Expression of Hox, Cdx, and Six3/6 genes in the hoplonemertean Pantinonemertes californiensis offers insight into the evolution of maximally indirect development in the phylum Nemertea
© Hiebert and Maslakova. 2015
Received: 5 February 2015
Accepted: 15 July 2015
Published: 4 August 2015
Maximally indirect development via a pilidium larva is unique to the pilidiophoran clade of phylum Nemertea. All other nemerteans have more or less direct development. The origin of pilidial development with disjunct invaginated juvenile rudiments and catastrophic metamorphosis remains poorly understood. While basal members of the phylum, the Palaeonemertea, do not appear to have ever had a pilidium, certain similarity exists in the development of the Pilidiophora and the sister clade, the Hoplonemertea. It is unclear whether this similarity represents the homology and whether pilidial development evolved before or after pilidiophorans diverged from hoplonemerteans. To gain insight into these questions, we examined the expression of Hox, Cdx, and Six3/6 genes in the development of the hoplonemertean Pantinonemertes californiensis and expression of Six3/6 in the pilidium of Micrura alaskensis. To further characterize the function of larval structures showing expression of these genes, we examined the serotonergic nervous system and cell proliferation in P. californiensis.
We show that Hox and Cdx genes, which pattern the pilidial imaginal discs giving rise to the juvenile trunk, are expressed in paired posterior epidermal invaginations in P. californiensis larvae. We also show that Six3/6 patterns both the pilidial cephalic discs, which give rise to the juvenile head, and a pair of anterior epidermal invaginations in hoplonemertean development. We show that anterior invaginations in larval P. californiensis are associated with a pair of serotonergic neurons, and thus may have a role in the development of the juvenile nervous system. This is similar to the role of cephalic discs in pilidiophoran development. Finally, we show that four zones of high cell proliferation correspond to the paired invaginations in P. californiensis, suggesting that these invaginations may play a similar role in the development of the hoplonemertean juvenile to the role of imaginal discs in the pilidium, which also exhibit high rates of cell proliferation.
Expression of Hox, Cdx, and Six3/6 genes supports the homology between the imaginal discs of the pilidium and the paired larval invaginations in hoplonemerteans. This suggests that invaginated juvenile rudiments (possible precursors to pilidial imaginal discs) may have been present in the most recent common ancestor of the Pilidiophora and Hoplonemertea.
Development in nemerteans, a phylum of mostly marine predatory worms, ranges from direct with a crawl-away juvenile, to mostly direct with a planktonic juvenile-like larva, to a maximally indirect with a unique planktonic larva known as the pilidium [1, 2]. The pilidium is a feeding larva, which has a distinctly different body plan from that of the adult. The juvenile develops inside the pilidium from a set of initially isolated rudiments called imaginal discs [see 3 and references therein]. The imaginal discs grow and fuse together around the larval stomach over a span of weeks or months. Once the juvenile is completely formed inside the pilidium, the larva undergoes a catastrophic metamorphosis, in which the juvenile escapes from and devours the larval body [3–5]. The pilidial life cycle is found in a single clade of nemerteans, the Pilidiophora [6, 7]. However, when, in relation to the major nemertean lineages, and how this metamorphic life history arose remains unclear .
The basal lineage, Palaeonemertea, exhibits no traces of pilidial development. Palaeonemerteans typically possess so-called planuliform (superficially planula-like) planktonic larvae that develop into juveniles without a conspicuous metamorphosis. Maslakova et al. [8, 9] found that the larva of the paleonemertean Carinoma tremaphoros has a “hidden” prototroch derived from the classical spiralian trochoblast lineage, and this prototroch is lost early in larval development. This suggests that a kind of trochophore larva (such as found in related spiralian phyla, e.g., annelids and mollusks) may have characterized ancestral nemerteans. The pilidium, on the other hand, appears to have evolved within nemerteans, after the split between the Palaeonemertea and the Neonemertea, which comprise the Hoplonemertea and the Pilidiophora [6, 7, 10].
