Annelids along with many other protostome invertebrates share the presence of a brain and ventral longitudinal nerve cords. While homology of these parts of the central nervous system within annelids is widely accepted, it is still a matter of debate, how the annelid trunk nervous system relates to that of other bilaterians. Highly similar mediolateral pattering of nerve cord development between annelids and distantly related bilaterians suggest a general homology [19,20,31]. The structural diversity of bilaterian nervous systems and molecular data on nervous system patterning from some groups were, however, also interpreted in favor of independent nerve cord evolution [15]. Molecular aspects of early neurogenesis have been studied in several species of Spiralia [21–24,26,32–34], but not with focus on the pioneer neurons prefiguring the main scaffold of the later nervous system. Data on the first steps in nervous system formation are thus highly interesting from both, a developmental and an evolutionary point of view.
Neurogenesis in Malacoceros fuliginosus – variation of a common theme
The basic processes of neurogenesis, i.e. specification of neurogenic areas, progression of progenitors, specification of precursors and differentiation of neurons in M. fuliginosus largely follows a conserved pattern observed in many organisms. The first of the studied proneural genes showing up in prospective neuroectoderm is an ortholog of SoxB, whose function in specification and proliferation of neural progenitors has been documented throughout the animal kingdom [35,36]. SoxC, known to promote cell-cycle exit follows little later [37]. Then, Prox1 and Elav, typical markers of postmitotic precursors [38–40] become expressed and finally markers for neural differentiation like Synaptotagmin and Rab3. The observed co-expression of succeeding markers in several cells suggests that this set of genes labels continuous lineages of developing cells. Other common neurogenic factors, such as Achaete-scute, NeuroD, Neurogenin and Olig are also broadly expressed in the neuroectoderm of M. fuliginosus. This set of genes is in a similar temporal order involved in neurogenesis in many animal groups [1,2,34,41].
A more detailed comparison to other representatives of annelids mainly reveals differences in the time course of neural development along the anterior-posterior axis. Compared to Platynereis dumerilii and Capitella teleta, neurogenesis progresses at more similar speed in the anterior, the trunk and the posterior in M. fuliginosus. Differentiation markers Synaptotagmin and Elav show up in the anterior region in Platynereis dumerilii and M. fuliginosus around 14 hpf and also onset of Prox expression may be similar, as it is already expressed in several cells in 12 hpf in Platynereis dumerilii [42]. However, in the trunk of Platynereis dumerilii SoxB is most broadly expressed around 24 hpf and stays active until 55 hpf [24], while we find broadest expression in the trunk of M. fuliginosus around 14 hpf and downregulation already around 18 hpf. The differentiation markers Elav and Synaptotagmin are reported from 32 hpf onwards in the trunk of Platynereis dumerilii [19], while they show up already at 14-18 hpf in the trunk of M. fuliginosus. In Capitella teleta SoxB, Prox and several bHLH proneural genes show up much later in the trunk and the posterior than in the anterior [22].
The special case of the posterior pioneer neuron of the ventral nerve cord
Owing to their easy identification and tracking, the function and features of pioneer neurons are best studied in arthropods, vertebrates and nematodes, and their role in formation of the axonal scaffold is established [4,43–45]. Several factors set them apart from follower and other neurons: from their precise positional cues to distinct molecular profiles and pathfinding abilities [46,47]. In particular, studies in C. elegans have shown that the positioning of pioneer neurons is different from other neurons as they considerably deviate from optimized neuronal wiring principle thereby suggesting strong underlying developmental constraint [48]. Moreover, different pioneers can be specified by different transcription factors and also have different developmental mechanisms [49,50].
In the large taxon of Lophotrochozoa and Spiralia, the molecular development of the neurons which pioneer the main routes of the developing nervous system has not yet been analyzed in detail. By correlating immunohistochemical data on nervous system differentiation with gene expression analysis we found that development of the first neuron showing up in the posterior deviates considerably from that of other neurons. This neuron probably takes in a very important role in the development of the nervous system of M. fuliginosus, since its axons pioneer the ventral nerve cord. This particular neuron starts differentiating very early evidenced by atub-LIR from 8 hpf and expression of the synaptic genes Synaptotagmin and Rab3, which are known to be involved in neurotransmitter release, from 12 hpf onwards.We have no evidence, but we also cannot rule out that the posterior pioneer neuron or its pre-differentiated form is expressing or maternally inheriting SoxB in very early stages, since we did not have a specific marker for this cell during the first 8 h of development. But from 8 hpf onwards, where the cell can be identified by its tubulin expression, SoxB cannot be detected within the cell. Further, our data suggests that this cell does not express SoxC, Achaete-scute, NeuroD, Neurogenin, Olig, Prox1 and Elav which are likely expressing in most if not all other neurons
The detection of Mfu-Brn3 in the posterior neuron starting from 10 hpf corresponds to the stage where sensory cilia are developing. Brn3 has been identified in sensory neuron development in several bilaterian taxa suggesting an evolutionary conserved role [51–53], therefore it is likely that Mfu-Brn3 confers a sensory function for the posterior neuron. Functional studies in C. elegans and mice have shown the importance of Brn3 as a terminal selector in differentiation of neurons and maintenance of neuronal identity [54]. The continuous expression of Mfu-Brn3 in the posterior neuron and other cells suggests a similar role in M. fuliginosus.
