The first pioneer neurons with long neurites appear at the anterior and the posterior pole of the larvae
The early development of the M. fuliginosus larva is highly synchronous and therefore allows for precise staging across batches. Throughout the early development up to stages of 24 hpf, the larva is surrounded by a thick ornamented chorion of the egg (Fig. 1a-d, e). The chorion later becomes an integral part of the cuticle, which at 48 hpf already has a smooth surface (Fig. 1f). Cilia of the prototroch and the apical tuft penetrate the chorion from 7 hpf onwards. Pigmented eyes appear at around 14 hpf (Fig. 1b-d), and chaetae start penetrating the chorion around 24 hpf (Fig. 1e) and form prominent bundles at 48 hpf (Fig. 1f). Immunohistochemistry shows that the prototroch and the telotroch are formed by bundles of cilia (Fig. 1g-m), which in later stages is less obvious by external examination (Fig. 1e, f). The prototroch band is not continuous on the dorsal body surface from the time of its emergence (Fig. 1e-i). The nervous system development is relatively fast, and due to a low yolk content of the larvae, a detailed investigation is possible by immunohistochemical stainings. For this purpose, we performed antibody stainings against acetylated alpha-tubulin (a-tub) from 7 hpf onwards, where only the cilia of the prototroch and an apical tuft are visible.
Studying stages in short time intervals allowed us to identify the first appearing pioneer neurons that send out the first neurites and initiate the formation of the early neuronal scaffold. The first neuron to send out axonal processes is a single posterior pioneer neuron (PPN), which starts to differentiate around 8 hpf with the accumulation of dense microtubules (data not shown) and acquires a distinct morphology by 9 hpf (Figs. 1g, 2a, 3, Additional file 1). This bifurcating cell projects two axons proceeding towards the prototroch from 12 hpf onwards (Fig. 1h), which extends already one third to the prototroch at 14 hpf (Figs. 1i, 3, Additional files 2, 3, 4). During this phase, the PPN also acquires a curved morphology (Fig. 2b) with few sensory cilia extending outwards on the ventral side. The axons of the PPN reach the prototroch area around 19 hpf (Additional files 8, 9, Fig. 3). At around 16 hpf, a new neuron (without sensory cilia) and presumably a follower develops adjacent to the PPN (Fig. 2c). It extends axons along the processes formed by the PPN (Additional files 5, 6, 7). More follower neurons appear at 34 hpf, when a pair of weakly stained ciliated sensory cells are visible on either side of the PPN and become more prominent at 36 hpf (Fig. 2d).
In the anterior end, the first neurons can be observed from 9 hpf underneath the apical tuft (Figs. 1g, 3, Additional file 1), but we could not identify extending axons before 14 hpf. At this time, several cells lie underneath the apical tuft, but only one sends a short axon towards the prospective brain neuropil (Fig. 3, Additional files 2, 3, 4). At the same time, the prototroch nerve starts to develop from a pair of cells situated adjacent to the ventromedial prototroch cells. We name these cells as the prototroch nerve forming neurons (PNNs). These cells have a triangular morphology and sensory cilia projecting externally. Interestingly, for a few hours, only one of these cells sends out processes extending on either side along the prototrochal cells (Figs. 2h, i, 3, Additional files 5, 6, 7). From 19 hpf onwards, the other cell, which only has thin neurites projects onto the passing connectives from the adjacent cell (Figs. 2e, f, 3, Additional files 8, 9, 10, 11, 12). On the other hand, the surrounding prototroch cells seem to send extensions towards the prototroch nerve (Additional files 5, 6, 7, 8, 9, 10, 11, 12). As the prototroch is discontinuous on the dorsal side, the prototroch nerve extends only until the dorsal-most prototroch cell without forming a complete ring.
At 16 hpf, a single ganglion cell appears on one side of the apical organ along with a descending axon (Fig. 3, Additional files 5, 6, 7), which becomes more prominent at 18 hpf (Fig. 2k). We name this pioneer as the apical neuron 1 (AN1). Around 19 hpf, this growing axon from AN1 traverses contralaterally up to the prototroch region (on the other side). It extends posteriorly towards the anteriorly traveling neurite of the PPN (Figs. 2l, 3, Additional files 8, 9) and thereby closing the gap between anterior and posterior parts of the developing VNC. Meanwhile, at the same time, the growing axons from PNNs travel posteriorly and join the developing VNC (Fig. 2e).
