BACKGROUND
neurogenesis is a complex process that involves the formation of a vast array of neuronal and glial cell types that must be produced in the correct numbers and at appropriate positions.
genetic and molecular studies mainly conducted in drosophila and vertebrates have shown that genes encoding transcription factors of the basic helix-loop-helix  class play pivotal roles in various steps of neurogenesis, including commitment of neural precursors , specification of particular neuronal identities, and neuronal differentiation  <cit> .
most of the genes encoding bhlh transcription factors and which are involved in neural development , belong to five of the numerous phylogenetically-defined bhlh families, achaete-scute and four families of atonal-related genes, neurogenin, atonal, olig , and neurod  <cit> .
while some of the neural bhlh genes show strikingly similar functions in drosophila and vertebrates  <cit> , there are also profound differences between them  <cit> .
first, in vertebrates, genes of the neurogenin family  are required for the formation of the precursors of many neural cells of both the peripheral and central nervous systems   <cit>  while their single drosophila ortholog, tap/biparous, has no proneural role and is expressed in a few differentiating neural cells  <cit> .
in drosophila, the main proneural bhlh genes for the cns belong to the achaete-scute family and are also involved, together with atonal family genes, in the formation of the sensory organs  <cit> .
vertebrate achaete-scute and atonal genes probably also have proneural functions but in a much more limited set of cells, in particular in the cns  <cit> .
second, vertebrate proneural genes contribute to the specification of progenitor-cell identity  <cit> .
a clear example of such a function is provided by the dorsal embryonic spinal cord, in which math <dig> , ngn <dig>  and mash <dig>  are required for the correct specification of discrete dorsoventral progenitor domains that produce distinct types of interneurons  <cit> .
mash <dig> has also been shown to have instructive roles in the specification of noradrenergic, gabaergic, and serotonergic neurons in various positions in the brain and the spinal cord  <cit> .
finally, ngn <dig> has a key role for motor neurons formation in the ventral spinal cord  <cit> .
such important roles in neuronal specification for proneural bhlh genes in the cns are not found in drosophila  <cit> .
third, bhlh genes that have important functions during vertebrate neurogenesis do not have orthologs in drosophila.
many vertebrate neurons require the function of genes, which belong to the neurod family, for their proper differentiation and survival  <cit> .
genes of the olig family  have key roles in the specification of motor neurons, dorsal interneurons, and oligodendrocytes in the vertebrate cns  <cit> .
olig and neurod genes do not exist in drosophila   <cit> .
given these differences and in order to decipher which aspects of the functions of neural bhlh genes are ancestral to bilaterians and which are derived characters specific to some bilaterian lineages, we have isolated and studied these genes in the polychaete annelid, platynereis dumerilii, which belongs to a different branch  of the bilaterians tree than drosophila  and vertebrates  and is therefore evolutionary distant to both these organisms  <cit> .
in addition, platynereis is considered to have retained some bilaterian ancestral features, making it a useful model for comparative developmental biology .
here, we report the expression patterns of platynereis orthologs of the most important neural bhlh genes, including the olig and neurod genes not found in drosophila.
our data suggest that platynereis bhlh genes have both proneural and neuronal specification functions, in a way more akin to the vertebrate situation than to that of drosophila.
these data indicate that these functions were already established in urbilateria, the last common ancestor of all bilaterians.
RESULTS
brief overview of the formation of the platynereis larval nervous system
platynereis displays an indirect development which gives rise to a ciliated trochophore larva that subsequently metamorphoses into a juvenile worm  <cit> .
the formation of the platynereis larval trunk nervous system has been thoroughly described, up to the late trochophore stage , using whole-mount in situ hybridization  with rna antisense probes corresponding to platynereis neuronal differentiation genes, such as elav , synaptotagmin , tryptophane hydroxylase , and vesicular acetylcholine transporter   <cit> .
in order to give an overview of larval neurogenesis, which is important to understand the next parts of this article, we show here some wmish for these previously characterized genes in  <dig> hpf to  <dig> hpf larvae and extend the published description by looking at juvenile worms .
a simple larval nervous system first differentiates during the early  to late trochophore  stages: a few cells expressing pdu-elav are observed on the ventral side of the  <dig> hpf and  <dig> hpf larvae  and these cells give rise to two bilateral anterior groups and one posterior group of pdu-syt-expressing neurons .
different neuronal subtypes can be identified, such as serotonergic  and cholinergic .
from  <dig> hpf, a large number of cells enter neural differentiation and express first pdu-elav  and then pdu-syt .
these neurons form the ventral nerve cord  of the  <dig> hpf juvenile worm .
many of the formed neurons are cholinergic neurons as seen by the massive expression of pdu-vacht  in the vnc, except in its medialmost part where several serotonergic neurons differentiate .
outside the vnc, peripheral neurons are also found mainly associated with the appendages .
identification of platynereis atonal- and achaete-scute-related bhlh genes
by sequence similarity searches on an expressed sequence tag  collection   <cit> , we identified several platynereis dumerilii bhlh genes among which some show strong sequence similarity to either achaete-scute- or atonal-related genes.
