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Among animals, diploblasts contain two germ layers, endoderm and ectoderm, while triploblasts have a distinct third germ layer called the mesoderm. Spiralians are a group of triploblast animals that have highly conserved development: they share the distinctive spiralian cleavage pattern as well as a unique source of mesoderm, the ectomesoderm. This population of mesoderm is distinct from endomesoderm and is considered a hallmark of spiralian development, but the regulatory network that drives its development is unknown. Here we identified ectomesoderm-specific genes in the mollusc Tritia (aka Ilyanassa) obsoleta through differential gene expression analyses comparing control and ectomesoderm-ablated embryos, followed by in situ hybridization of identified transcripts. We identified a Tritia serpin gene (ToSerpin1) that appears to be specifically expressed in the ectomesoderm of the posterior and head. Ablation of the 3a and 3b cells, which make most of the ectomesoderm, abolishes ToSerpin1 expression, consistent with its expression in these cells. Morpholino knockdown of ToSerpin1 causes ectomesoderm defects, most prominently in the muscle system of the larval head. This is the first gene identified that is specifically implicated in spiralian ectomesoderm development.

Caudal homeobox genes are found across animals, typically linked to two other homeobox genes in what has been called the ParaHox cluster. These genes have been proposed to pattern the anterior-posterior axis of the endoderm ancestrally, but the expression of Caudal in extant groups is varied and often occurs in other germ layers. Here we examine the role of Caudal in the embryo of the mollusc Tritia (Ilyanassa) obsoleta. ToCaudal expression is initially broad, then becomes progressively restricted and is finally only in the developing hindgut (a.k.a. intestine). Knockdown of ToCaudal using morpholino oligonucleotides specifically blocks hindgut development, indicating that despite its initially broad expression, the functional role of ToCaudal is in hindgut patterning. This is the first functional characterization of Caudal in an animal with spiralian development, which is an ancient mode of embryogenesis that arose early in bilaterian animal evolution. These results are consistent with the hypothesis that the ancestral role of the ParaHox genes was anterior-posterior patterning of the endoderm.

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.

Spiralian development is shared by several protostome phyla and characterized by regularities in early cleavage, fate map, and larva. Experimental evidence from multiple spiralian species implicates cells in the D quadrant lineage as the organizer of future axial development of the embryo. However, the mechanisms by which the D quadrant is specified differ between species with equal and unequal spiral cleavage. Equally cleaving mollusc embryos establish the D quadrant via cell–cell interactions between the micromeres and macromeres at the 24- to 36-cell stage. In unequally cleaving embryos, the D quadrant is established at the 4-cell stage via asymmetries in the first 2 cell divisions. We have begun to explore the molecular mechanisms of D quadrant patterning in spiralians. Previously, we showed that, in the unequally cleaving embryo of the mollusc Ilyanassa obsoleta, the MAPK pathway is activated and functionally required in 3D and also in the micromeres known to require a signal from 3D. Here, we examine the role of MAPK signaling in 4 spiralians with equal cleavage. In 3 equally cleaving molluscs, the chiton Chaetopleura, the limpet Tectura, and the snail Lymnaea, the MAPK pathway is activated in the 3D cell but not in the overlying micromeres. In the equally cleaving embryo of the polychaete annelid Hydroides, MAPK activation was not detected in the 3D macromere but was observed in one of its daughter cells, 4d. In addition, inhibiting Tectura MAPK activation disrupts differentiation of 3D and cells induced by it, supporting a functional role for MAPK in axis specification in equally cleaving spiralians. Thus, MAPK signaling may have a conserved role in the D quadrant organizer cell 3D in molluscs. However, there have been at least 2 evolutionary changes in the activation of the MAPK pathway during spiralian evolution. MAPK function in the Ilyanassa micromeres is a recent cooption and, since the divergence of annelids and molluscs, there has been a shift in onset of MAPK activation between 3D and 4d. We propose that this latter shift correlates with a change in the timing of specification of the secondary embryonic axis.