Unfolding the ventral nerve center of chaetognaths

Background Chaetognaths are a clade of marine worm-like invertebrates with a heavily debated phylogenetic position. Their nervous system superficially resembles the protostome type, however, knowledge regarding the molecular processes involved in neurogenesis is lacking. To better understand these processes, we examined the expression profiles of marker genes involved in bilaterian neurogenesis during post-embryonic stages of Spadella cephaloptera. We also investigated whether the transcription factor encoding genes involved in neural patterning are regionally expressed in a staggered fashion along the mediolateral axis of the nerve cord as it has been previously demonstrated in selected vertebrate, insect, and annelid models. Methods The expression patterns of genes involved in neural differentiation (elav), neural patterning (foxA, nkx2.2, pax6, pax3/7, and msx), and neuronal function (ChAT and VAChT) were examined in S. cephaloptera hatchlings and early juveniles using whole-mount fluorescent in situ hybridization and confocal microscopy. Results The Sce-elav + profile of S. cephaloptera hatchlings reveals that, within 24 h of post-embryonic development, the developing neural territories are not limited to the regions previously ascribed to the cerebral ganglion, the ventral nerve center (VNC), and the sensory organs, but also extend to previously unreported CNS domains that likely contribute to the ventral cephalic ganglia. In general, the neural patterning genes are expressed in distinct neural subpopulations of the cerebral ganglion and the VNC in hatchlings, eventually becoming broadly expressed with reduced intensity throughout the CNS in early juveniles. Neural patterning gene expression domains are also present outside the CNS, including the digestive tract and sensory organs. ChAT and VAChT domains within the CNS are predominantly observed in specific subpopulations of the VNC territory adjacent to the ventral longitudinal muscles in hatchlings. Conclusions The observed spatial expression domains of bilaterian neural marker gene homologs in S. cephaloptera suggest evolutionarily conserved roles in neurogenesis for these genes among bilaterians. Patterning genes expressed in distinct regions of the VNC do not show a staggered medial-to-lateral expression profile directly superimposable to other bilaterian models. Only when the VNC is conceptually laterally unfolded from the longitudinal muscle into a flat structure, an expression pattern bearing resemblance to the proposed conserved bilaterian mediolateral regionalization becomes noticeable. This finding supports the idea of an ancestral mediolateral patterning of the trunk nervous system in bilaterians. Supplementary Information The online version contains supplementary material available at 10.1186/s13064-024-00182-6.