Similar to the palaeonemerteans, the Hoplonemertea, the sister clade to the Pilidiophora, develop via a planuliform larval stage, which may be encapsulated or free-swimming [1, 2, 11]. However, the larvae of hoplonemerteans differ from those of palaeonemerteans in a number of ways. None of the studied hoplonemertean larvae have a recognizable prototroch (such as was found in Carinoma). Instead, they have features that are rather reminiscent of pilidia. Many species were found to have a transitory larval epidermis composed of large ciliated cells that are gradually displaced (resorbed or shed) by the much smaller cells of the definitive epidermis [12, 13]. This loss of larval epidermis may be homologous to pilidial metamorphosis [2, 14, 15]. Maslakova  coined the term “decidula” for the hoplonemertean larvae to emphasize the process of ontogenetic loss of the larval epidermis. Some decidulae have been noted to have bilaterally symmetrical epidermal invaginations whose fate or function is not always clear (Maslakova and von Döhren ). Hiebert et al.  hypothesized that some of these invaginations may be homologous to imaginal discs of the pilidium. Thus, it is possible that pilidial development, in some form, has evolved before the split between the Hoplonemertea and the Pilidiophora, and these invaginations represent vestiges of imaginal discs. Alternatively, pilidial development may have evolved at the base of the Pilidiophora, from the kind of development found in extant hoplonemerteans, and these structures in hoplonemertean development represent precursors to pilidial imaginal discs. Regardless of which is correct, until now we lacked other evidence of homology beyond vague morphological similarity.
Here, we provide additional evidence of homology between pilidial imaginal discs and paired invaginations of the hoplonemertean decidula based on the expression of the axial patterning genes Hox and Cdx in the hoplonemertean species P. californiensis and expression of Six3/6 in pilidial and hoplonemertean development. To better illustrate organogenesis, larval anatomy, and to understand the function of these invaginations in hoplonemertean development we also examined the structure of the serotonergic nervous system and cell proliferation patterns in P. californiensis larvae.
Collection of adults and larval culturing
Adult hoplonemerteans, P. californiensis, were collected under rocks along the high intertidal above a mudflat in Coos Bay near Glasgow, OR. Reproductive adults were identified in the field, as gametes are visible through the body wall (pink oocytes, white sperm), and transported in 50-mL plastic tubes to the Oregon Institute of Marine Biology in Charleston, OR. Adults were kept inside individual 50-mL conical tubes with a few milliliters of 0.45 µm-filtered sea water (FSW) in the flowing sea-water table at ambient sea temperature. Water was changed every few days. Spawning, in some cases, was observed immediately after collection, or days to weeks later following a water change. But spawning also occurred without any observed change in conditions. Gametes were successfully obtained July–September of 2009–2013. Our observations of spawning suggest that timing may be influenced by phase of the moon, but we have not rigorously tested this hypothesis. On a number of occasions, the worms released gametes during 2–3 days around the time when the absolute heights of the two daily low tides were similar to each other. Spawned oocytes were resuspended in FSW in glass custard dishes and fertilized with a few drops of dilute sperm suspension. Fertilized eggs were kept at ambient seawater temperature (12–16°C). After hatching (1 day post-fertilization) swimming larvae were transferred into a clean custard dish with fresh FSW. Water was changed every few days by reverse filtration. Collection, fertilization, and larval culture methods for the pilidiophoran Micrura alaskensis were as described in Maslakova  and Hiebert and Maslakova .
Isolation and identification of Hox, Cdx, and Six3/6 sequences
Primers used for Hox amplification
Nested RACE (5′ end):
Nested RACE (gene specific):
Hox and Cdx gene orthology was determined by phylogenetic analysis. Hox and Cdx fragments from P. californiensis were aligned with Cdx and Hox complements of a deuterostome (Branchiostoma floridae), two ecdysozoans (Tribolium castaneum, Drosophila melanogaster), and five lophotrochozoans (an annelid Capitella teleta, a bryozoan Bugula turrita, a nemertean Micrura alaskensis, a brachiopod Lingula anatina, and a mollusk Euprymna scolopes) (see Additional file 1: Table S1). Bayesian inference analysis was conducted using MrBayes version 3.2.1 [18, 19]. Hox fragments were aligned using the homeodomain and the 12 3ʹ amino-acids since this upstream flanking region shows some sequence conservation. The analysis was done with the Rtrev amino-acid model with a gamma-shaped distribution of rates across sites. Drosophila melanogaster Even skipped (eve) was specified as the outgroup. The analysis was done with five heated chains with 5,000,000 generations and was sampled every 500 generations. Four independent runs were conducted. The first 25% samples from the cold chain were discarded as burn-in. Trees were visualized and manipulated using FigTree version 1.4.0 and Adobe Illustrator version 17.1.0.