At 21 hpf, more cells appear symmetrically around the apical organ and traverse contralaterally along the neurites established by the AN1, which persists (Figs. 2f, 3, Additional files 10, 11, 12). The crisscross of neurites originating alongside the apical organ creates a plexus, which becomes the larval brain neuropil (Figs. 2f, 3). By 24 hpf, the AN1 becomes less prominent and is not easily identifiable as more and more differentiated cells start to innervate the brain neuropil (Figs. 2g, 3, Additional files 13, 14, 15, 16). The PNNs, however, are still identifiable at 24 hpf (Fig. 2g), which by 34 hpf becomes less conspicuous (data not shown).
Around 24 hpf, a weakly stained commissure is arising in the anterior trunk region that connects the two VNC neurite bundles (Figs. 1l, 3, Additional files 13, 14, 15, 16). The nervous system represented by the 34 hpf stage is of a typical annelid trochophore composed of an apical tuft, prototroch, telotroch, and neuronal elements such as central cephalic neuropil, a prototroch nerve ring (semi-circular) and VNC (Fig. 1m). This basic neuronal architecture continues into the later larval stages and likely becomes part of the adult nervous system. In the anterior end, numerous sensory cells develop throughout the head region, with projections reaching the brain neuropil (Fig. 1m).
Development of serotonergic and FMRFamidergic neurons
For comparative purposes, we studied immunoreactivity against serotonin (5-HT) and FMRFamide, which are commonly used markers in studies on invertebrate neural development. 5-HT and FMRF are important neurotransmitters in many animals [33, 34]. While 5-HT and FMRFamide are strongly expressed in the apical neuropil and the trunk in 48 hpf stages (Fig. 2m, n), only a few serotonergic and FMRFamidergic cells are present in earlier stages. FMRFamide is detectable first at 14 hpf as a single, ciliated flask-shaped weakly labeled cell, which lies slightly left and dorsal from the apical tuft cell (Figs. 2o, 3, Additional files 2, 3). This cell develops an axon that is later projecting into the area of the apical neuropile and is accompanied by a similar second cell on the right body side at 24 hpf (Figs. 2p, 3, Additional files 14, 16). Already at 21 hpf, a second pair of FMRFamidergic ciliated flask-shaped cells also sending axons in the apical neuropil become visible ventrally from the apical tuft cell (Fig. 3, Additional file 11) and are visible as well at 24 hpf (Figs. 2r, 3, Additional files 14, 16). 5-HT can be detected from 21 hpf onwards in a pair of cells ventral to the apical tuft cell, which likewise are ciliated and flask-shaped and send their axons into the forming apical neuropil (Figs. 2s, 3, Additional file 10). Up to the 24 hpf stage, neither -FMRFamide nor 5-HT is detectable in the mid or posterior body region. In all investigated stages, only a very small fraction of the identified neurons and none of the described neurons pioneering the ventral nerve cord, the prototroch, or the connection between the apical plexus and the VNC contains FMRFamide or 5-HT.
The first neurons show synaptic activity likely from 12 hpf onwards
To get an idea when the developing neurons are entering differentiation and are getting functional on the molecular level, we screened the transcriptome resources of M. fuliginosus and public sequence databases for orthologs to synaptotagmin-1 (Syt1), which is a conserved Ca2+ sensor for fast synaptic vesicle exocytosis in many neurons of metazoans, and ras-related protein 3 (Rab3), which regulates synaptic vesicle fusion. Two sequences were found, which after reciprocal blast against Genbank gave only Syt1 and Rab3 sequences as the first 100 hits, and we named them Mfu-Syt1 and Mfu-Rab3.
In order to find an ortholog for the RNA binding protein Elav1, which is a common marker for postmitotic neuronal precursor cells in many metazoans, we ran a maximum-likelihood tree of metazoan Elav and CUGBP Elav-like genes. As in many other lophotrochozoans, in M. fuliginosus, we found two CUGBP Elav-like sequences, and two Elav sequences, both of which have RRM RNA binding motifs. One sequence (Mfu-Elav2) groups with other lophotrochozoan sequences (Additional file 17) confirming the existence of a lophotrochozoan specific Elav2 gene [21, 35]. The other one (Mfu-Elav1) clusters with high support with metazoan Elav1. Since the expression of Elav2 is not much studied and is not specific to neurons in Capitella teleta [21] and Sepia officinalis [35], we investigated only the expression of Mfu-Elav1.
Similarly, we ran an analysis of metazoan POU genes to identify the M. fuliginosus ortholog to POU4, which is an important regulator of terminal differentiation in many neurons of Metazoa. We found several POU gene sequence in the transcriptomic resources of M. fuliginosus, all containing a POU-specific domain. Only one, Mfu-POU4, belongs to the well supported POU4 clade (Additional file 18). Likewise, most other lophotrochozoans have only one POU4 gene. If there are more, then they are the result of species-specific gene duplications.