Background
There is a tremendous diversity of body plans in adult and developing bilaterian animals [1].For a long time, the condensed central nervous systems (CNS) of diverse evolutionary lineages have been considered as an evolutionarily conserved organ system among bilaterians.For the majority of bilaterian clades neuronal condensations in form of glomeruli, ganglia, and brains or nerve cords are well documented, although also organisms with a nerve net as an uncondensed nervous system such as Acoelomorpha do exist [2,3].This diversity is also reflected during the ontogeny of these organisms and while embryos may initially possess one or more paired nerve cords, these may have fused secondarily, for example, to a single median nerve cord in adults of some taxa [3].Interestingly, even in morphologically diverse nervous systems, similar molecular signatures have been observed during nervous system (NS) development [4][5][6].These consistently observed similarities in the gene expression profiles during NS development have been proposed as one of the most concrete pieces of evidence of a single evolutionary origin of the bilaterian nerve cord [7][8][9].The expression patterns of the transcription factor encoding genes foxA, nkx2.1/nkx2.2,nkx6, pax6, pax3/7, and msx have shown similar staggered mediolateral patterns in the developing nerve cord of some bilaterian representatives, including vertebrates, the fruit fly Drosophila melanogaster, and the annelid Platynereis dumerilii [reviewed in 2,4].In addition, the spatial organization of their expression patterns corresponds to the mediolateral regionalization of different neuronal cell subtypes along the neural midline of the developing CNS.Serotonergic neurons form along the medial domain (nk2.2+ / nk6 + ), cholinergic neurons and interneurons at the intermediate domain (pax6 + /nk6 + and pax6 + /pax3/7 + , respectively), and sensory neurons at the lateral domain (msx + /pax3/7 + ) [5,6,8,9].The mediolaterally staggered expression patterns in these neuronal cell populations have been a pivotal basis for proposing the hypothesis of a medially condensed ventral nerve cord that features a mediolateral patterning system in the bilaterian ancestor [4][5][6][7]9].However, this mediolateral patterning has been argued to be absent in the annelid Owenia fusiformis, the rotifer Epiphanes senta, the nemertean Lineus ruber [10], the nematode Caenorhabditis elegans [11], onychophorans [12][13][14], and the hemichordate enteropneust Saccoglossus kowalevskii [15][16][17].In addition, in several other phylogenetically informative taxa, including acoelomorphs and brachiopods, mediolateral patterning has not been (or only partially) observed [5,10].The lack of mediolateral patterning in the latter taxa has led to the argument that nerve cords evolved independently in several bilaterian lineages ( [10,18] but see [5] for an alternative standpoint).Additional arguments against the evolutionary conservation of nerve cords among Bilateria are the divergent ontogenetic histories of the latter in phylogenetically distantly related taxa such as annelids, vertebrates, or arthropods.While the early neuroectoderm of e.g.hemichordates and chordates undergoes an infolding process into a tube [19], among panarthropods neuroblasts delaminate from the neuroectoderm in the region giving rise to the head and the ventral nerve cords [20].Subsequent fates of these neuroblasts may, however, differ considerably.In spiralians such as annelids or mollusks, early neuroectoderm invaginates and forms nerve cords and ganglia of varying numbers and of different organizations in different representatives [3].
Although the currently available data either substantiate or refute the hypothesis that mediolateral patterning during CNS development is conserved in Bilateria, our understanding of the ancestral states of neuroectoderm mediolateral patterning and the origin of nerve cords remains incomplete.This is principally due to the lack of information on the molecular architecture of key bilaterian groups, especially those that exhibit unique CNS features and represent an evolutionarily relevant taxonomic branch.Therefore, further investigation into additional bilaterian clades and developmental stages is imperative to infer the evolutionary history of the CNS.
