Ectomesoderm and epithelial-mesenchymal transition-related genes in spiralian development.

BACKGROUND: Spiralians (e.g., annelids, molluscs, and flatworms) possess two sources of mesoderm. One is from endodermal precursors (endomesoderm), which is considered to be the ancestral source in metazoans. The second is from ectoderm (ectomesoderm) and may represent a novel cell type in the Spiralia. In the mollusc Crepidula fornicata, ectomesoderm is derived from micromere daughters within the A and B cell quadrants. Their progeny lie along the anterolateral edges of the blastopore. There they undergo epithelial-mesenchymal transition (EMT), become rounded and undergo delamination/ingression. Subsequently, they assume the mesenchymal phenotype, and migrate beneath the surface ectoderm to differentiate various cell types, including muscles and pigment cells. RESULTS: We examined expression of several genes whose homologs are known to regulate Type 1 EMT in other metazoans. Most of these genes were expressed within spiralian ectomesoderm during EMT. CONCLUSIONS: We propose that spiralian ectomesoderm, which exhibits analogous cellular behaviors to other populations of mesenchymal cells, may be controlled by the same genes that drive EMT in other metazoans. Perhaps these genes comprise a conserved metazoan EMT gene regulatory network (GRN). This study represents the first step in elucidating the GRN controlling the development of a novel spiralian cell type (ectomesoderm). Developmental Dynamics 247:1097-1120, 2018. © 2018 Wiley Periodicals, Inc.

Beyond the sea: Crepidula atrasolea as a spiralian model system.

This paper introduces the black-footed slipper snail, Crepidula atrasolea, as a new model for biological studies in the Spiralia. C. atrasolea is a calyptraeid gastropod, and congener of the Atlantic slipper snail, C. fornicata. Like C. fornicata, C. atrasolea shares a sedentary, filter-feeding, protandrous lifestyle, but is preferable as a developmental model because of its short generation time, year-round reproduction, and direct development. In our lab, individuals go from egg to reproductive females in under six months, as compared to an estimated 1-2 years for C. fornicata. Here we provide details for collecting and transporting animals, setting up inland aquaria, and maintaining laboratory colonies of C. atrasolea. We also describe early development, which is similar to that in other calyptraeids. Females brood encapsulated embryos for three weeks, which hatch as "crawl-away" juveniles. We also present a developmental transcriptome for C. atrasolea, covering early cleavage through late organogenesis stages, as a useful tool for future studies of gene expression and function. We provide this information to the broader developmental community to facilitate widespread use of this system.

Developmental transition to bilaterally symmetric cell divisions is regulated by Pax-mediated transcription in embryos of the leech Helobdella austinensis.

The leech embryo develops by spiral cleavage, and establishes the symmetry properties of its adult body plan through the bilaterally symmetric divisions of mesodermal proteloblast DM″ and ectodermal proteloblast DNOPQ‴. We here show that transcriptional inhibitors α-amanitin and actinomycin D specifically disrupt the symmetry and orientation of these two proteloblast cell divisions while having no apparent effect on the timing or geometry of other divisions. Transcriptional inhibition had a similar effect on both proteloblasts, i.e. cytokinesis was highly asymmetric and the cleavage plane roughly orthogonal to that seen during normal development. These findings suggest that zygotic gene product(s) are required, either directly or indirectly, for the correct placement of the proteloblast cleavage furrow. The same phenotypes were also observed following in vivo expression of dominant-negative Pax gene constructs. These dominant-negative phenotypes depended on protein/DNA interaction, and could be rescued by coexpression of full length Pax proteins. However, symmetric cleavage of the mesodermal proteloblast was rescued by full length constructs of either Hau-Paxß1 or Hau-Pax2/5/8, while only Hau-Paxß1 rescued the symmetry of ectodermal cleavage. We conclude that both proteloblasts need Pax-mediated transcription to adopt their normally symmetric cleavage patterns, but differ in terms of the specific Pax proteins required. The implication of these findings for the evolution of spiral cleavage is discussed.

Regional differences in BMP-dependence of dorsoventral patterning in the leech Helobdella.

In the leech Helobdella, the ectoderm exhibits a high degree of morphological homonomy between body segments, but pattern elements in lateral ectoderm arise via distinct cell lineages in the segments of the rostral and midbody regions. In each of the four rostral segments, a complete set of ventrolateral (O fate) and dorsolateral (P fate) ectodermal pattern elements arises from a single founder cell, op. In the 28 midbody and caudal segments, however, there are two initially indeterminate o/p founder cells; the more dorsal of these is induced to adopt the P fate by BMP5-8 emanating from the dorsalmost ectoderm, while the more ventral cell assumes the O fate. Previous work has suggested that the dorsoventral patterning of O and P fates differs in the rostral region, but the role of BMP signaling in those segments has not been investigated. We show here that suppression of dorsal BMP5-8 signaling (which effects a P-to-O fate change in the midbody) has no effect on the patterning of O and P fates in the rostral region. Furthermore, ectopic expression of BMP5-8 in the ventral ectoderm (which induces an O-to-P fate change in the midbody) has no effect in the rostral region. Finally, expression of a dominant-negative BMP receptor (which induces a P-to-O fate change in the midbody) fails to affect O/P patterning in the rostral region. Thus, the rostral segments appear to use some mechanism other than BMP signaling to pattern O and P cell fates along the dorsoventral axis. From a mechanistic standpoint, the OP lineage of the rostral segments and the O-P equivalence group of the midbody and caudal segments constitute distinct developmental modules that rely to differing degrees on positional cues from surrounding ectoderm in order to specify homonomous cell fates.