Phylogenetic analysis was also performed to determine Six3/6 gene orthology. Six family sequences from representative taxa were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/; accession numbers listed in Additional file 1: Table S2), including those from three deuterostomes (Mus musculus, Saccoglossus kowalevskii, Strongylocentrotus purpuratus), one ecdysozoan (Drosophila melanogaster), three lophotrochozoans (a brachiopod Terebratalia transversa, a mollusk Lottia gigantea, the annelids Capitella teleta and Platynereis dumerilii), and a cnidarian (Nematostella vectensis). These sequences were aligned with the Six3/6 sequences from M. alaskensis and P. californiensis, using MUSCLE v3.8 . Alignment was checked by eye. Bayesian analysis and tree visualization was conducted as described above.
Larval fixation and in situ hybridization
Prior to fixation, larvae were relaxed in 1:1 mix of 0.37 M MgCl2:FSW for 10 min. Larvae were fixed overnight at 4°C in 4% paraformaldehyde made up from 16% paraformaldehyde (Electron Microscopy Sciences) in FSW, then washed three times in 1× phosphate-buffered saline, pH 7.4 (PBS, Fisher Scientific) with 0.2% Triton X-100 (Fisher Scientific). Larvae were then washed twice with deionized water, dehydrated in a series of methanol (25, 50, 75, 100%), and stored at −20°C in 100% methanol. Probe preparation and in situ hybridization was conducted as described in Hiebert and Maslakova . In short, larvae were rehydrated in PBS, acetylated with triethanolamine and acetic anhydride, re-fixed with paraformaldehyde, and hybridized in a formamide buffer with 1 ng/µl dioxygenin (DIG)-labeled RNA probe at 63°C for 2–3 days. Excess probe was washed away in low concentration saline sodium citrate. The remaining bound probe was labeled with anti-DIG alkaline phosphatase, which was allowed to react in the dark with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate for 1 h to overnight until blue-purple color developed. Larvae were mounted in 80% glycerol in PBS. Larvae were imaged with a Leica DFC400 digital color camera mounted on an Olympus BX51 microscope equipped with differential interference contrast optics. Ten to twenty specimens were examined for each gene and stage. Some in situ-stained larvae were mounted with propidium iodide in 80% gylcerol in 1× PBS on poly-l-lysine-coated coverslips for confocal microscopy. Coverslips were dipped into a 0.1% poly-l-lysine (Sigma) solution and allowed to dry.
Larvae were fixed as above and rehydrated from methanol into phosphate-buffered saline (PBS) via 5-min changes in 60% methanol, 30% methanol, and then PBS. Larvae were permeabilized with three 10-min washes in PBS with 0.1% Triton X-100 (PBT). To block non-specific staining, larvae were incubated in 5% normal goat serum in PBT for 2 h at room temperature. Normal goat serum was washed out with three 10-min washes in PBT. Larvae were incubated overnight at 4°C in one of two primary antibodies diluted 1:500 in PBT: rabbit-anti-5HT (Immunostar, Cat #20080) or rabbit-anti-phospho-histone H3 (Ser10) (Millipore, Cat #06-570). Larvae were then washed in three 10-min changes in PBT and incubated for 2 h at room temperature in goat-anti-rabbit 488 secondary antibody (Molecular Probes, Cat #A11008) diluted 1:600 in PBT. Larvae were then washed in three 10-min changes of PBS and mounted on poly-l-lysine-coated coverslips using CFM-2 mounting media (CitiFlour LTD).
Confocal microscopy and analysis
Confocal images were obtained with an Olympus FluoView 1000 laser scanning confocal system (Olympus America, Center Valley, PA, USA) mounted on an Olympus IX81 inverted microscope with a UPlanFLN 40 × 1.3 NA oil lens or a PlanApoN 60× 1.42 NA oil lens. Images were processed with ImageJ v. 1.45b (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Overlays were made using Photoshop CC (2014, Adobe).