Seemingly specification of neural fate of VNC pioneer depends on a highly deviant gene regulatory network devoid of a broad spectrum of genes involved in neurogenesis of other neurons. The only neural transcription factor we found expressed is Brn3, which likely acts on a low hierarchical level. Patterning processes highly relevant for the development of nervous system like Wnt and BMP signaling are known to drive neural fate determination by acting on the expression of Sox genes and other early neural determinants. Since the ventral nerve cord pioneer does not express those genes, it will be very interesting to find out, whether fate determination in this specific cell may depend less on cell-cell signaling, but rather on inherited cell-intrinsic properties. Such kind of cell-autonomous fate determination is generally an important mode of specification during development of spirally cleaving embryos of lophotrochozoans like annelids, molluscs and nemerteans and other Spiralia like flatworms [55–58]. mRNA segregation during several rounds of asymmetric cell divisions is supposed to be the main mechanism for passing on maternal factors [59–62] and probably is most relevant for specification of early differentiating cells, before regulative mechanisms mediated by extrinsic factors take over during later development of the embryos.
The direct influence of cell-intrinsic properties on nervous system development in Spiralia, however, is poorly understood. Notably, most evidence has recently been provided from annelids, where data point towards cell-autonomous specification of neurons in the anterior region of the embryo. Correlation of 3D cell-lineage data of the trochophore episphere with gene expression data in Platynereis dumerilii revealed that many of the later appearing neurons with bilateral symmetry and similar features do not share corresponding lineages in left- right opposing quadrants suggesting a position related conditional specification [42]. In difference, early differentiating neurons of the apical organ do not originate from bilateral symmetrical clones and do not express several transcription factors involved in head regionalization pointing towards cell-autonomous specification. In Capitella teleta, separation of the micromeres 1a-1d from the rest of the embryo in the 8-cell stage of Capitella teleta leads to head-only partial larva with cells expressing the neuronal marker Elav, seen as evidence for cell-autonomous specification of the respective neural fate [63]. Notably, the single ganglion cell in the future apical organ pioneering the anterior part of the circumoesophageal connective in M. fuliginosus also does not have a bilateral symmetrical counterpart, which might as well indicate cell-autonomous specification. But this remains speculative and needs further investigation. Our data, however, point for the first time towards possible cell autonomous specification of neural fate in the posterior end of a spiralian embryo, i.e. in the posterior pioneer neuron of the ventral nerve cord. This is not in contradiction with observations reported from Capitella teleta suggesting conditional specification of trunk neurons [63]. Here, no signs of trunk neurons were detected in partial larvae derived from the micromeres 1a-1d plus cell 2d, whose progeny in complete embryos gives rise to the trunk neuroectoderm. We suggest that while development of the vast majority of trunk neurons depends on extrinsic factors in annelids [19–21,24,31,63], a posterior ventral nerve cord pioneer which may be subject to cell-autonomous specification is secondarily missing in Capitella teleta (see discussion beyond).