the predicted amino acid sequence of the bhlh domains were aligned with those of a sample of metazoan bhlh genes identified in a previous study  <cit> .
multiple phylogenetic reconstructions show that we identified orthologs of the neurogenin/biparous, achaete-scute/ascl/ash, olig, and neurod genes .
we named these genes pdu-ngn, pdu-ash, pdu-olig, and pdu-neurod, respectively.
for each family, we found a single platynereis member but we cannot exclude that additional members do exist, although duplicated evolutionary-conserved genes are rare in platynereis  <cit> .
we also included in our analysis the previously identified atonal ortholog   <cit>  in order to study platynereis representatives for all the main families of bhlh genes involved in neural determination and specification in vertebrates and drosophila  <cit> .
we also identified beta <dig> and mist genes in platynereis , but these genes were not further characterized as their drosophila and vertebrate orthologs do not have well defined functions in neurogenesis.
we used wmish to monitor the expression of pdu-ngn, pdu-ash, pdu-olig, pdu-ath, and pdu-neurod during platynereis development and focused on possible expressions during trunk nervous system formation for which a good characterization exists  <cit> .
all the genes are also expressed in the head, probably in the brain and/or sensory organs, but these expressions were not further characterized.
platynereis atonal- and achaete-scute-related bhlh genes are expressed during trunk nervous system formation
the five bhlh genes are already expressed in  <dig> hpf larvae.
pdu-neurod is expressed in a broad ventral ectodermal domain  which corresponds to the larval neuroectoderm.
this expression domain is similar to that of platynereis orthologs of bilaterian conserved neuroectodermal genes, such as soxb .
pdu-ngn and pdu-ash are expressed in a few cells located on the ventral side of the larvae, in particular around the stomodaeal area .
this expression nicely prefigures the distribution of differentiating neurons that is observed in slightly later stages , suggesting that the two genes are expressed in precursors of the larval central nervous system.
pdu-ath and pdu-olig are expressed in a few lateral cells we were unable to identify .
in  <dig> hpf larvae, pdu-neurod is still expressed in a broad ventral ectodermal domain  which includes the prospective vnc region in which pdu-ngn and pdu-ash become widely expressed .
at this stage, the expression of pdu-soxb strongly decreases in the same region .
in vertebrates, the transition from neuroectodermal cells to committed progenitors is linked to the activation of the expression of bhlh proneural genes and a concomitant repression of the expression of soxb genes .
our data suggest that a similar transition occurs around  <dig> hpf in the platynereis larvae.
however, at this stage, only very few cells express pdu-elav , indicating that the pdu-ngn and pdu-ash expressing precursors are still not engaged towards differentiation.
both pdu-ngn and pdu-ash are also expressed in more lateral cells that may correspond to peripheral nervous system precursors .
pdu-ath and pdu-olig continue to be expressed in lateral cells, with pdu-olig expressed in three bilateral small stripes .
in  <dig> hpf to  <dig> hpf larvae, pdu-neurod continues to be broadly expressed in the ventral ectoderm .
expression of pdu-neurod is restricted to the superficial layer of the ectoderm .
from  <dig> hpf to  <dig> hpf larvae, pdu-ngn is largely expressed in the whole prospective vnc region as well as in some more lateral cells .
pdu-ngn is expressed in a salt and pepper manner, with highly-expressing cells interspersed with weakly-expressing ones.
in  <dig> hpf and  <dig> hpf larvae, denes et al.
<cit>  showed that the prospective platynereis vnc region is multilayered with, from superficial  to more internal , a proliferating progenitor zone , a post-mitotic progenitor zone , and a differentiation zone .
to define in which zone of the prospective vnc pdu-ngn is expressed, we performed double wmish  <cit>  for pdu-ngn, on one hand, and pdu-elav or pdu-syt, on the other hand, and visualized the labellings with confocal microscopy .
we found that pdu-ngn is mainly expressed in superficial cells in contrast to pdu-elav and pdu-syt .
we used 3d reconstructions of confocal stacks to perform virtual cross-sections of the vnc region : we found that pdu-ngn is expressed in the apicalmost cells of the prospective vnc with very little overlap with pdu-elav and no overlap with pdu-syt.
we therefore conclude that pdu-ngn is expressed in undifferentiated neural precursors and mainly in the proliferating ones.
pdu-ash and pdu-olig, at  <dig> and  <dig> hpf, are expressed in the medialmost part of the prospective vnc region as well as in more lateral cells .
as for pdu-ngn, pdu-ash and pdu-olig are expressed in proliferating progenitors, as determined by double wmish .
the expression of pdu-ash and pdu-olig in the medialmost part of the prospective vnc region is similar to that of the platynereis nk <dig>  gene and corresponds to the region from which serotonergic neurons will emerge   <cit> .
we confirmed by double wmish that the serotonergic neurons  are located below the pdu-ash-expressing precursors .
at  <dig> hpf, pdu-ash shows the same expression pattern than in the previous stages, but pdu-olig is no more expressed in the prospective vnc region .