Development of P. californiensis
Serotonergic nervous system in P. californiensis larvae
The central nervous system of rudiment-stage P. californiensis larvae is a blueprint of the adult nervous system. It consists of paired cerebral ganglia (two dorsal and two ventral) joined into a brain ring around the proboscis via ventral and dorsal cerebral commissures, and the two lateral nerve cords. Lateral nerve cords originate from the ventral cerebral ganglia. The nervous system occupies the majority of space between the body wall (epidermis + body wall muscles) and the gut. Phalloidin labeling helps to visualize the fibrous core (consisting of axons) of the cerebral ganglia and the lateral nerve cords, but not the ganglionic portion (i.e., the cell bodies), which surrounds the fibrous core (Fig. 1c–e). To help visualize at least some of the cell bodies, here we describe the serotonergic component of the nervous system.
Early-rudiment-stage larvae have apical neurons, a caudal neuron, and a number of additional neurons in the regions of the developing cerebral ganglia and lateral nerve cords (Fig. 2b). By late-rudiment stage, larvae have lost the apical and caudal neurons (Fig. 2c). Most serotonergic neurons are situated laterally along the developing lateral nerve cords at this stage (Fig. 2c). The serotonergic neurons form a connection between the lateral nerve cords in the posterior half of the larva. Additional paired neurons are present posterior to and lateral to this connection (Fig. 2c).
Although serotonergic neurons comprise only one component of the nervous system, they illustrate the position and relative proportion of the nervous system elements in the larval body, specifically the two large groups of cells at the anterior portion of the lateral nerve cords. This is important because we later describe the expression of several gene markers in these regions.
Hox, Cdx, and Six3/6 sequences
Six Hox-containing contigs and one with Cdx were recovered from the developmental transcriptome of P. californiensis. Long coding sequences were isolated for all seven genes. Based on Bayesian phylogenetic analysis (see Additional file 2), P. californiensis has Hox gene representatives from six paralog groups (PGs): PG1 (Labial/Lab), PG2 (Proboscipedia/Pb), PG3 (Hox3), PG4 (Deformed/Dfd), PG6 (Lox5), and PG9–PG13 (Post2). The paralog group, length of predicted open reading frame, and GenBank accession number for each gene are listed in Additional file 1: Table S3. Both the M. alaskensis and P. californiensis Six family genes recovered from the respective transcriptomes fall into the Six3/6 clade (see Additional file 3).
Hox and Cdx expression in the development of P. californiensis
Six3/6 expression in nemertean larvae
Cell proliferation in the invagination stage P. californiensis
We isolated six Hox genes from P. californiensis: Labial, Proboscipedia, Hox3, Deformed, Lox5, and Post2. This is fewer than the number of Hox genes found in the pilidiophoran Micrura alaskensis . We were not able to isolate Sex combs reduced, Antennapedia, or Lox4 from P. californiensis. These three genes are found in M. alaskensis  and are typical for other spiralians. This suggests that either these genes are not present in P. californiensis, are not involved in developmental processes, or are expressed at a level too low to detect at the stages we examined.
For most of the Hox genes examined in P. californiensis, expression first occurs in two lateral patches near the posterior end in the invagination-stage larvae. Later, expression appears to be restricted to the lateral nerve cords. So it appears that Hox genes pattern the trunk, as is the case for many other metazoans. While most Hox genes examined show a bilateral pattern of expression from the invagination stage onward, PcLox5 has a non-canonical pattern of expression, in which a number of additional cells express this gene early in development. This pattern suggests that PcLox5 may have been co-opted for some other use in early larval development, but still maintains its AP-patterning function later in development. PcLox5 does not show non-canonical expression in the pilidiophoran Micrura alaskensis, so this may be a trait unique to the hoplonemerteans or to P. californiensis, specifically.
