Chaetopterus pergamentaceus body plan
We analyzed organ system organization and differentiated tissue types in C. pergamentaceus larvae by labeling for nuclei with Hoechst (Fig. 2d, e), muscle fibers (Fig. 2f, g) and cell cortices (Fig. 2h, i) with phalloidin, and neuronal processes with the cross-reactive antibody, anti-FMRFamide (Fig. 2j, k). Specifically, the distinct stomodeum, midgut, and hindgut compartments are visible in animals labeled for nuclei (Fig. 2d, e) and filamentous actin (Fig. 2h, i). Phalloidin staining also allows for the visualization of the larval body wall musculature. L2 larvae have numerous circumferential and longitudinal muscle fibers throughout the body. Notably, there are two prominent, bilaterally symmetric longitudinal muscles that straddle the dorsal midline and span the length of the body (Fig. 2f, g, i; white arrow heads). Also visible by actin staining is the position of the apical tuft, and its attachment point is clearly located on the dorsal, anterior surface of the head (Fig. 2f, g, i). Actin labeling is similarly used to identify the attachment point of the apical tuft in the larvae of nemerteans [42,43,44]. In the head of C. pergamentaceus larvae, there is a bilateral pair of cerebral ganglia visible via the spatial expression of COE; a transcription factor involved in neural specification [45]. We further characterized the organization of the nervous system by visualizing a subset of nerves using an anti-FMRFamide antibody. There is a medial cluster of FMRFamide immunoreactive cells positioned between the cerebral ganglia (Fig. 2j, k; cbr). From the cerebral commissure, a pair of neuronal processes circumvents the stomodeum as the circumesophageal connective (Fig. 2j, k; cc). The pair of anti-FMRFamide immunoreactive processes is visible along the length of the ventral side of the trunk as the main connective (Fig. 2j, k; mc) and terminates in the pygidium. The two longitudinal connectives are positioned closer together in the midbody and posterior end relative to their lateral position in the head and anterior portion of the trunk. This more medial position of the connectives begins at the approximate position of the posterior face of the midgut. A subesophageal commissure is also visible, just posterior to the stomodeum (Fig. 2j; sc). These features are bilaterally symmetric and mark the ventral side of the larva. In addition, the subesophageal commissure and the FMRFamide immunoreactive cells in the head mark the anterior of the larva.
Together, the anterior apical tuft, stomodeum and cerebral commissure, along with the posterior hindgut, are all morphological features that enable the detection of an anterior–posterior axis. The dorsal position of both the apical tuft attachment point and the pair of longitudinal muscles, along with both the ventral stomodeum opening and ventral neuronal processes, likewise indicate a clear dorsal–ventral axis. Although L2 larvae have bilaterally symmetric eye spots, we could not reliably detect them since exposure to detergents during the antibody labeling process causes the loss of eye pigment. Bilateral symmetry is therefore confirmed by the presence of the pair of ventral neuronal processes and the pair of dorsal longitudinal muscles.
Inhibition of the ERK/MAP kinase pathway results in abnormal gut and muscle formation
To investigate the identity and timing of the signaling pathway involved in axis specification in C. pergamentaceus, we exposed embryos at early cleavage stages to small chemical inhibitors of specific signaling pathways. Experiments were performed at various time intervals and drug concentrations (Fig. 3). Initial treatments included exposure to each drug at concentrations ranging from 5 to 50 μM. The lowest concentration at which there was a consistently reproducible larval phenotype was selected for detailed analysis. Exposures above 20 μM with any drug tested resulted in arrested development. For all experiments, embryos were exposed to concentrations of DMSO equivalent to experimental conditions to control for nonspecific effects of the solvent.
Experimental evidence showing that activation of the ERK/MAPK pathway is associated with patterning of the dorsal–ventral axis of some spiralians led us to investigate the role of this pathway during early development in C. pergamentaceus using the small molecule inhibitor U0126. U0126 functions by inhibiting the kinase activity of both MEK1 and MEK2 [46]. As a result, MEK1/2 is unable to phosphorylate and activate MAPK, and thereby inhibits the ERK/MAP kinase pathway. In experiments investigating the effects of inhibiting the ERK/MAP kinase pathway with U0126, exposures were conducted during two independent time intervals: the 4 cell to late cleavage stage and from late cleavage stage to early gastrula (Fig. 3). The duration of each time interval was 90 min, during which approximately five cleavage divisions occurred. Following drug exposure, embryos were raised in sea water to larval stage L2, and then scored for axial anomalies.