A member of the six gene family promotes the specification of P cell fates in the O/P equivalence group of the leech Helobdella.

The lateral ectoderm of the leech embryo arises from the o and p bandlets, two parallel columns of blast cells that collectively constitute the O/P equivalence group. Individual blast cells within this equivalence group become committed to alternative O or P developmental pathways in accordance with their respectively ventrolateral or dorsolateral position (Weisblat and Blair, 1984). We here describe a novel member of the Six gene transcription factor family, Hau-Six1/2A, which contributes to the patterning of these cell fates in the leech Helobdella sp. (Austin). During embryogenesis Hau-Six1/2A expression is restricted to the dorsolateral column of p blast cells, and thus correlates with P cell fate over most of the body's length. Experimental manipulations showed that Hau-Six1/2A expression is induced in p blast cells by the interaction with the adjoining q bandlet. In addition, misexpression of Hau-Six1/2A in the ventrolateral o blast cells by injection of an expression plasmid elicited the dorsolateral P cell fates ectopically. These data imply that Hau-Six1/2A is a component of the molecular pathway that normally distinguishes O and P cell fates within this equivalence group. Genomic analysis revealed that the Six1/2 subfamily has expanded to a total of six genes in Helobdella. The pattern of Hau-Six1/2A expression during later embryogenesis suggested that this gene may have lost ancestral function(s) and/or acquired novel roles in association with the gene duplications that produced this expansion.

Paxbeta: a novel family of lophotrochozoan Pax genes.

Paxbeta is a novel gene family restricted to the bilaterian superphylum Lophotrochozoa. The Paxbeta paired domain is highly diverged from other bilaterian Pax genes, and we were unable to identify an unambiguous sister-group by phylogenetic sequence analysis. However, conservation of a paired domain intron suggests that the Paxbeta genes arose by duplication and divergence from an ancestral Pax2/5/8 gene. We have identified Paxbeta genes in annelids, molluscs, a nemertean, a brachiopod, and a flatworm, indicating that the founding gene duplication occurred before the separation of those taxa and therefore early in the lophotrochozoan radiation. The leech Helobdella has two genes in this family, Hau-Paxbeta1 and -Paxbeta2, which are expressed during embryonic development. Hau-Paxbeta1 is present in the zygote as a localized maternal transcript, and both show dynamic patterns of apparently zygotic expression during later stages. These later expression patterns differ significantly, but both genes are expressed broadly in the mesoderm and also in the central nervous system during organogenesis.

Hau-Pax6A expression in the central nervous system of the leech embryo.

The leech Helobdella sp. (Austin) has two genes of the Pax6 subfamily, one of which is characterized in detail. Hau-Pax6A was expressed during embryonic development in a pattern similar to other bilaterian animals. RNA was detected in cellular precursors of the central nervous system (CNS) and in peripheral cells including a population associated with the developing eye. The CNS of the mature leech is a ventral nerve cord composed of segmental ganglia, and embryonic Hau-Pax6A expression was primarily localized to the N teloblast lineage that generates the majority of ganglionic neurons. Expression began when the ganglion primordia were four cells in length and was initially restricted to a single cell, n(s).a, whose descendants will form the ganglion's anterior edge. At later stages, the Hau-Pax6A expression pattern expanded to include additional CNS precursors, including some descendants of the O teloblast. Expression persisted through the early stages of ganglion morphogenesis but disappeared from the segmented body trunk at the time of neuronal differentiation. The timing and iterated pattern of Hau-Pax6A expression in the leech embryo suggests that this gene may play a role in the segmental patterning of CNS morphogenesis.

Hau-Pax3/7A is an early marker of leech mesoderm involved in segmental morphogenesis, nephridial development, and body cavity formation.

