The Nodal signaling pathway controls left-right asymmetric development in amphioxus

Abstract

Background: Nodal is an important determinant of the left-right (LR) body axis in bilaterians, specifying the right side in protostomes and non-chordate deuterostomes as opposed to the left side in chordates. Amphioxus represents an early-branching chordate group, rendering it especially useful for studying the character states that predate the origin of vertebrates. However, its anatomy, involving offset arrangement of axial structures, marked asymmetry of the oropharyngeal region, and, most notably, a mouth positioned on the left side, contrasts with the symmetric arrangement of the corresponding regions in other chordates.

Results: We show that the Nodal signaling pathway acts to specify the LR axis in the cephalochordate amphioxus in a similar way as in vertebrates. At early neurula stages, Nodal switches from initial bilateral to the left-sided expression and subsequently specifies the left embryonic side. Perturbation of Nodal signaling with small chemical inhibitors (SB505124 and SB431542) alters expression of other members of the pathway and of left/right-sided, organ-specific genes. Upon inhibition, larvae display loss of the innate alternation of both somites and axons of peripheral nerves and loss of left-sided pharyngeal structures, such as the mouth, the preoral pit, and the duct of the club-shaped gland. Concomitantly, the left side displays ectopic expression of otherwise right-sided genes, and the larvae exhibit bilaterally symmetrical morphology, with duplicated endostyle and club-shaped gland structures.

Conclusions: We demonstrate that Nodal signaling is necessary for establishing the LR embryonic axis and for developing profound asymmetry in amphioxus. Our data suggest that initial symmetry breaking in amphioxus and propagation of the pathway on the left side correspond with the situation in vertebrates. However, the organs that become targets of the pathway differ between amphioxus and vertebrates, which may explain the pronounced asymmetry of its oropharyngeal and axial structures and the left-sided position of the mouth.

Keywords: Amphioxus; Embryonic development; Left-right asymmetry; Mouth opening; Nodal signaling.

Figure 1

The left-right asymmetric characters of amphioxus. (A, B) Dorsal views of mid-neurula (N2) and late neurula (N3), marked with m-actin to display asymmetric arrangement of somites. White dotted lines mark the boundary of each somital segment. The asterisk (*) marks the anterior; ‘L’ represents the left side and ‘R’ the right side of the embryo. Scale bar, 100 μm. (C) The myomeres (phalloidin staining) and axon bundles (acetylated α-tubulin staining) of amphioxus larva (L2) are positioned asymmetrically. White dotted lines mark myomere boundaries, and white arrows mark connections of peripheral axon bundles to the neural tube. Scale bar, 50 μm. (D) Left lateral view of L2-stage larva. Scale bar, 100 μm. en, endostyle; csg, club-shaped gland; fgs, first gill slit; pp, preoral pit. (E) Left lateral view of L2-stage larva focused on the right side. The club-shaped gland is marked with FoxE4 riboprobe. Dashed arrows mark the section planes in (G-J). Pharyngeal organs are labeled as described for G–J. Scale bar, 100 μm. (F) Left lateral view of L2-stage larva focused on the left-sided mouth opening (yellow arrowhead). Scale bar, 100 μm. (G-J) Sections of L2-stage larva from (E) showing asymmetrical positioning of pharyngeal organs. ‘n’ marks the notochord and the sections are seen from the posterior direction. The preoral pit (G, black arrowhead) and the mouth (I, yellow arrowhead) open on the left. The endostyle (H, black arrow) forms on the right. The dorsal part of the club-shaped gland (I, blue double arrowhead) is situated on the right, while its ventral part (duct of the club-shaped gland) extends to the left side (H, I, blue arrowhead). The first gill slit (I, J, cyan arrowhead) opens to the right side. Each section is 10 μm thick. Scale bar, 25 μm.

Figure 2

Left-right asymmetric expression of developmental regulatory genes during amphioxus embryogenesis. All images are of B. floridae, taken from the dorsal view. ‘L’ marks the left side, ‘R’ marks the right side, and the asterisk (*) marks the anterior. Double arrowheads indicate stronger expression patterns, and single arrowheads indicate relatively weaker expression patterns. White arrowheads indicate the midline. Scale bar, 100 μm. (A-F) At the onset of neurulation (stage N0), Nodal and Gdf1/3 are expressed symmetrically on both the left and the right sides of the embryo. Lefty is expressed on the left side, while Cerberus is expressed on the right side; these are the first signs of asymmetrical gene regulation. Pitx is expressed on the left side of a fraction of embryos, suggesting that Pitx is just starting to be transcribed. (G-K) At the early neurula stage (N1), Nodal becomes expressed preferentially on the left side, while Gdf1/3, Lefty, and Pitx are expressed exclusively on the left side. Additionally, Lefty is also expressed at the midline. Cerberus transcripts are found on the right side and also at the midline. (L-P) At the mid-neurula stage (N2), Nodal, Gdf1/3, Lefty, and Pitx are expressed exclusively on the left side while Cerberus is expressed at the midline. (Q-S) At the late neurula stage (N3), some organ-specific genes already display asymmetric expression. Expression of FoxE4, Nkx2.1, and Hand is biased towards the right side.