in  <dig> hpf to  <dig> hpf larvae, pdu-ath is only expressed in a few cells located on both sides of the prospective vnc region .
at  <dig> hpf, the five genes are expressed in more limited sets of cells in the vnc region and in cells associated with the parapodes .
in addition, pdu-ngn, pdu-ash, pdu-olig, and pdu-neurod are expressed in posteriorly-located internal cells  whose position roughly corresponds to that of cells expressing platynereis hunchback  <cit>  and stem-cells markers, such as platynereis piwi and vasa  <cit> .
the cells expressing pdu-ngn, pdu-ash, pdu-olig, and pdu-neurod may therefore belong to the posterior subterminal growth zone that will allow the posterior addition of new segments to the existing ones  in a sequential manner throughout most of the life of the animal  <cit> .
discussion
olig and neurod genes belong to the ancestral bilaterian toolkit of neural developmental genes
in this article, we report, for the first time, the characterization of several atonal- and achaete-scute-related bhlh genes from a lophotrochozoan species, the annelid platynereis dumerilii.
our phylogenetic analyses demonstrate that we have identified neurogenin, achaete-scute, olig, and neurod orthologs in addition to a previously characterized atonal gene .
neurogenin, achaete-scute, and atonal genes have been found in many diverse species, including drosophila melanogaster, caenorhabditis elegans, and several vertebrates, and shown to be involved in neural development in all these species .
we found a similar situation in platynereis as the three genes display specific expressions during neurogenesis , confirming their evolutionary-wide implication in neurogenesis in bilaterian animals.
neurod genes have been shown to be important neuronal differentiation genes in vertebrates .
a putative neurod gene  has been described in the nematode caenorhabditis elegans and is involved in several steps of the formation of the motor neurons  <cit> .
neurod genes cannot be found in the genomes of drosophila melanogaster and of the urochordate ciona intestinalis  <cit> .
our phylogenomic analysis  indicates that neurod genes are in fact widely found in bilaterians and that their absence in drosophila melanogaster and ciona intestinalis results from a rather specific loss in these species.
we found the platynereis neurod gene to have a broad and early neuroectodermal expression, which is completely different from the neuronal expression of vertebrate neurod genes and the neural subtype-specific expression of caenorhabditis cnd- <dig>  it is therefore difficult to infer from these data any putative ancestral expression or function of neurod.
more precise inference will await additional data from other species.
olig genes were thought to be vertebrate-specific genes as they are not found in the drosophila melanogaster, caenorhabditis elegans, and ciona intestinalis genomes  <cit> .
in fact, olig genes are found in several non-vertebrate species .
however, we cannot find such genes in any of the sequenced arthropod and nematode genomes , suggesting that olig genes have been lost quite early during the evolution of these phyla.
we found the platynereis olig gene to be specifically expressed during nervous system formation and that its expression shows similarities to that of vertebrate olig genes .
we therefore conclude that olig, together with neurogenin, achaete-scute, atonal, and neurod genes belong to the ancestral bilaterian toolkit of neural developmental genes.
vertebrate-like expression of platynereis neurogenin suggests a major proneural role and provides insights into the evolution of the proneural function in bilaterians
one striking difference between vertebrate and drosophila neurogenesis is the differential use of neurogenin genes in these species.
indeed, neurogenin genes are the key proneural genes in the vertebrate cns while it is not the case for their drosophila counterpart .
in drosophila, and probably more generally in arthropods, the proneural function in the cns is mainly performed by the achaete-scute genes .
these genes have a much more limited proneural functions in vertebrates .
given these differences, it is challenging to infer what was the ancestral situation, i.e.
what were the main proneural genes acting in urbilateria, the last common ancestor of all bilaterians.
our data on platynereis help to answer this question.
we found that platynereis neurogenin  has an expression suggestive of a wide proneural function in the developing trunk cns which is similar to that of the vertebrate neurogenin genes.
indeed, pdu-ngn expression arises at early stages of neural development, overlapping that of the neuroectodermal marker soxb, and preceding the expression of the differentiation marker elav.
the expression domain of pdu-ngn is large and covers the whole prospective cns region  as well as more lateral regions which probably correspond to the pns.
pdu-ngn is expressed in a salt and pepper manner which is often found for proneural genes as a consequence of lateral inhibition processes .
finally, pdu-ngn is expressed in the apicalmost part of the forming cns, which has been shown to include proliferative neuroectodermal cells and neural precursors  <cit> .
pdu-ngn is therefore expressed at the right time, the right place, and the right manner to be the major proneural gene for the formation of the platynereis trunk nervous system, like its orthologs in vertebrates.
this similarity between vertebrates  and an annelid  suggests that the broad proneural function of neurogenin genes is ancestral to bilaterians and has been lost in the evolutionary lineage leading to present-day insects .
this does not necessarily mean that proneural function of achaete-scute genes is a derived character: indeed, such function is found in vertebrates and in arthropods, and the expression of pdu-ash is not incompatible with this function, as it is expressed in early stages of vnc formation .