Little is also known about early chaetognath neurogenesis.In S. cephaloptera, the neural progenitors that give rise to the VNC have been first observed in the anterolateral regions of the ectoderm during early embryogenesis (Fig. 7 d) [42].These neurogenic cells multiply their nuclei and the latter eventually sink inwards and form the progenitor cells of the VNC (referred to as ganglion cells by [42]).Subsequently, these ganglion cells separate from the row of ectodermal cells, aggregate into a mass of cells in the ventrolateral region of the mesodermal cell bands (future longitudinal muscle), and successively form two lateral somata clusters as apparent in hatchlings (Fig. 1e).
The nervous system of the chaetognath hatchling resembles that of the adult to some extent (Fig. 1a, b, d,  h).Both possess a dorsal cerebral ganglion that is connected to a ventrally positioned ganglion (VNC) along the trunk (Fig. 1d, e, h, i) [23,25,43].As the hatchling matures, the rudimentary loop-like cephalic ganglion becomes structurally more complex and develops additional cephalic ganglia, including the vestibular and esophageal ganglia.The VNC features a central neuropil bordered by two bands of somata clusters on the lateral sides (lateral somata clusters) and small clusters of cells on its medioventral side (medioventral somata clusters) [23,25].The VNC is already well established at hatching [25].While it wraps around the centrally positioned muscle and gut bundle in hatchlings (Fig. 1e), it eventually becomes restricted to the midventral region underneath the longitudinal muscle bands of the adult trunk (Fig. 1g) [23,25,43].Chaetognaths display typical protostomal neuroarchitecture such as the circumoral nervous system; however, the adult brain exhibits two components (i.e.anterior and posterior brain) that form two circumoral nerve rings connected to the ventral ganglia of the head and the trunk [25,43].In addition, arrow worms also possess a number of unique neural structures, organizational patterns, and developmental features that are remarkably different from other gnathiferans.For example, they possess a neuro-muscular boundary that lacks specialized junctions of axonal varicosities, presynapses and the underlying muscles and is separated by a thick extracellular matrix [23,25].They also feature an intraepidermal ventral nerve center [25], and other apomorphic traits known from early neurogenesis which do not appear to be closely related to traits of either deuterostomes or other protostomes [25].
So far, the morphology of the chaetognath nervous system has been adequately described [43][44][45][46] and there have been comparative morphological analyses of neural features (e.g.ventral nerve cord) between chaetognaths and other taxa, such as annelids and arthropods [10][11][12].However, our understanding of their neurogenesis and neural patterning, especially the molecular regulatory circuitry, underlying these processes, remains incomplete.
To further explore the molecular aspects of nervous system development in chaetognaths, we performed an in-depth characterization of evolutionarily highly conserved genes involved in bilaterian neurogenesis in the early post-embryonic stages of Spadella cephaloptera.We determined whether these axial patterning genes are regionally expressed along the mediolateral axis of the developing nerve cord similar to what is observed in diverse bilaterian representatives identified in [16,26].We first screened for gene orthologs of foxA, nkx2.2, pax6, pax3/7, msx, ChAT, VAChT, and elav in the draft transcriptome of S. cephaloptera [47] and examined their spatial expression using in situ hybridization on hatchlings and early juveniles.By comparing our findings with data from other bilaterians, we were able to identify similarities in the expression patterns of these gene markers in the CNS.Ultimately, this provided insights into the putative roles of these genes during late neural development of chaetognaths.All the above-mentioned genes play important roles in early neural patterning, a process that this study could not document due to the unsuccessful in situ hybridization experiments on earlier encapsulated chaetognath embryos.Nevertheless, our findings show that these transcription factors probably play an evolutionarily conserved role during the formation of the CNS in chaetognath hatchlings, similar to other bilaterians.At first glance, chaetognath mediolateral gene expression patterns appear partially scrambled compared to those of other bilaterian models.However, in chaetognath hatchlings, the conceptual unfolding of the VNC resembles the postulated ancestral bilaterian staggered array, thus reinforcing the view that this mediolateral array is part of a key ancestral process during the formation of the trunk nervous system in bilaterians.