Figure 3

Left-right asymmetric expression of developmental regulatory genes during larval stages. Images are of B. lanceolatum at the early larval stage (L1) and the open mouth stage (L2) from either left lateral view or dorsal view. ‘L’ marks the left side and ‘R’ marks the right side; anterior is to the left. Black arrowheads indicate the preoral pit, black arrows mark the mouth region, white arrows point to the club-shaped gland, and white arrowheads mark the endostyle. Scale bar, 100 μm. (A-G, A’-G’) At L1, Pitx is expressed broadly in the left anterior pharyngeal region spanning the area of the prospective preoral pit and the mouth. Lhx3 expression is confined to the prospective preoral pit, while Dkk1/2/4 is expressed focally in the regions destined to form both the preoral pit and the mouth. FoxE4 and Krox transcripts are restricted to the prospective club-shaped gland, where the expression pattern of Krox is nested within that of FoxE4. FoxQ1 is expressed anterior to FoxE4 in the prospective endostyle region and also at other regions within the pharynx. Nkx2.1 transcripts are confined solely to the endostyle region. (H-N, H’-N’) The L2 stage exhibits well-developed pharyngeal structures, with expression patterns similar to those at L1. Pitx and Dkk1/2/4 continue to be expressed in the preoral pit and the mouth, while Lhx3 expression is confined solely to the preoral pit. FoxE4 and Krox are expressed in the club-shaped gland, with FoxE4 marking the whole structure, while Krox transcripts are confined to its dorsal part. In addition to its expression elsewhere in the pharynx, FoxQ1 is co-expressed with Nkx2.1 in the endostyle.

Figure 4

Inhibition of the Nodal signaling pathway changes the expression pattern of left-right regulatory genes. Images are of B. floridae taken from the dorsal view. Double arrowheads mark strong expression and single arrowheads mark weak expression. Scale bar, 100 μm. (A-D) At the onset of neurulation (N0), embryos treated with Nodal inhibitors exhibit bilateral expression of Nodal and Gdf1/3, albeit diminished as compared to controls. Similarly, the strong unilateral expression of Lefty and Cerberus in controls is converted into reduced bilateral expression by each treatment. (E-I) At the early neurula stage (N1), Nodal expression remains bilateral but is greatly diminished in the treated embryos. Expression of genes expressed on the left side (Gdf1/3, Lefty, and Pitx) is lost in the treated embryos, while Cerberus expression becomes bilaterally symmetrical. (J-M) At the mid-neurula stage (N2), Nodal, Gdf1/3, Lefty, and Pitx expression cannot be detected in the treated embryos. (N-P) At the late neurula stage (N3), the right-sided bias in expression of FoxE4, Nkx2.1, and Hand is abolished by the treatment, and these genes become symmetrically expressed on both left and right sides.

Figure 5

Inhibition of Nodal signaling alters the left-right asymmetric arrangement of muscle segments and nervous system. All images are taken from the dorsal view. ‘L’ marks the left side, ‘R’ marks the right side, and asterisks (*) mark the anterior. The notochord is marked with an ‘n’. Dashed lines mark somite borders and white arrows mark axons of the peripheral nerves. Scale bar, 50 μm. (A, B) B. belcheri embryos were stained with phalloidin to mark somite outlines. Morphological asymmetry is barely visible at N2, but asymmetrical arrangement of the somites is apparent by the N3 stage. Treatment causes the staggered arrangement of somites to become symmetrical. (C-E) Expression of B. floridae m-actin confirms the asymmetrical arrangement of the somites, and expression of Hu/Elav and ERR reveals staggered arrangement of neurons in the central nervous system. In the treated embryos, the staggered expression patterns of m-actin, Hu/Elav, and ERR along the anterior-posterior axis become bilaterally symmetrical. (F-G) Representative B. belcheri larvae stained with phalloidin (green) and acetylated α-tubulin (red). At larval stages, inhibition of Nodal signaling results in symmetrization of the myotomes and of axons of the peripheral nerves.