it is therefore conceivable that both neurogenin and achaete-scute were acting as proneural genes in urbilateria, the former being the predominant one.
platynereis bhlh genes may have neural specification functions in the cns – evolution of neural specification in bilaterians
another striking difference between vertebrates and drosophila is the differential use of bhlh genes in neural subtype specification in the cns.
in vertebrate, neural bhlh genes such as olig and achaete-scute genes are important to specify many types of neural cells which are produced in the cns, while it is not the case in the drosophila cns .
this raises the possibility that the functions of bhlh genes in neural subtype specification may largely represent vertebrate innovations.
alternatively, they could be bilaterian ancestral functions that have been lost in the evolutionary lineage leading to drosophila.
we found that two platynereis bhlh genes, pdu-ash and pdu-olig, are expressed in a way suggestive of an involvement in neural specification in the cns.
both genes are expressed in the medialmost part of the cns, they are first co-expressed and in a second time, only pdu-ash continues to be expressed.
interestingly, these expression profiles coincide with those of vertebrates olig and achaete-scute genes which are also expressed in medial parts of the neural tube .
importantly, this similarity is probably meaningful as the overall organization of the platynereis cns bears striking resemblances with that of vertebrates, as seen by the conserved expression domains of many neural patterning and differentiation genes between platynereis and vertebrates  <cit> .
the cells expressing pdu-ash and pdu-olig correspond to a domain of the vnc characterized by the co-expression of platynereis nk <dig>  and nk <dig> genes  <cit> .
in vertebrates, such as mammals, ascl1/mash <dig> is also expressed in a nk <dig> /nk <dig> positive medial domain  and controls the formation of serotonergic neurons from this domain in the hindbrain  <cit> .
strikingly, serotonergic neurons in platynereis also emerge from a medial domain of the cns, corresponding to cells expressing nk <dig>  and achaete-scute.
we therefore suggest that pdu-ash is involved in the specification of serotonergic neurons in platynereis and that this may represent an ancestral function of achaete-scute genes in bilaterians.
in vertebrates, such as mammals, olig genes have important roles in neural specification.
olig <dig> gene is expressed in the so-called pmn domain of the spinal cord, located slightly more laterally than the p <dig> domain .
this domain corresponds to a nk6/pax6-positive domain from which originate hb9-positive cholinergic motor neurons and oligodendrocytes, the specification of both of these cells types being controled by olig <dig>  this nk6/pax6-positive domain also exists in platynereis and, as in vertebrates, hb9-positive cholinergic neurons emerge from this domain  <cit> .
pdu-olig is, however, not expressed in this domain  and is therefore much probably not involved in motor neurons specification.
however its co-expression with pdu-ash at some but not all stages of vnc formation, in the nk6/nk <dig> -positive domain, suggests it may contribute to the diversification of neural cell types from this domain.
we suggest that different neural cell types may form in the prospective platynereis vnc first from the pdu-ash/pdu-olig-positive cells and then from the pdu-ash-positive  cells.
this suggestion is based on the fact that, in vertebrates, different combinations of neural bhlh have been shown to control the formation of different cell types from a single medio-lateral domain  <cit> .
our data suggest that some neural bhlh genes are involved in neural subtype specification in platynereis, like in vertebrates, and that this may therefore represent an ancestral feature of bilaterians.
in insects, such as drosophila, the specification functions of neural bhlh genes  have been lost.
this may be related to the fact that insects have evolved a divergent way to pattern their cns: while in vertebrates and platynereis the cns is subdivided into large domains from which emerge specific neural cell types, insects have shifted to a mainly cell-to-cell-based process in which neuroblasts  will express different combinations of developmental genes that control their identity.
CONCLUSIONS
we have identified, for the first time in a lophotrochozoan species, platynereis dumerilii, orthologs of the most important neural bhlh genes known in vertebrates, including the olig and neurod genes not found in drosophila.
we have performed a detailed analysis of the expression patterns of these platynereis bhlh genes and we show that all these genes are expressed during neurogenesis.
our analysis suggests that the platynereis bhlh genes have both proneural and neuronal specification functions, in a way more akin to the vertebrate situation than to that of drosophila.
our data suggest that these functions are ancestral to bilaterians.

