. Phospho-smad staining indicates that the pathway is active in all lineages during organizer signaling, but activation is strongest on the dorsal side. Knockdown of IoDpp by morpholino oligos prevents the development of all structures that require organizer signaling and ventralizes the embryo. Ectopic activation of the pathway can induce eyes and external shell, which require organizer signaling. These results indicate that Dpp/BMP2-4 signaling is a key part of the spiralian organizer and suggest similarity with other metazoan organizers. However, the fact that IoDpp/BMP2-4 is inducing, rather than repressing, the neuroectoderm is a surprising difference that may be conserved among spiralians. These results connect the spiralian organizer to this general aspect of secondary axis patterning but highlight the significant variation across animals in effects of the pathway on particular cell types and tissues.

The Snail Ilyanassa: A Reemerging Model for Studies in Development

Ilyanassa obsoleta is a marine gastropod that is a long-standing and very useful model for studies of embryonic development. It is especially important as a model for the spiralian development program, a distinctive mode of early development shared by a large group of animal phyla, but poorly understood. Ilyanassa adults are readily obtainable and easy to keep in the laboratory, and they produce large numbers of embryos throughout most of the year. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. In this article, we present an overview of aspects of its biology and use as a model organism.