Figure 6

Inhibition of Nodal signaling disrupts formation of the left-sided mouth and causes duplication of the right-sided pharyngeal structures in amphioxus larvae. (A, A’) Dorsal views of the pharyngeal region of B. lanceolatum L2 larvae stained for β-catenin and acetylated α-tubulin. ‘L’ indicates the left side and ‘R’ indicates the right side; anterior is to the left. Following administration of SB505124, the larvae exhibit loss of the preoral pit (pp, black arrowhead), the mouth (m, black arrow), and the first gill slit (fgs, white double arrow) and ectopic development of the club-shaped gland (csg, white arrow) and the endostyle (en, white arrowhead). (B-H, B’-H’) In situ hybridization of the treated B. lanceolatum larvae revealed loss of expression of Pitx, Lhx3, and Dkk1/2/4 and bilateral expression of Krox, FoxE4, FoxQ1, and Nkx2.1 concomitant with the duplication of the right side. (I-K, I’-K’) View from the left side displaying loss of the duct of the club-shaped gland (white double arrowhead) upon treatment with SB505124 as deduced from the expression of Krox, FoxE4, and Nkx2.1. (L-O, L’-O’) Transverse sections of B. floridae larvae through the pharynx showing the loss of Pitx expression concomitant with the loss of both the preoral pit (black arrowhead) and the mouth (black arrow) and the ectopic left-sided expression of FoxE4 and Nkx2.1 concomitant with the ectopic development of the dorsal part of the club-shaped gland (white arrowhead) and the endostyle (white arrow) after treatment with SB505124. (P, P’) Snapshots of the 3D model of B. floridae larva reveal consistent morphological changes with B. lanceolatum larvae for both the normal and treated embryos. The snapshots are taken from the left lateral view. Asterisks (*) mark the anterior. Green, preoral pit; yellow, mouth region; blue, endostyle; red, club-shaped gland; purple, gill slits. Scale bars, 25 μm.

Figure 7

Time-restricted inhibition of Nodal signaling reveals that LR asymmetry is specified at neurula stages. (A) Inhibition of Nodal signaling by SB505124 results in three larval (L2) phenotypes, designated as ‘wild-type’, ‘mild’, and ‘strong’, and defined by differential expression of Lhx3 as a marker of the preoral pit and Krox as a marker of the dorsal part of the club-shaped gland. The mild phenotype involves a loss of the ventral portion of the pharynx (small arrowheads) and reduced Lhx3 expression, indicative of a reduced preoral pit (arrowhead). The strong phenotype involves a loss of the ventral portion of the pharynx (small arrowheads), complete loss of the preoral pit, and an ectopic club-shaped gland on the left side (arrow). (B) Long-term treatments were performed to demonstrate that Nodal inhibition during the neurula stages results in altered phenotypes. (C) Short-term treatments were performed to demonstrate that Nodal inhibition results in altered phenotypes, especially when administered at the N0 to N2 stages. All images are from B. lanceolatum.

Figure 8

Model of the Nodal signaling pathway during establishment of the LR asymmetry in amphioxus. Dashed lines mark proposed interactions based on the data from other chordates. Gray text denotes inhibition of expression of the respective factors. During neurulation, Nodal and its co-ligand Gdf1/3 are expressed symmetrically. However, right-sided inhibition of Nodal by Cerberus results in propagation of the pathway on the left side, where Nodal activates expression of Nodal, Gdf1/3, Lefty, and Pitx and inhibits expression of Cerberus. We propose that during the course of later development, Pitx interacts with organ-specific factors and promotes left-side specific morphogenesis. Anteriorly, Pitx is proposed to interact with Pit1 and to activate expression of Lhx3 to promote differentiation of cell types within the preoral pit. Slightly more posterior, Pitx is proposed to regulate expression of Dkk1/2/4, which inhibits Wnt/β-catenin signaling, and thus promotes fusion of ectoderm and endoderm epithelia during morphogenesis of the mouth opening. The mechanism underlying activation of expression of factors responsible for right-sided morphogenesis is currently unknown. csg, club-shaped gland; en, endostyle; fgs, first gill slit; m, mouth; pp, preoral pit.

Figure 9

Hypothetical scenario of the role of Pitx in LR asymmetry and mouth development across metazoans. Green text marks LR asymmetric expression; magenta text marks median expression. In protostomes, echinoderms and hemichordates, the Nodal-Pitx pathway regulates determination of the right side. In cephalochordates, tunicates and vertebrates, on the other hand, this pathway determines the left side due to inversion of the body axes, an event that supposedly occurred at the base of chordate phylogeny. Concomitantly, new Pitx expression downstream of BMP signaling occurred at the anterior neural boundary (this interaction remains to be confirmed in amphioxus). Mouth development seems to be regulated differently between different deuterostome lineages. BMP signaling suppresses oral development in echinoderms and hemichordates, while it promotes oral development through Pitx in tunicates and vertebrates. In the cephalochordate amphioxus, the Nodal-Pitx pathway regulates LR asymmetry and is also responsible for development of the mouth, whose relation to the mouth of other chordates is contested.