Mfu-Elav1 and Mfu-Syt1 were first detected at 12hpf (Fig. 4a, d). At this stage, Mfu-Elav1 is only restricted to the anterior region close to the apical tuft, whereas Mfu-Syt1 is expressed near the apical region, in a pair of bilateral cells and in the posterior region (Fig. 4a, d). While the expression of Mfu-Syt1 in the bilateral cells corresponds to the developing eye photoreceptors (data not shown), the posterior region corresponds to the PPN, as shown by co-staining with ac-tubulin (Fig. 4i). Shortly later, at 14 hpf, Mfu-Elav1 also starts expressing in the posterior region but not in the PPN. At the same stage, Mfu-Rab3 starts expressing in few cells spanning the anterior, middle, and posterior regions in a similar pattern to Mfu-Syt1, nonetheless, in more cells (Fig. 4g). From 18hpf onwards, several cells throughout the body express Mfu-Elav1, while Mfu-Syt1 and Mfu-Rab3 are mainly restricted to the anterior and posterior regions. The dense staining of Mfu-Syt1 and Mfu-Rab3 in the anterior region reflects the many neurons contributing to the central neuropil (Fig. 4f, h). In the posterior region, Mfu-Syt1 is only expressed in the PPN, whereas Mfu-Rab3 is expressed in the PPN and cells adjacent to it (Fig. 4h). By 48 hpf, more neurons in the anterior, oral, and along the developing VNC express Mfu-Syt1 and Mfu-Rab3 (Additional file 24).
The terminal selector POU4 (Mfu-POU4) starts to express from 10hpf in two cells, one in the anterior region and one in the posterior region (Fig. 4j). The expression in the posterior region very likely corresponds to the PPN as determined by the position and morphology of the cell. At 14hpf, an additional pair of cells lying adjacent to the posterior neuron start to express Mfu-POU4 (data not shown). In subsequent stages, the expression of Mfu-POU4 expands mainly in the anterior peripheral cells (from 12hpf) and along the trunk (from 18hpf) (data not shown). The expression of Mfu-POU4 in the PPN is maintained until the 24 hpf, after which it gets weaker.
Evolution of M. fuliginosus Sox, Prospero and bHLH genes
Sox, Prospero, and bHLH genes are important regulators of neurogenesis in many metazoans. Several genes of these groups duplicated and diversified during evolution, and many organisms have several copies of specific genes. To clarify the orthology relationships of the genes we found in the transcriptome resources of M. fuliginosus, we performed broad phylogenetic analyses with a focus on a good taxon sampling in Lophotrochozoa. Prospero1 (Prox1) genes are fairly distinct from other homeobox genes. Thus, we chose not to include any outgroup for tree inference. In our unrooted tree, the single Mfu-Prox1 groups with high support with other lophotrochozoan Prox1 genes (Additional file 19), and, thus, they are regarded as orthologs. We did not find more than one Prox1 in any lophotrochozoan.
bHLH genes are a large group of genes sharing the basic helix-loop-helix motif. Many genes of the bHLH subgroup A family: Achaete scute (ASC), Oligo, Beta3, Neurogenin, NeuroD, and Atonal play important roles in neural development. Based on a first analysis, which in addition to the above-mentioned genes includes many other bHLH group A genes, we obtained 6 Achaete scute (ASC), 3 Oligo, one each of Beta3, Atonal, Neurogenin and NeuroD sequences for M. fuliginosus (Additional file 20). For gene annotation, we adopted the terminology of [36]. The gene numbers in the specific families are similar as in other lophotrochozoans for which genomic data exist, and all families are well supported in the tree with the exception of NeuroD and Oligo, which are paraphyletic. Since within this assemblage Mfu-NeuroD clusters well-supported with NeuroD from other lophotrochozoans, we regard it as being an ortholog to those. Based on our tree, we could not resolve the 1:1 orthologs of the three M. fuliginosus Oligo genes. Since not many comparative data exist on Oligo, we did not perform further analyses and named the genes Mfu-OligoA, Mfu-OligoB, and Mfu-OligoC. We recorded clear expression patterns for Mfu-OligoA, Mfu-OligoB, Mfu-NeuroD, Mfu-neurogenin, and Mfu-ASCa1, Mfu-ASCa2, Mfu-ASCa3, and Mfu-ASCa4.