Animal collection, husbandry, and fixation
Live adult specimens of Spadella cephaloptera (Busch, 1851) were collected in the intertidal zone off the shore of Roscoff, France, and maintained in aquaria with a blend of artificial and natural sea water (kept at 18°C and 35 ppt) in the aquatic research facility of the University of Vienna.Mature individuals (n ~ 35) were collected from tanks and transferred to culture plates (14.1 cm radius Petri dishes) and fed with Artemia sp.nauplii until egglaying.One-day post-hatching individuals (8 -20h old hatchlings; 1 dph) and early juveniles (7 -10 dph) were manually collected from the culture plates and fixed in MEM-PFA (0.5 M MOPS, 5 mM EGTA, 10 mM MgSO 4 , 2.5M NaCl, and 4% paraformaldehyde) for one hour at room temperature.They were washed three times for five minutes each in PBTw (1x PBS, pH 7.5 with 0.1% Tween-20), followed by three 15-minute washes with 100% methanol, and were then stored at -20°C in methanol.In situ hybridization experiments have been carried out on encapsulated prehatching embryos without success because it was not possible to remove or open the egg capsule without destroying the embryo.Consequently, this study only focused on early post-embryonic stages.

Sequencing and riboprobe synthesis
Different developmental stages of S. cephaloptera were sacrificed and pooled for total RNA extraction using RNAqueous ™ -Micro Total RNA Isolation Kit (Invitrogen GmbH, Karlsruhe, Germany), which was then used for cDNA synthesis using First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche Molecular Biochemicals, Vienna, Austria).
Primers were designed for the identified transcripts using Geneious Prime or Primer-BLAST (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast/).Reverse primers were synthesized with a T7 promoter sequence (5′-TAA TAC GAC TCA CTA TAG GG-3′) at the 5' end.To generate the DNA template for the riboprobe, PCR experiments were performed using these gene-specific primers and Taq 2X Master Mix (New England Biolabs).PCR amplicons were purified with QIAquick PCR Purification Kit (Qiagen Vertriebs GmbH, Vienna, Austria) and were sent to Microsynth Austria for Sanger sequencing to validate their identity.The resulting electropherograms were visually inspected with Geneious Prime.Digoxigenin (DIG)labeled antisense riboprobes were generated using the T7 polymerase and DIG RNA labeling mix kit (Roche Diagnostics, Mannheim, Germany).Total riboprobe concentrations were estimated using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA).All primer sequences and their annealing temperatures are available in Additional file 2.

Microscopy and image processing
Samples were imaged on a Leica TCS SP5 confocal microscope (Leica Microsystems, Heidelberg, Germany).A 633 nm gas laser was used for the fluorescent scanning of Fast Blue stains and the 405 nm laser was used to visualize the DAPI-stained perikarya.Fiji [54] was used to view and adjust confocal images for brightness and contrast and to perform z-stack projections of the optical sections.IMARIS (v 3.4.1)was also used for 3D visualization and to capture images of the orthogonal sections (lateral and transverse planes).Figure assembly and schematic drawings were prepared with Inkscape (https:// inksc ape.org).All anatomical identifications used here were based on the descriptions of [23][24][25][55][56][57].

Gene orthologs and phylogenetic analyses
Putative orthologs of foxA, nkx2.2, pax6, pax3/7, msx, ChAT, VAChT, and elav were identified in the draft transcriptome of S. cephaloptera.Only Sce-chat, Sce-nkx2.2and Sce-msx showed an incomplete open reading frame, while the remaining genes were full-length.Phylogenetic analyses based on a Bayesian approach confirmed the orthology of S. cephaloptera sequences to their bilaterian orthologs (see Additional file 3).

Neural regions of the Spadella hatchling
To further describe the neural domains of the Spadella hatchlings, we examined the expression patterns of the marker for postmitotic neuronal precursor cells in many bilaterians [58,59].In the head of hatchlings, Sce-elav transcripts are detected in the cells of the developing brain (Fig. 2a, d, j), in a pair of somata clusters bordering the esophagus (presumptive developing vestibular ganglia) (Fig. 2k), in the eyes (Fig. 2a, d, k, circles), and at a low level in cells of the corona ciliata (Fig. 2a, l, dashed outline).In the trunk, Sce-elav expression is observed in cells of the lateral and medioventral somata clusters (Fig. 2b, e -h, m).The expression in the tail is detected in cells of lateral ciliary sense organs (Fig. 2c, i, h).

Expression of neural mediolateral patterning genes in Spadella
The expression patterns of mediolateral patterning genes in hatchlings and juveniles of S. cephaloptera were determined using fluorescent whole-mount in situ hybridization (WMFISH).Dorsal scans are presented as z-stack projections, while lateral and transverse sections are shown as one optical section.All additional figures in the succeeding sections have been deposited in the Supplementary material file.