Paired anterior invaginations have been documented in the development of many hoplonemerteans and palaeonemerteans, but their function and fate are uncertain [see the discussion in 12, 13]. Maslakova and von Döhren  showed that a pair of anterior invaginations in the hoplonemertean Paranemertes peregrina gave rise to cerebral organs, but this was not observed in P. californiensis . In other species of hoplonemerteans and palaeonemerteans, various authors hypothesized, but did not show definitively, that various anterior invaginations may be rudiments of various other structures, including the nervous system, cerebral organs, frontal organ, apical organ, proboscis, or the rhynchodeum [9, 15, 22–25]. We find that Six3/6, a broadly conserved gene involved in anterior neural ectoderm specification in bilaterians , is expressed in both the cephalic discs of M. alaskensis and near the anterior invaginations of P. californiensis. This suggests that the anterior invaginations of P. californiensis and, possibly, of some other hoplonemerteans and even palaeonemerteans may be homologous to pilidial cephalic discs, as was first hypothesized by Hiebert et al.  (see Fig. 6). Anti-serotonin antibody labeling suggests that the anterior invaginations of P. californiensis are associated with serotonergic neurons and may be involved in the sensory function of the larva, or the development of the adult serotonergic nervous system, or both. This supports the idea that the anterior invaginations contribute to the head region of the hoplonemertean juvenile, similar to the role of the cephalic imaginal discs in pilidiophorans, which give rise to the head of the juvenile inside of the pilidium. Six3/6 expression is also observed in some cells located near the rim of the apical organ of the pilidium (Fig. 6c). No expression was observed in the apical organ of P. californiensis at the invagination stage, but Six3/6 is expressed near the apical organ at the rudiment stage (Fig. 6b). It is parsimonious to assume that the apical plate of pilidiophorans and hoplonemerteans is homologous (since it is present in larvae of all extant nemertean taxa and was likely present in the most recent common ancestor of all nemerteans). The apical plate of the pilidium is destroyed during metamorphosis and does not participate in the formation of the adult. Similarly, the apical plate in hoplonemerteans is gradually remodeled during transition to the juvenile life (adults have no apical plate). In P. californiensis, the expression exists in two broad lateral domains at the invagination stage in regions that appear to persist through to juvenile stages, which is more similar to the cephalic-disc expression in the pilidium than the apical organ expression. Thus, we are fairly certain that the appropriate comparisons should be: first, between the expression in the apical plates of both species; and second, between the expression in the anterior invaginations in the hoplonemertean development and the cephalic discs in the pilidium.
Developmental homology between pilidial imaginal discs and invaginations of the decidula larva is also supported by the patterns of cell division. Anti-phospho-histone antibody labeling shows that the two pairs of invaginations in the hoplonemertean larva contain many dividing cells. Thus, hoplonemertean larval invaginations are sites of cell proliferation, much like the axils and the imaginal discs in the pilidium . One might speculate that it may be functionally advantageous to tuck away the growth zones into some sort of invaginated rudiment(s) of a swimming larva. The decidula’s epidermis comprises multiciliated cells, which cannot divide [see discussion in 27]. If proliferative zones which consist of non-ciliated or monociliated cells were left on the surface, they would disrupt the pattern of ciliation and thus might affect larval swimming.
The finding that pilidial imaginal discs have a likely homolog in the hoplonemerteans has important implications for the origins of the pilidium. These results suggest that invaginated rudiments of some sort were likely present in the hoplonemertean–pilidiophoran ancestor. We suggest that the decidula’s invaginations may be more representative of the ancestral condition than the pilidial imaginal discs, because the presence of the imaginal discs in the ancestral larva would require a severe reduction of pilidial features within the Hoplonemertea. Supporting this case is the fact that the typical planktotrophic pilidium appears to have been lost several times in pilidiophoran evolution, being replaced with highly reduced lecithotrophic forms [11, and references therein]. All studied secondarily reduced pilidia retain imaginal discs and catastrophic metamorphosis [28–30]. If the hoplonemertean–pilidiophoran ancestor was more pilidium-like, we would expect that more prominent imaginal discs and metamorphosis would be present in the extant hoplonemerteans.
Hox, Cdx, and Six3/6 gene expression as well as cell proliferation patterns support the homology between pilidial imaginal discs and hoplonemertean larval invaginations (Fig. 7). This suggests that the common ancestor of pilidiophorans and hoplonemerteans likely developed tucked-away juvenile rudiments of some sort. Invaginated rudiments in the hoplonemertean–pilidiophoran ancestor may represent the precursors to imaginal discs in the pilidium.
LSH: data collection and analysis, figure preparation, manuscript writing, critical revision, and final approval of the manuscript. SAM: conception and design, manuscript writing, critical revision, financial support, and final approval of the manuscript. All authors read and approved the final manuscript.
This work was supported by the National Science Foundation Graduate Research Fellowship DGE-0829517 to LSH and NSF Grant IOS-1120537 to SAM.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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