 neurogenesis is a complex process that involves the formation of a vast array of neuronal and glial cell types that must be produced in the correct numbers and at appropriate positions. genetic and molecular studies mainly conducted in drosophila and vertebrates have shown that genes encoding transcription factors of the basic helix-loop-helix class play pivotal roles in various steps of neurogenesis, including commitment of neural precursors, specification of particular neuronal identities, and neuronal differentiation. most of the genes encoding bhlh transcription factors and which are involved in neural development, belong to five of the numerous phylogenetically-defined bhlh families, achaete-scute and four families of atonal-related genes, neurogenin, atonal, olig, and neurod. while some of the neural bhlh genes show strikingly similar functions in drosophila and vertebrates, there are also profound differences between them. first, in vertebrates, genes of the neurogenin family are required for the formation of the precursors of many neural cells of both the peripheral and central nervous systems while their single drosophila ortholog, tap/biparous, has no proneural role and is expressed in a few differentiating neural cells. in drosophila, the main proneural bhlh genes for the cns belong to the achaete-scute family and are also involved, together with atonal family genes, in the formation of the sensory organs. vertebrate achaete-scute and atonal genes probably also have proneural functions but in a much more limited set of cells, in particular in the cns. second, vertebrate proneural genes contribute to the specification of progenitor-cell identity. a clear example of such a function is provided by the dorsal embryonic spinal cord, in which math, ngn and mash are required for the correct specification of discrete dorsoventral progenitor domains that produce distinct types of interneurons. mash has also been shown to have instructive roles in the specification of noradrenergic, gabaergic, and serotonergic neurons in various positions in the brain and the spinal cord. finally, ngn has a key role for motor neurons formation in the ventral spinal cord. such important roles in neuronal specification for proneural bhlh genes in the cns are not found in drosophila. third, bhlh genes that have important functions during vertebrate neurogenesis do not have orthologs in drosophila. many vertebrate neurons require the function of genes, which belong to the neurod family, for their proper differentiation and survival. genes of the olig family have key roles in the specification of motor neurons, dorsal interneurons, and oligodendrocytes in the vertebrate cns. olig and neurod genes do not exist in drosophila. given these differences and in order to decipher which aspects of the functions of neural bhlh genes are ancestral to bilaterians and which are derived characters specific to some bilaterian lineages, we have isolated and studied these genes in the polychaete annelid, platynereis dumerilii, which belongs to a different branch of the bilaterians tree than drosophila and vertebrates and is therefore evolutionary distant to both these organisms. in addition, platynereis is considered to have retained some bilaterian ancestral features, making it a useful model for comparative developmental biology. here, we report the expression patterns of platynereis orthologs of the most important neural bhlh genes, including the olig and neurod genes not found in drosophila. our data suggest that platynereis bhlh genes have both proneural and neuronal specification functions, in a way more akin to the vertebrate situation than to that of drosophila. these data indicate that these functions were already established in urbilateria, the last common ancestor of all bilaterians.  brief overview of the formation of the platynereis larval nervous system platynereis displays an indirect development which gives rise to a ciliated trochophore larva that subsequently metamorphoses into a juvenile worm. the formation of the platynereis larval trunk nervous system has been thoroughly described, up to the late trochophore stage, using whole-mount in situ hybridization with rna antisense probes corresponding to platynereis neuronal differentiation genes, such as elav, synaptotagmin, tryptophane hydroxylase, and vesicular acetylcholine transporter. in order to give an overview of larval neurogenesis, which is important to understand the next parts of this article, we show here some wmish for these previously characterized genes in hpf to hpf larvae and extend the published description by looking at juvenile worms. a simple larval nervous system first differentiates during the early to late trochophore stages: a few cells expressing pdu-elav are observed on the ventral side of the hpf and hpf larvae and these cells give rise to two bilateral anterior groups and one posterior group of pdu-syt-expressing neurons. different neuronal subtypes can be identified, such as serotonergic and cholinergic. from hpf, a large number of cells enter neural differentiation and express first pdu-elav and then pdu-syt. these neurons form the ventral nerve cord of the hpf juvenile worm. many of the formed neurons are cholinergic neurons as seen by the massive expression of pdu-vacht in the vnc, except in its medialmost part where several serotonergic neurons differentiate. outside the vnc, peripheral neurons are also found mainly associated with the appendages. identification of platynereis atonal- and achaete-scute-related bhlh genes by sequence similarity searches on an expressed sequence tag collection, we identified several platynereis dumerilii bhlh genes among which some show strong sequence similarity to either achaete-scute- or atonal-related genes. the predicted amino acid sequence of the bhlh domains were aligned with those of a sample of metazoan bhlh genes identified in a previous study. multiple phylogenetic reconstructions show that we identified orthologs of the neurogenin/biparous, achaete-scute/ascl/ash, olig, and neurod genes. we named these genes pdu-ngn, pdu-ash, pdu-olig, and pdu-neurod, respectively. for each family, we found a single platynereis member but we cannot exclude that additional members do exist, although duplicated evolutionary-conserved genes are rare in platynereis. we also included in our analysis the previously identified atonal ortholog in order to study platynereis