Expression of Sce-foxA
A large region of the head shows strong Sce-foxA expression: the mesodermal-derived cells of the developing pharynx and esophagus; a bulbous-shaped structure, resembling the perioral epidermis in adults, that seemingly envelopes the inner regions of the head where most of the cephalic muscles develop (Fig. 3a; Additional file 5: Fig. S9a, b, e-g, red arrowheads); and a pair of cell clusters (Fig. 3a; Additional file 5: Fig. S9b, e, dashed oval), which presumably corresponds to the developing vestibular and esophageal ganglia, laterally flanking the esophagus (solid lines).In addition, Sce-foxA + cells are also located in the ventral-most region of the head and posterior to the future buccal cavity (Additional file 5: Fig. S9c).In the trunk, Sce-foxA is expressed in the medioventral neurons (Fig. 3a, c; Additional file 5: Fig. S9i' , k) and in distinct lateral neuronal cells that are arranged in two rows parallel to the gut: a more lateral domain (Fig. 3c; Additional file 5: Fig. S9i) and a ventral domain proximal to the midline, slightly dorsal to the medioventral neuronal cells (Additional file 5: Fig. S9i').The more lateral rows (Additional file 5: Fig. S9i) include large cells that feature an inconspicuously large nucleus interspersed within the smaller neuronal cells (Additional file 5: Fig. S9 j, j').The gene transcripts are also distinguished along the entire trunk portion of the gut (Additional file 5: Fig. S9i, arrows, k) and in a group of cells medioposterior to the somata of the ventral nerve center (Additional file 5: Fig. S9i' , l, arrowhead).In early juveniles, Sce-foxA is evidently expressed in the anterior-most organs of the head, including the cerebral ganglion (Fig. 6a-c; Additional file 5: Fig. S9m, q, s, t), the vestibular ganglia (Additional file 5: Fig. S9n, r, dashed outlines), and the cells of the structure surrounding the buccal cavity (more specifically perioral epidermis) where the teeth develop (Fig. 6ac; Additional file 5: Fig. S9n -r).FoxA expression is also detected in cells of the lateral plates (Additional file 5: Fig. S9o) and the ventral-most cells of the head, posterior to the mouth opening (Additional file 5: Fig. S9p).In the VNC, Sce-foxA is expressed in the medioventral neurons and, unlike in hatchlings, the expression increases in the lateral somata cluster (Fig. 6a-c).No expression is observed in the tail except for the ventral cells medially situated on the posterior border of the VNC, where the presumptive anal pore is positioned (Additional file 5: Fig. S9u-w, arrowhead).

Expression of Sce-nkx2.2
In the neural domains, Sce-nkx2.2 is expressed in cells of the lateral somata clusters and is less pronounced in the medioventral somata clusters (Fig. 3d-f; Additional file 6: Fig. S10h, i).Low Sce-nkx2.2expression was observed in the anterior head region where the cerebral ganglion is developing (Additional file 6: Fig. S10a, d, g).A pair of cell clusters lateral to the oral cavity is also Sce-nkx2.2positive (Additional file 6: Fig. S10a, b, d, white arrowheads).Additionally, the expression is prominent along the digestive tract, spanning from the esophageal region (posterior to the future mouth) and extending throughout the entire intestine (Fig. 3d; Additional file 6: Fig. S10b, f, g,  i).Sce-nkx2.2 is also detected in the ventral cells situated on the trunk-tail boundary (Additional file 6: Fig. S10h, i, arrowhead).No Sce-nkx2.2signal was observed in the early juvenile (data not shown).

Expression of Sce-pax6
Sce-pax6 is expressed in the dorsal anterolateral portion of the head (Fig. 4a; Additional file 8: Fig. S11a, c, f ).Transcripts were also detected in sensory structures between the two concentric rings of the corona ciliata, the eyes, and the lateral sensory organs of the trunk and tail (Fig. 4a; Additional file 7: Fig. S11a, d, h, i).In the trunk, Sce-pax6 is expressed in cells of the lateral somata clusters (Fig. 4a  -c; Additional file 7: Fig. S11a, b, g) but not in the mediodorsal region of the VNC (Fig. 4b).Similar expression domains are generally observed in early juveniles (Fig. 6 g-i; Additional file 7: Fig. S11j-s) except for a weaker signal in the eyes and corona ciliata (Additional file 7: Fig. S11j, o, p).Sce-pax6 is still evident in the anterolateral portions of cephalic ganglia (Fig. 6g; Additional file 7: Fig. S11j).In addition, Sce-pax6 is strongly expressed in the anterior head (presumably the anterior-most part of the brain flanked by a pair of frontal connectives (Additional file 7: Fig. S11m), the posterior brain adjacent to the retrocerebral organ (Additional file 7: Fig. S11j), in cells of the presumptive esophageal ganglia (Additional file 7: Fig. S11k, n, arrowheads), in cells lining the buccal area (presumably perioral epidermis; Additional file 7: Fig. S11l, o), and in medioventral neuronal cells (Additional file 7: Fig. S11q').In the tail, a few ciliary fence organs also express Sce-pax6 (Additional file 7: Fig. S11r, s), although slightly less intense than in 1 dph individuals.