Two genes of the Pax III subfamily, Hau-Pax3/7A and -Pax3/7B, were identified from the leech Helobdella, and the expression and function of Hau-Pax3/7A in development are described. Leech embryos undergo spiral cleavage, then produce a set of teloblastic stem cells that generate segmented mesoderm and ectoderm. Hau-Pax3/7A is present as a maternal transcript in both ectodermal and mesodermal progenitors, but this pool of early RNA disappears and is replaced by a pattern of zygotic transcription restricted to the blast cell progeny of the mesodermal M teloblasts. Each mesodermal blast cell clone goes through multiple phases of Hau-Pax3/7A expression, the last of which is associated with the organogenesis of the nephridia and other segment-specific structures. Morpholino-mediated knockdown of Hau-Pax3/7A expression causes the mesodermal blast cell clones to undergo irregular patterns of morphogenesis that disrupt the segmental organization of the germinal plate, and interferes with both the specification and morphological differentiation of the mesodermal nephridia. Knockdown of Hau-Pax3/7A in the mesoderm can also lead to abnormalities in the formation of the dorsal cavities, possibly through indirect effects of this germ layer on neighboring tissues. This is the first report of broad mesodermal Pax III expression outside of chordates, and raises the possibility that such expression may be a primitive trait inherited from the last common ancestor of the bilaterian superphyla.

Evolutionary diversification of specification mechanisms within the O/P equivalence group of the leech genus Helobdella.

Developmental fates and cell lineage patterns are highly conserved in the teloblast lineages that give rise to the segmental ectoderm of clitellate annelids. But previous studies have shown that the pathways involved in specification of the ventrolateral O lineage and the dorsolateral P lineage differ to some degree in distantly related clitellate species such as the leeches Helobdella and Theromyzon, and the sludgeworm Tubifex. To examine this developmental variation at a lower taxonomic level, we have explored the specification pathways of the O and P lineages in the leech genus Helobdella. In leech, the O and P lineages arise from a developmental equivalence group of O/P teloblasts. In this study, we demonstrate that the cell-cell interactions involved in cell fate specification of the O/P equivalence group differ among three laboratory colonies of closely related species. In two populations, the Q lineage is necessary to specify the P fate in the dorsalmost O/P lineage, but in the third population the P fate can be specified by a redundant pathway involving the M lineage. We also observe interspecific variation in the role played by cell interactions within the O/P equivalence group, and in the apparent significance of extrinsic signals from the micromere cell lineages. Our data suggest that cell fate specification in the O/P equivalence group is a complex process that involves multiple cell-cell interactions, and that the developmental architecture of the O/P equivalence group has undergone evolutionary diversification in closely related species, despite maintaining a conserved morphology.

A distinct patterning mechanism of O and P cell fates in the development of the rostral segments of the leech Helobdella robusta: implications for the evolutionary dissociation of developmental pathway and morphological outcome.

Despite a high degree of homonomy in the segmental organization of the ectoderm, the body plan of the leech is divided into two zones based on the distinct cell lineage patterns that give rise to the O/P portion of the segmental ectoderm. In the midbody and caudal segments, each segmental repeat of ectoderm arises in part from one 'o' blast cell and one 'p' blast cell. These two blast cells are positionally specified to distinct O and P fates, and give rise to differentiated descendant cells called O and P pattern elements, respectively. In the rostral segments, each segmental repeat of O and P pattern elements arises from a single 'op' blast cell. Based on their developmental fates and their responses to the ablation of neighboring cells, the granddaughters of the primary op blast cell are categorized into two O-type cells and two P-type cells. The O-type cells do not require the presence of the rest of the op blast cell clone for their normal development. By contrast, normal development of the P-type cells depends upon interactions with the other OP sublineages. Additional experiments showed that the O-type cells are the source of a repressive signal involved in the normal fate specification of the P-type cells. Our data suggest that the cell interactions involved in fate specification differ substantially in the rostral and midbody segments, even though the set of differentiated descendants produced by the rostral OP pathway and the midbody O and P pathways are very similar.

A hedgehog homolog regulates gut formation in leech (Helobdella).

Signaling by the hedgehog (hh)-class gene pathway is essential for embryogenesis in organisms ranging from Drosophila to human. We have isolated a hh homolog (Hro-hh) from a lophotrochozoan species, the glossiphoniid leech, Helobdella robusta, and examined its expression by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. The peak of Hro-hh expression occurs during organogenesis (stages 10-11). No patterned expression was detected within the segmented portion of the germinal plate during the early stages of segmentation. In stage 10-11 embryos, Hro-hh is expressed in body wall, foregut, anterior and posterior midgut, reproductive organs and in a subset of ganglionic neurons. Evidence that Hro-hh regulates gut formation was obtained using the steroidal alkaloid cyclopamine, which specifically blocks HH signaling. Cyclopamine induced malformation of both foregut and anterior midgut in Helobdella embryos, and no morphologically recognizable gonads were seen. In contrast, no gross abnormalities were observed in the posterior midgut. Segmental ectoderm developed normally, as did body wall musculature and some other mesodermal derivatives, but the mesenchymal cells that normally come to fill most of the coelomic cavities failed to develop. Taken with data from Drosophila and vertebrates, our data suggest that the role of hh-class genes in gut formation and/or neural differentiation is ancestral to the bilaterians, whereas their role in segmentation evolved secondarily within the Ecdysozoa.