Position and role of pioneer neurons in Lophotrochozoa
Insights into the role of pioneer neurons in early nervous system development of lophotrochozoans and other Spiralia rely mainly on immunohistochemical analysis of nervous system differentiation. Similar as in M. fuliginosus the first appearing neurons in many organisms were found in the anterior and/or the posterior region of the embryos. Observed variability in position, neurotransmitter and projection raised many questions on whether formation of the nervous system progresses in these animals rather from anterior to posterior, vice versa or from both sides and from central to the periphery or the other way round. A comparison of data is biased by the fact that many studies focus mainly on the small subset of serotonergic and FMRFamidergic neurons, while our data show that in M. fuliginosus the earliest differentiating pioneer neurons are devoid of these neurotransmitters and many other neurons are already present before the first serotonergic and FMRFamidergic neurons are discernable. Nevertheless, evolutionary conservation of some patterns can be inferred. A posterior ventral nerve cord pioneer probably has been present in the last common ancestor of the two major polychaete subgroups, which are Errantia and Sedentaria [64]. A posterior bifurcating neuron highly similar to the cell we investigated in Malacoceros fuliginosus has been described from the errant polychaetes Phyllodoce maculata [65] and Platynereis dumerilii [66,67] and the sedentary polychaete Pomatoceros lamarckii [68]. In all four species the respective cell is located on the very posterior tip of the developing larva, sends a bundle of cilia to the exterior and two very long axons anteriorly along the future ventral nerve cord. These similarities strongly suggest homology. Thorough electron microscopy based characterization of neural circuitry in 3 days old larvae of Platynereis dumerilii revealed that this cell beside its pioneering role also takes in a sensory-motor function by directly synapsing to multiciliated cells of the prototroch, which propel the larva through the water [69]. We could trace the axons of the posterior pioneer to the level of the prototroch making a similar function conceivable in M. fuliginosus. Neurotransmitter content of the posterior pioneer neuron seems to vary in polychaetes. While it is serotonergic in Phyllodoce maculata [65] and Pomatoceros lamarckii [68], it contains serotonin and FMRFamide in Platynereis dumerilii [67,69] and none of these transmitters in M. fuliginosus, even though the expression of Synaptotagmin and Rab3 suggest the capacity of transmitter release. Accordingly, presence of this cell may be overlooked, if studies on nervous system development rely mainly on stainings against few neurotransmitters like anti-FMRFamide-LIR and anti-5HT-LIR and not on broad neuronal markers and also, if time intervals between studied stages are too big to trace the outgrowth of the first neurites. A posterior ventral nerve cord pioneer is likely lost in Capitella teleta, where close examination of neural development provides no hints on presence of such a cell[70], while this is difficult to judge for few other annelid trochophores, where less detailed data exist.
In the anterior region we found two different kinds of pioneer neurons in M. fuliginosus. While the prototroch ring nerve is pioneered by axons of two peripheral sensory cells, the nerves running posteriorly from the apical plexus associated with the apical organ is pioneered by neurites of a ganglion cell. This challenges the view that the CNS of annelid trochophores is generally prefigured only by peripheral sensory cells [65,67,71,72]. As in the case of the posterior pioneer neuron, these early anterior pioneer neurons of M. fuliginosus cannot be identified by studying anti-serotonin or anti-FMRFamide immunoreactivity. In accordance, cell-lineage data from Platynereis dumerilii show that several acetyl-cholinergic neurons appear much earlier in the anterior region than the first serotonergic neurons and that the former form already a considerable mesh of neurites 30 h after fertilization [42]. Data from Capitella teleta show that the first differentiating neurons in the apical region are not peripheral sensory cells, but form within the future brain [70].
In conclusion, it is likely that in the ancestor of errant and sedentary annelids the first neurons pioneering the scaffold of the later CNS arose at the anterior as well as the posterior pole. While in the posterior only peripheral sensory cells take in this function, in the anterior probably both peripheral sensory cells and central ganglion cells are involved. This may be valid for annelids in general, though the situation in the few groups of basally branching needs further investigation. The question in which direction CNS formation is progressing in annelids encompasses different aspects which are not necessarily linked to each other. For the differentiation of the vast majority of CNS neurons, existing data point towards an anterior-posterior progression. On the other side, the suggested ancestral presence of pioneers in both the anterior and the posterior means that scaffold formation of the CNS started from both sides. From which side the first neurites extend faster in extant representatives is a matter of the exact onset of neurite outgrowth from the respective poles. This may be strongly affected by even small heterochronic changes in development underlying the observable variation between species.
If further evidence will arise that cell-autonomous specification drives formation of the first differentiating neurons in the anterior as well as in the posterior of annelid trochophores, scaffold formation of the whole annelid CNS may be under strong influence of neurons specified by maternally inherited factors. No molecular data exist from lophotrochozoans or Spiralia other than annelids on the neurogenesis and specification of early CNS pioneer neurons. Yet, a large number of immunohistochemical studies suggest that the first neurites prefiguring the main routes of the nervous system likewise develops from few early developing cells in mollusks, nemerteans, and lophophorates. Obtaining deep data on pioneer specification, their cell-lineage and role in axonal pathfinding in spiral cleaving animals may thus be highly informative for a better understanding of nervous system development and evolution.