To get insights into the relationship of the M. fuliginosus ASC genes to those studied in other species, especially C. teleta and P. dumerilii, we generated a further tree based on deeper sampling and a more ASC specific and longer alignment. It corroborates the three big metazoan clades ASCc, ASCb containing vertebrate ASC3–5, and ASCa containing arthropod ASC T1-T8 and vertebrate ASC1–2 (Additional file 21). M. fuliginosus has representatives of all ASC groups as do other lophotrochozoans. Four M. fuliginosus sequences belong to the clade ASCa. The evolution of this group is in lophotrochozoans seemingly more complex than hitherto anticipated, since many species have several gene copies, many of which are poorly studied. Mfu-ASCa1 and Mfu-ASCa2 are closely related to P. dumerilli Achaete-scute 1 and to C. teleta Achaete scute 2 (Additional file 21), but also to two more transcripts from P. dumerilii, which we received from the Genbank TSA database, for which no cellular expression data exist, but according to PdumBase [37] are highly expressed in larvae from early on. To give a final answer on clear orthology relationships amongst these 6 genes is not possible based on our tree. It also remains unclear whether Mfu-ASCa3 has an ortholog in P. dumerilii and C. teleta. Mfu-ASCa4 clusters with another transcript of P. dumerilii, which according to PdumBase is highly expressed in early larvae.
SoxB and SoxC are important regulators of early neurogenesis. Still, the evolution and orthology relationships of SoxB genes in Lophotrochozoa and the relationship to vertebrate SoxB1 and SoxB2 is poorly understood [22, 24, 38]. This may be due to low taxon sampling in the respective phylogenetic analyses. Thus, we performed a broad taxon sampling, generated a first unrooted tree across most existing Sox genes, and found SoxC, SoxD, SoxE, SoxF, and at least a major part of SoxB well supported (Additional file 22). A second analysis focusing specifically on SoxB provides clear evidence that the SoxB1 group predates Bilateria and that Mfu-SoxB1 is orthologous to SoxB1 from C. teleta, P. dumerilii, and Crassostrea gigas (Additional file 23). Mfu-SoxB2 likewise falls in a well-supported group, which contains the vertebrate SoxB2 representatives Sox-21 and Sox-14 and also C. teleta SoxB and P. dumerilii SoxB, which we, accordingly, regard as SoxB2 orthologs. The basal branching of the SoxB group remains, however, elusive. We could not detect the expression of Mfu-SoxB2 in the stages analyzed, but we got clear expression patterns for Mfu-SoxB1 and Mfu-SoxC.
Expression of several neural developmental genes starts early at the anterior and posterior pole and is highly dynamic on the cellular level
To determine the regions of neuronal specification during early development, we studied the expression of the aforementioned neural and bHLH class proneural genes. The bHLH class of proneural genes is known to be expressed in a transient manner in developing neurons [39]. Therefore, the expression of bHLH genes, but also SoxB1, SoxC, and Prox1, was investigated at 1-h intervals starting from 2 hpf stage. We could not detect any expression of the genes studied at 2hpf and 3hpf. The first gene being expressed is Mfu-SoxB1, which at 4 hpf is only confined to few cells in the animal pole (Fig. 5). At 5 hpf, the expression of Mfu-SoxB1 extends throughout the animal pole cells while excluding the larger cells at the vegetal pole (Fig. 5). Shortly later, at 6 hpf stage, the expression of Mfu-SoxB1 spans throughout the body, and from 7 hpf stage, it is observed more prominently in the vegetal region. Around 12 hpf stage onwards, the expression of Mfu-SoxB1 gets more segregated, which may hint to the emergence of dedicated regions of proliferation and differentiation (Additional file 25). From 18 hpf, however, it remains mainly restricted to the anterior domain, and this pattern continues into later stages (Additional file 25).
Mfu-SoxC appears around 4 hpf in 2–3 cells near the animal pole. By 5 hpf stage, more cells start expressing Mfu-SoxC (Fig. 5). From 9 hpf stage onwards, some expression can be detected close to the posterior region, albeit transiently, but no expression was detected in the position of the PPN (Fig. 5). In general, expression of Mfu-SoxC remains mainly confined to the anterior and mid domains with expression in the trunk region starting later from 12 hpf stage (Fig. 5, Additional file 25). The expression of Mfu-SoxC in later stages (24 and 48 hpf) is similar to that of Mfu-SoxB1, as both are mostly restricted to the anterior domain (Additional file 25).
Mfu-Prox1 is expressed from 6 hpf in few cells in the mid-region. In subsequent stages, the expression also continues to expand mainly in the anterior and mid domains. From 12 hpf, the expression of Mfu-Prox1 extends along the trunk and posterior regions and is mainly detected in pairs of cells (Additional file 25).