representatives for all the main families of bhlh genes involved in neural determination and specification in vertebrates and drosophila. we also identified beta and mist genes in platynereis, but these genes were not further characterized as their drosophila and vertebrate orthologs do not have well defined functions in neurogenesis. we used wmish to monitor the expression of pdu-ngn, pdu-ash, pdu-olig, pdu-ath, and pdu-neurod during platynereis development and focused on possible expressions during trunk nervous system formation for which a good characterization exists. all the genes are also expressed in the head, probably in the brain and/or sensory organs, but these expressions were not further characterized. platynereis atonal- and achaete-scute-related bhlh genes are expressed during trunk nervous system formation the five bhlh genes are already expressed in hpf larvae. pdu-neurod is expressed in a broad ventral ectodermal domain which corresponds to the larval neuroectoderm. this expression domain is similar to that of platynereis orthologs of bilaterian conserved neuroectodermal genes, such as soxb. pdu-ngn and pdu-ash are expressed in a few cells located on the ventral side of the larvae, in particular around the stomodaeal area. this expression nicely prefigures the distribution of differentiating neurons that is observed in slightly later stages, suggesting that the two genes are expressed in precursors of the larval central nervous system. pdu-ath and pdu-olig are expressed in a few lateral cells we were unable to identify. in hpf larvae, pdu-neurod is still expressed in a broad ventral ectodermal domain which includes the prospective vnc region in which pdu-ngn and pdu-ash become widely expressed. at this stage, the expression of pdu-soxb strongly decreases in the same region. in vertebrates, the transition from neuroectodermal cells to committed progenitors is linked to the activation of the expression of bhlh proneural genes and a concomitant repression of the expression of soxb genes. our data suggest that a similar transition occurs around hpf in the platynereis larvae. however, at this stage, only very few cells express pdu-elav, indicating that the pdu-ngn and pdu-ash expressing precursors are still not engaged towards differentiation. both pdu-ngn and pdu-ash are also expressed in more lateral cells that may correspond to peripheral nervous system precursors. pdu-ath and pdu-olig continue to be expressed in lateral cells, with pdu-olig expressed in three bilateral small stripes. in hpf to hpf larvae, pdu-neurod continues to be broadly expressed in the ventral ectoderm. expression of pdu-neurod is restricted to the superficial layer of the ectoderm. from hpf to hpf larvae, pdu-ngn is largely expressed in the whole prospective vnc region as well as in some more lateral cells. pdu-ngn is expressed in a salt and pepper manner, with highly-expressing cells interspersed with weakly-expressing ones. in hpf and hpf larvae, denes et al. showed that the prospective platynereis vnc region is multilayered with, from superficial to more internal, a proliferating progenitor zone, a post-mitotic progenitor zone, and a differentiation zone. to define in which zone of the prospective vnc pdu-ngn is expressed, we performed double wmish for pdu-ngn, on one hand, and pdu-elav or pdu-syt, on the other hand, and visualized the labellings with confocal microscopy. we found that pdu-ngn is mainly expressed in superficial cells in contrast to pdu-elav and pdu-syt. we used 3d reconstructions of confocal stacks to perform virtual cross-sections of the vnc region: we found that pdu-ngn is expressed in the apicalmost cells of the prospective vnc with very little overlap with pdu-elav and no overlap with pdu-syt. we therefore conclude that pdu-ngn is expressed in undifferentiated neural precursors and mainly in the proliferating ones. pdu-ash and pdu-olig, at and hpf, are expressed in the medialmost part of the prospective vnc region as well as in more lateral cells. as for pdu-ngn, pdu-ash and pdu-olig are expressed in proliferating progenitors, as determined by double wmish. the expression of pdu-ash and pdu-olig in the medialmost part of the prospective vnc region is similar to that of the platynereis nk gene and corresponds to the region from which serotonergic neurons will emerge. we confirmed by double wmish that the serotonergic neurons are located below the pdu-ash-expressing precursors. at hpf, pdu-ash shows the same expression pattern than in the previous stages, but pdu-olig is no more expressed in the prospective vnc region. in hpf to hpf larvae, pdu-ath is only expressed in a few cells located on both sides of the prospective vnc region. at hpf, the five genes are expressed in more limited sets of cells in the vnc region and in cells associated with the parapodes. in addition, pdu-ngn, pdu-ash, pdu-olig, and pdu-neurod are expressed in posteriorly-located internal cells whose position roughly corresponds to that of cells expressing platynereis hunchback and stem-cells markers, such as platynereis piwi and vasa. the cells expressing pdu-ngn, pdu-ash, pdu-olig, and pdu-neurod may therefore belong to the posterior subterminal growth zone that will allow the posterior addition of new segments to the existing ones in a sequential manner throughout most of the life of the animal. discussion olig and neurod genes belong to the ancestral bilaterian toolkit of neural developmental genes in this article, we report, for the first time, the characterization of several atonal- and achaete-scute-related bhlh genes from a lophotrochozoan species, the annelid platynereis dumerilii. our phylogenetic analyses demonstrate that we have identified neurogenin, achaete-scute, olig, and neurod orthologs in addition to a previously characterized atonal gene. neurogenin, achaete-scute, and atonal genes have been found in many diverse species, including drosophila melanogaster, caenorhabditis elegans, and several vertebrates, and shown to be involved in neural development in all these species. we found a similar situation in platynereis as the three genes display specific expressions during neurogenesis, confirming their evolutionary-wide implication in neurogenesis in bilaterian animals. neurod genes have been shown to be important neuronal differentiation genes in vertebrates. a putative neurod gene has been described in the nematode caenorhabditis elegans and is