Embryos exposed to 0.2% DMSO during the interval between the 4 cell stage and the early gastrula stage result in phenotypically normal larvae (Fig. 4a–d; n = 3 technical replicates). These larvae have differentiated cell types, and were morphologically analyzed following visualization of nuclei, filamentous actin, and anti-FMRFamide immunoreactive cells. Identifiable anterior features such as the stomodeum (Fig. 4a and c), apical tuft attachment point (Fig. 4b and c), and the cerebral commissure (Fig. 4d) are present. A posterior hindgut is also present (Fig. 4a and c). A dorsal–ventral axis is detectable via the presence of the dorsal position of the apical tuft attachment point (Fig. 4b and c) and the pair of dorsal longitudinal muscles (Fig. 4b), while ventral is identifiable by the presence of a stomodeum, and ventral neuronal processes (Fig. 4d). Bilateral symmetry is also detectable by the presence of the bilateral pair of dorsal longitudinal muscles (Fig. 4b), and the pair of ventral neuronal processes circumventing the stomodeum and midgut (Fig. 4d). All three body axes were detectable in 99% (n = 69/70) of larvae.
Larvae resulting from exposure to 20 μM U0126 during the 4 cell to late cleavage stage (Fig. 4e–h) are phenotypically abnormal, but have differentiated cell types and possess all three body axes (n = 3 technical replicates). Anteriorly, the stomodeum (Fig. 4e and g), apical tuft attachment point (Fig. 4f), and cerebral commissure (Fig. 4h) are present. However, a gut with a clear tripartite organization is not visible, and a hindgut is not detectable (Fig. 4e and g). Instead, larvae exhibit an abnormal gut cavity in place of a midgut (Fig. 4e–g; agc). The dorsal apical tuft attachment point (Fig. 4f) along with the ventral opening of the stomodeum (Fig. 4e, g), and the ventral pair of circumesophageal and main connectives (Fig. 4h) indicate presence of a dorsal–ventral axis. Dorsal longitudinal muscles are not detected and there is a general disorganization of the muscle fibers (Fig. 4f and g). Bilateral symmetry is seen via the ventral pair of circumesophageal and main connectives circumventing the stomodeum and abnormal gut cavity (Fig. 4h). Altogether, despite abnormalities in gut formation and muscle organization, an anterior–posterior axis, dorsal–ventral axis, and bilateral symmetry are detectable in 98% (n = 98/100) of the resulting larvae.
Similarly, larvae resulting from exposure to 20 μM U0126 during the interval between late cleavage stage and early gastrula exhibit abnormal phenotypes and have differentiated cell types and possess all three body axes (Fig. 4i–l) (n = 3 technical replicates). The anterior stomodeum (Fig. 4i, k), apical tuft attachment point (Fig. 4j and k), and cerebral ganglia (Fig. 4l) are all present. These larvae exhibit a single abnormal gut cavity in place of a midgut (Fig. 4i and k), and there is no detectable hindgut (Fig. 4i and k). The dorsal apical tuft attachment point (Fig. 4j and k), together with the ventrally opened stomodeum (Fig. 4i, k) and the ventral pair of circumesophageal and main connectives (Fig. 4l) indicate the presence of a dorsal–ventral axis. Notably, actin fibers are generally disorganized, and there are few muscles present (Fig. 4j). The ventral pair of circumesophageal and main connectives circumventing the stomodeum and abnormal gut cavity (Fig. 4l) indicates bilateral symmetry. Altogether, all three body axes are detectable in 98% (n = 111/113) of resulting larvae.
An omnibus Chi square test of homogeneity was used to compare the proportions of larvae with and without a dorsal–ventral axis resulting from experimental and control conditions. Our analysis shows no statistically significant difference (p > 0.05) in the proportions of larvae with and without a dorsal–ventral axis following U0126 exposures at either developmental interval in comparison to those larvae resulting from DMSO control conditions. These results indicate that exposure to U0126 prior to gastrulation does not affect dorsal–ventral axis formation.
BMP inhibition with DMH1 or dorsomorphin does not affect axes formation
BMP signaling has been shown to mediate organizing signaling and dorsal–ventral axis formation in some mollusks, a sister taxa to annelids [36, 37]. Since C. pergamentaceus is an early branching annelid, we were interested in investigating whether C. pergamentaceus uses the same molecular signal to mediate organizing activity as mollusks. We inhibited the BMP signaling pathway using two different chemical inhibitors, DMH1 and dorsomorphin dihydrochloride (Fig. 3). The chemical inhibitor DMH1 functions by preventing the phosphorylation of the BMP type 1 receptor ALK2 [47]. Similarly, dorsomorphin dihydrochloride functions by preventing the phosphorylation of BMP type I receptors, ALK2 and ALK3 [48].