The expression of proneural genes Mfu-ASCa1 and Mfu-Ngn were detected from 5 hpf. While Mfu-ASCa1 is expressed in two cells in the anterior region, Mfu-Ngn was detected in both the anterior and posterior poles (Fig. 5). At 6 hpf Mfu-ASCa1 and now, Mfu-NeuroD also starts to express in the posterior region (Fig. 5). The posterior expression signal of all three genes corresponds to the position of PPN we observed by immunohistochemistry from 9 hpf onwards. Expression of both Mfu-ASCa1 and Mfu-Ngn in the posterior cell is more transient than Mfu-NeuroD, whose expression was observed from 6 hpf stage until around 8 hpf stage (Fig. 5). From 9 hpf stage onwards, none of the proneural genes were detected in the posterior cell, whereas expression domains expand in the anterior region (Fig. 5, Additional file 26).
Other Achaete scute genes – Mfu-ASCa2, Mfu-ASCa3, and Mfu-ASCa4 also begin expressing from 7 to 10 hpf stage onwards in an apparent non-overlapping manner (Additional file 26). The expression of Mfu-OligoA and Mfu-OligoB were detected from 9 to 10 hpf in a few cells in the anterior and along the developing trunk (Additional file 26). The Mfu-Oligo genes are expressed in restricted domains in comparison to other bHLH proneural genes. Around the stages 12–14 hpf, several proneural genes display an expanding pattern of expression (Additional file 26). In summary, the expression of proneural genes is dynamic, and many display broad patterns in later stages of development (Additional file 26).
The first cell expressing proneural genes in the posterior region appears after 7 cleavages and is a descendant of the 2d blastomere
Having detected the expression of proneural genes from 5 hpf onwards at the position where the PPN differentiates, we were interested in tracing the clonal origin of the cell at the posterior pole. For this purpose, we fixed embryos from 30 min to 6 h post-fertilization at every 10 min intervals. At least 100 specimens were used during each fixation. We then stained the nuclei with DAPI and recorded z-stacks of at least five larvae from each fixation (a total of 165 embryos). Since the pace of development differed to some extent between embryos, we ordered the image stacks up to the 64-cell stage not by time, but by the number of nuclei. We counted cleaving cells as two. At 6 hpf in the posterior pole, two small blastomeres are visible residing between two larger cells (Fig. 6s’, t, t’). We traced the posterior most of these cells back through development as being a descendant of the D quadrant. Daughter cells were named according to the spindle orientation and the relative position of the cells to each other along the animal-vegetal axis. Thirty minutes after fertilization, the zygote contains two polar bodies along with the male and female pronucleus (Fig. 1a, a’, Additional file 27). Twenty minutes later, the first cleavage generates a larger CD and a smaller AB cell (Fig. 6b, b’, b″, c, c′, Additional files 28, 29). The next cleavage gives rise to the 4-cell stage with a D-cell being considerably larger in size than the A, B, and C-cell (Fig. 6d, d’, d”, e, e’, Additional files 30, 31). The spindle orientation of the first two cleavages is perpendicular to the animal-vegetal axis and tilts upwards. The D-cell is the first cell that enters the third cleavage around 1 h 50 min post-fertilization is dextral and gives rise to a smaller animal 1d and a larger vegetal 1D-cell (Fig. 6f, f’, f″, g, g’, Additional files 32, 33). The next cleavage is sinistral and gives rise to the cells 2D and 2d (Fig. 6h, h’, h″, Additional file 34). Notably, in the 16-cell stage, the animal 2d cell is larger than the vegetal 2D cell (Fig. 6k, k’, Additional file 35). Cleavage 5 and 6 generate first the cell 2d2 on the right body side (Fig. 6l, l’, l”, 6m, m’, Additional files 36, 37), which then divides into 2d21 remaining on the right body side and 2d22 with a more central position (Fig. 6n, n’, n″, Additional file 38). The 2d22 travels further posterior until it reaches the posterior-most region in the 64-cell stage, which is around 5 hpf (Fig. 6o, o’, 6p, p′, Additional files 39, 40). Cleavage 7, finally, generates the two small blastomeres 2d221, which takes in the most posterior position in the embryo and 2d222, which lies a bit more vegetal and anterior (Fig. 6r, r’, r”, 6s, s′, Additional files 41, 42). At 6 hpf, the cells 2d221 and 2d222 are flanked left and right by two cells with large nucleus (Fig. 6t, t’). These nuclei are also useful landmarks after fluorescence in-situ hybridization. Between the large nuclei, the Mfu-NeuroD expression signal is surrounding a small nucleus, which corresponds to the position of 2d221 (Fig. 6u). Slightly anterior and vegetal is a second small nucleus that corresponds to the position of the cell 2d222 and is not surrounded by expression signal (Fig. 6u’).