involved in several steps of the formation of the motor neurons. neurod genes cannot be found in the genomes of drosophila melanogaster and of the urochordate ciona intestinalis. our phylogenomic analysis indicates that neurod genes are in fact widely found in bilaterians and that their absence in drosophila melanogaster and ciona intestinalis results from a rather specific loss in these species. we found the platynereis neurod gene to have a broad and early neuroectodermal expression, which is completely different from the neuronal expression of vertebrate neurod genes and the neural subtype-specific expression of caenorhabditis cnd- it is therefore difficult to infer from these data any putative ancestral expression or function of neurod. more precise inference will await additional data from other species. olig genes were thought to be vertebrate-specific genes as they are not found in the drosophila melanogaster, caenorhabditis elegans, and ciona intestinalis genomes. in fact, olig genes are found in several non-vertebrate species. however, we cannot find such genes in any of the sequenced arthropod and nematode genomes, suggesting that olig genes have been lost quite early during the evolution of these phyla. we found the platynereis olig gene to be specifically expressed during nervous system formation and that its expression shows similarities to that of vertebrate olig genes. we therefore conclude that olig, together with neurogenin, achaete-scute, atonal, and neurod genes belong to the ancestral bilaterian toolkit of neural developmental genes. vertebrate-like expression of platynereis neurogenin suggests a major proneural role and provides insights into the evolution of the proneural function in bilaterians one striking difference between vertebrate and drosophila neurogenesis is the differential use of neurogenin genes in these species. indeed, neurogenin genes are the key proneural genes in the vertebrate cns while it is not the case for their drosophila counterpart. in drosophila, and probably more generally in arthropods, the proneural function in the cns is mainly performed by the achaete-scute genes. these genes have a much more limited proneural functions in vertebrates. given these differences, it is challenging to infer what was the ancestral situation, i.e. what were the main proneural genes acting in urbilateria, the last common ancestor of all bilaterians. our data on platynereis help to answer this question. we found that platynereis neurogenin has an expression suggestive of a wide proneural function in the developing trunk cns which is similar to that of the vertebrate neurogenin genes. indeed, pdu-ngn expression arises at early stages of neural development, overlapping that of the neuroectodermal marker soxb, and preceding the expression of the differentiation marker elav. the expression domain of pdu-ngn is large and covers the whole prospective cns region as well as more lateral regions which probably correspond to the pns. pdu-ngn is expressed in a salt and pepper manner which is often found for proneural genes as a consequence of lateral inhibition processes. finally, pdu-ngn is expressed in the apicalmost part of the forming cns, which has been shown to include proliferative neuroectodermal cells and neural precursors. pdu-ngn is therefore expressed at the right time, the right place, and the right manner to be the major proneural gene for the formation of the platynereis trunk nervous system, like its orthologs in vertebrates. this similarity between vertebrates and an annelid suggests that the broad proneural function of neurogenin genes is ancestral to bilaterians and has been lost in the evolutionary lineage leading to present-day insects. this does not necessarily mean that proneural function of achaete-scute genes is a derived character: indeed, such function is found in vertebrates and in arthropods, and the expression of pdu-ash is not incompatible with this function, as it is expressed in early stages of vnc formation. it is therefore conceivable that both neurogenin and achaete-scute were acting as proneural genes in urbilateria, the former being the predominant one. platynereis bhlh genes may have neural specification functions in the cns – evolution of neural specification in bilaterians another striking difference between vertebrates and drosophila is the differential use of bhlh genes in neural subtype specification in the cns. in vertebrate, neural bhlh genes such as olig and achaete-scute genes are important to specify many types of neural cells which are produced in the cns, while it is not the case in the drosophila cns. this raises the possibility that the functions of bhlh genes in neural subtype specification may largely represent vertebrate innovations. alternatively, they could be bilaterian ancestral functions that have been lost in the evolutionary lineage leading to drosophila. we found that two platynereis bhlh genes, pdu-ash and pdu-olig, are expressed in a way suggestive of an involvement in neural specification in the cns. both genes are expressed in the medialmost part of the cns, they are first co-expressed and in a second time, only pdu-ash continues to be expressed. interestingly, these expression profiles coincide with those of vertebrates olig and achaete-scute genes which are also expressed in medial parts of the neural tube. importantly, this similarity is probably meaningful as the overall organization of the platynereis cns bears striking resemblances with that of vertebrates, as seen by the conserved expression domains of many neural patterning and differentiation genes between platynereis and vertebrates. the cells expressing pdu-ash and pdu-olig correspond to a domain of the vnc characterized by the co-expression of platynereis nk and nk genes. in vertebrates, such as mammals, ascl1/mash is also expressed in a nk /nk positive medial domain and controls the formation of serotonergic neurons from this domain in the hindbrain. strikingly, serotonergic neurons in platynereis also emerge from a medial domain of the cns, corresponding to cells expressing nk and achaete-scute. we therefore suggest that pdu-ash is involved in the specification of serotonergic neurons in platynereis and that this may represent an ancestral function of achaete-scute genes in bilaterians. in vertebrates, such as mammals, olig genes have important roles in neural specification. olig gene is expressed in