Larvae resulting from exposure to 20 μM DMH1 (n = 3 technical replicates) during the interval between either the 4 cell to late cleavage stage (n = 78/81) or the late cleavage to early gastrula stage (n = 89/95) are phenotypically normal with no detectable abnormalities (Additional file 1: Figure S1E–L). Likewise, larvae resulting from exposure to 20 μM dorsomorphin dihydrochloride (n = 3 technical replicates) during either the 4 cell to late cleavage stage (n = 73/74) or the late cleavage to early gastrula stage (n = 115/117) are also phenotypically normal (Additional file 1: Figure S1M–T). There is no statistically significant difference (p > 0.05) between larvae exposed to DMH1 or dorsomorphin dihydrochloride and control larvae exposed to DMSO (omnibus Chi square test of homogeneity). As these exposures do not elicit detectable phenotypic effects in larvae, these inhibitors cannot be confirmed to be effective at inhibiting BMP signaling in C. pergamentaceus.
Inhibition of Activin/Nodal signaling results in abnormal axial development
The Activin/Nodal branch of the TGF-beta signaling pathway mediates dorsal–ventral axis patterning in the annelid C. teleta, [38, 39]. To determine if Activin/Nodal signaling also mediates dorsal–ventral axis patterning in C. pergamentaceus, we used the small molecule inhibitor SB431542, which functions by inhibiting the phosphorylation and activation of Activin/Nodal type I receptor, ALK4/5/7 [49]. Initial experiments indicated that exposures to 20 μM SB431542 during the interval between the 4 cell to late cleavage stage but not the late cleavage to early gastrula stage resulted in larvae with axial defects (data not shown). Therefore, subsequent drug exposure experiments were conducted to more accurately determine the timing of axis specification (Fig. 3). The original time interval (4 cell to late cleavage stage) was shortened by serially delaying when the inhibitor was added, therefore moving the beginning of the exposure timeframe forward one cleavage stage at a time. Specifically, the 4 cell to late cleavage time interval was subdivided as follows: 4–32 cell, 16–32 cell, and 32 cell to late cleavage stage (Fig. 3). The duration of these time intervals was approximately 45, 15, and 45 min, respectively (cleavage divisions occur at 15-min intervals). Embryos were visually monitored for the birth of each quartet prior to adding them to the inhibitor. This experimental design allowed us to be confident about the cell stage at which the inhibitor was added to the embryos, and accounts for any uncertainty regarding the time required for the chemical inhibitor to diffuse out of the embryonic tissue.
Exposures were also conducted during the late cleavage to early gastrula stage (approximately 90-min long) as a control time interval, during which anomalies in axis patterning were not expected. Embryos were exposed to concentrations of DMSO equivalent to experimental conditions to control for nonspecific effects of the solvent during the interval between the 4 cell stage and the early gastrula stage (3 h). Following drug exposure, embryos were raised to L2 larvae and morphologically analyzed.
A defect in dorsal–ventral axis formation occurs when embryos are exposed to SB431542 during a restricted time interval (Fig. 5). Control embryos exposed to 0.2% DMSO during the 4 cell to early gastrula stage result in 52/53 phenotypically normal larvae with differentiated cell types and three clearly distinguishable body axes (n = 3 technical replicates) (Fig. 5a–e). In contrast, embryos exposed to 20 μM SB431542 during the interval between the 4 and 32 cell stage result in severely abnormal larvae that have differentiated cell types and a circular morphology (n = 4 technical replicates) (Fig. 5f–j). In general, abnormal larvae are missing an identifiable stomodeum, midgut, and hindgut (Fig. 5f, g, i). However, larvae do possess a small abnormal internal lumen in the trunk (Fig. 5i; al). The number of actin fibers is reduced and the fibers present have a disorganized arrangement (Fig. 5h; white arrows). There is no detectable apical tuft attachment point (Fig. 5h). Although there are a few neuronal processes, these are disorganized (Fig. 5j; yellow arrows) and there is no identifiable cerebral commissure, subesophageal commissure, circumesophageal nerve or main connective nerves. As such, all three body axes were detectable in only 11% (n = 6/56) of larvae and were undetectable in 89% of cases (n = 50/56).