the so-called pmn domain of the spinal cord, located slightly more laterally than the p domain. this domain corresponds to a nk6/pax6-positive domain from which originate hb9-positive cholinergic motor neurons and oligodendrocytes, the specification of both of these cells types being controled by olig this nk6/pax6-positive domain also exists in platynereis and, as in vertebrates, hb9-positive cholinergic neurons emerge from this domain. pdu-olig is, however, not expressed in this domain and is therefore much probably not involved in motor neurons specification. however its co-expression with pdu-ash at some but not all stages of vnc formation, in the nk6/nk -positive domain, suggests it may contribute to the diversification of neural cell types from this domain. we suggest that different neural cell types may form in the prospective platynereis vnc first from the pdu-ash/pdu-olig-positive cells and then from the pdu-ash-positive cells. this suggestion is based on the fact that, in vertebrates, different combinations of neural bhlh have been shown to control the formation of different cell types from a single medio-lateral domain. our data suggest that some neural bhlh genes are involved in neural subtype specification in platynereis, like in vertebrates, and that this may therefore represent an ancestral feature of bilaterians. in insects, such as drosophila, the specification functions of neural bhlh genes have been lost. this may be related to the fact that insects have evolved a divergent way to pattern their cns: while in vertebrates and platynereis the cns is subdivided into large domains from which emerge specific neural cell types, insects have shifted to a mainly cell-to-cell-based process in which neuroblasts will express different combinations of developmental genes that control their identity.  we have identified, for the first time in a lophotrochozoan species, platynereis dumerilii, orthologs of the most important neural bhlh genes known in vertebrates, including the olig and neurod genes not found in drosophila. we have performed a detailed analysis of the expression patterns of these platynereis bhlh genes and we show that all these genes are expressed during neurogenesis. our analysis suggests that the platynereis bhlh genes have both proneural and neuronal specification functions, in a way more akin to the vertebrate situation than to that of drosophila. our data suggest that these functions are ancestral to bilaterians. 
we conclude that these features are ancestral to bilaterians and have been conserved in the vertebrates and annelids lineages, but have diverged in the evolutionary lineage leading to drosophila. in addition, we found that the platynereis neurod gene has a broad and early neuroectodermal expression, which is completely different from the neuronal expression of vertebrate neurod genes. we observed that all these genes have specific expression patterns during nervous system formation in platynereis. functional studies in model organisms, such as vertebrates and drosophila, have shown that basic helix-loop-helix proteins have important roles in different steps of neurogenesis, from the acquisition of neural fate to the differentiation into specific neural cell types. our data suggest that in platynereis, like in vertebrates but unlike drosophila, neurogenin is the main proneural gene for the formation of the trunk central nervous system, achaete-scute and olig genes are involved in neural subtype specification in the central nervous system, in particular in the specification of the serotonergic phenotype. our analysis suggests that the platynereis bhlh genes have both proneural and neuronal specification functions, in a way more akin to the vertebrate situation than to that of drosophila. to understand how the functions of neural bhlh genes have evolved among bilaterians, we have performed a detailed study of bhlh genes during nervous system development in the polychaete annelid, platynereis dumerilii, an organism which is evolutionary distant from both drosophila and vertebrates. we have studied platynereis orthologs of the most important vertebrate neural bhlh genes, i.e. achaete-scute, neurogenin, atonal, olig, and neurod genes, the latter two being genes absent of the drosophila genome. however, these studies highlighted many differences in the expression and function of orthologous bhlh proteins during neural development between vertebrates and drosophila.
 functional studies in model organisms, such as vertebrates and drosophila, have shown that basic helix-loop-helix proteins have important roles in different steps of neurogenesis, from the acquisition of neural fate to the differentiation into specific neural cell types. however, these studies highlighted many differences in the expression and function of orthologous bhlh proteins during neural development between vertebrates and drosophila. to understand how the functions of neural bhlh genes have evolved among bilaterians, we have performed a detailed study of bhlh genes during nervous system development in the polychaete annelid, platynereis dumerilii, an organism which is evolutionary distant from both drosophila and vertebrates.  we have studied platynereis orthologs of the most important vertebrate neural bhlh genes, i.e. achaete-scute, neurogenin, atonal, olig, and neurod genes, the latter two being genes absent of the drosophila genome. we observed that all these genes have specific expression patterns during nervous system formation in platynereis. our data suggest that in platynereis, like in vertebrates but unlike drosophila, neurogenin is the main proneural gene for the formation of the trunk central nervous system, achaete-scute and olig genes are involved in neural subtype specification in the central nervous system, in particular in the specification of the serotonergic phenotype. in addition, we found that the platynereis neurod gene has a broad and early neuroectodermal expression, which is completely different from the neuronal expression of vertebrate neurod genes.  our analysis suggests that the platynereis bhlh genes have both proneural and neuronal specification functions, in a way more akin to the vertebrate situation than to that of drosophila. we conclude that these features are ancestral to bilaterians and have been conserved in the vertebrates and annelids lineages, but have diverged in the evolutionary lineage leading to drosophila. 