Embryos exposed to 20 μM SB431542 during the interval between the 16–32 cell stage resulted in abnormal larvae, with elliptical morphology and differentiated cell types (n = 3 technical replicates) (Fig. 5k–o). Larvae are missing an identifiable stomodeum, midgut, and hindgut (Fig. 5k, l, n), but do possess an abnormal gastric lumen within the larval trunk (Fig. 5n; al). There are numerous actin fibers present that are highly disorganized (Fig. 5m; white arrows), and there is no detectable apical tuft attachment point. A cerebral commissure is detectable in these larvae indicating anterior polarity (Fig. 5o; cbr); however, the other anti-FMRFamide immunoreactive neuronal processes present in the trunk are disorganized (Fig. 5o; yellow arrows). All three body axes are detectable in 14% (n = 6/44) of larvae and undetectable in 86% (n = 38/44) of larvae.
Embryos exposed to 20 μM SB431542 during the interval between the 32 cell to late cleavage stage result in abnormal larvae with differentiated cell types (n = 3 technical replicates) (Fig. 5p–t). These larvae have an elliptical shape (Fig. 5p). An abnormal stomodeum opening is detected by DIC optics, and through nuclear and actin staining (Fig. 5p, q, s). Larvae also possess an abnormal gastric lumen within the larval trunk (Fig. 5q and s). There is no detectable apical tuft attachment point (Fig. 5r). Most of the actin fibers present are disorganized (Fig. 5r; white arrows); however, a partial pair of dorsal longitudinal muscles is detectable (Fig. 5r; white arrowheads). The cerebral commissure and main connectives are present and show bilateral symmetry (Fig. 5t). Altogether, these anti-FMRFamide immunoreactive components indicate anterior identity by the presence of the cerebral commissure as well as bilateral symmetry via the bilateral neuronal processes. Bilateral symmetry is further indicated via the anterior-most portion of the pair dorsal longitudinal muscles (Fig. 5r). A dorsal–ventral axis is present as evidenced by the combination of the dorsal longitudinal muscles on the opposite face of the larvae in relation to the stomodeum opening and the FMRFamide immunoreactive neuronal processes. As such, all three body axes are detectable 89% (n = 35/39) of resulting larvae, and undetectable in 10% (n = 4/39).
Last, embryos exposed to 20 μM SB431542 during the interval between the late cleavage and early gastrula stage result in the least phenotypically abnormal larvae of all the time intervals tested (n = 3 technical replicates) (Fig. 5u–y). The overall shape of these larvae resembles wild-type larvae (Fig. 5u). That is, they are elongated along one axis, with one end broader in width relative to the other end, and the widest part of the body is positioned approximately half way along the longest axis. In the gut, distinct stomodeum, midgut, and hindgut compartments are detectable, although the gut is not completely normal (Fig. 5v, x). An apical tuft attachment point (Fig. 5w), cerebral commissure (Fig. 5y), and bilateral main connectives (Fig. 5y) are all present. Some of the actin fibers present are disorganized (Fig. 5w; white arrows); however, in the anterior-most portion of the body there is a pair of dorsal longitudinal muscles (Fig. 5w; white arrowheads). These altogether indicate anterior identity via presence of the cerebral commissure (Fig. 5y) and apical tuft attachment point (Fig. 5w). The apical tuft attachment point and the partial formation of the dorsal longitudinal muscles indicate dorsal identity. The stomodeum opening and main neuronal connectives likewise indicate ventral identity. Bilateral symmetry is visible via the presence of a bilateral pair of main connectives and of dorsal longitudinal muscles in the anterior portion of the body. For this time interval, all three body axes are detectable in 96% (n = 48/50) of larvae and undetectable in only 4% (n = 2/50) of cases.
Experimental and control conditions were compared using an omnibus Chi square test of homogeneity followed by post-hoc pairwise comparisons using a z test of two proportions. The results of these tests indicate that exposures during the 4–32 cell stage and the 16–32 cell stage are not significantly different from each other (p > 0.05). However, both conditions result in a significantly lower proportion (p < 0.05) of larvae with a detectable dorsal–ventral axis when compared to larvae of the DMSO control condition, the 32 cell to late cleavage stage condition, or the late cleavage to early gastrula stage condition. Conversely, exposures during the 32 cell to late cleavage stage and the late cleavage to early gastrula stage are not statistically significantly different from each other (p > 0.05), nor from animals in the DMSO control condition. These data suggest that signaling via the Activin/Nodal pathway functions in dorsal–ventral axis formation prior to the 32 cell stage, likely completing its organizing activity at the end of the 16 cell stage.