Expression of Sce-pax3/7
Sce-pax3/7 is expressed in a pair of cells with large cell nuclei in the anteroventral tip of the lateral somata clusters of the hatchling (Fig. 4e, Additional file 8: Fig. S12a, b. b').In the VNC, the Sce-pax3/7 expression domain appears in two narrow longitudinal stripes that follow the contour of the entire inner side contour of the lateral somata clusters that flank the dorsal longitudinal muscles (Fig 4d-f; Additional file 8: Fig. S12c, f ), in presumptive mesodermal cells on both sides of the developing germ cells (Additional file 8: Fig. S12 c, d, g, h, arrow and arrowheads), and in cells situated on the ventral anterior tail, which could be the position for the future anus (Additional file 8: Fig. S12e -g, red arrowhead).Early juveniles exhibit Sce-pax3/7 expression in neural regions of the head, including the brain, vestibular ganglia, and esophageal ganglia (Fig. 6d; Additional file 8: Fig. S12i -k).In the lateral somata clusters, the expression becomes diffused (Fig. 6d-f; Additional file 8: Fig. S12k).Sce-pax3/7 is also detected in the presumptive specialized mesodermal cells comprising the posterior septum (Additional file 8: Fig. S12m -p) and in cells around the anal region (Additional file 8: Fig. S12l, n, p).

Expression of Sce-msx
Sce-msx expression domains in the head of the Spadella hatchling are observed in the dorsal cells slightly anterior to the eyes (Fig. 4g, h; Additional file 9: Fig. S13a -c, arrowheads) and in the ventral cells situated between corona ciliata and the eyes (Fig. 4g, h; Additional file 9: Fig. S13a, d, arrows).Scemsx is largely localized in the medio-dorsal region of the lateral somata clusters (Fig. 4g -i; Additional file 9: Fig. S13e, f, f ').This includes some distinguishable large Sce-msx + neuronal cells that are dorsally arranged in rows.These cells occupy a larger portion of the lateral somata cluster and appear to possess less distinct nuclei compared to the smaller surrounding cells (Additional file 9: Fig. S13g, g').In addition, Sce-msx transcripts are also present in the longitudinal muscle of the trunk, but more distinctly in the ventral longitudinal muscles (Fig. 4i; Additional file 9: Fig. S13f ' , arrowhead).Early juveniles exhibit Sce-msx expression in the brain and the vestibular and esophageal ganglia (Fig. 6j; Additional file 9: Fig. S13h, i).The lateral somata clusters have diffused Sce-msx expression (Fig. 6j; Additional file 9: Fig. S13h -j, j').In addition, Sce-msx appears to be absent in the longitudinal muscles of early juveniles.

Expression of the cholinergic markers Sce-ChAT and Sce-VAChT
The expression of Sce-ChAT and Sce-VAChT appears to be predominant in the medial (inner) cells of the lateral somata clusters, abutting the medial anterior neuropil and ventral longitudinal muscle (Fig. 5; Additional file 10: Fig. S14a, b; Additional file 11: Fig. S15a, b).Several Sce-ChAT + cells in the dorsolateral portion of the clusters are also identifiable in the hatchling (Additional file 10: Fig. S14a-c).In addition, Sce-ChAT is observed in encapsulated late embryos, where it is expressed in the medial lateral somata cluster (Additional file 10: Fig. S14d).Sce-VAChT expression is also present in cells lateral to the buccal opening (Additional file 11: Fig. S15b, c, arrowheads).Sce-ChAT and Sce-VAChT expression domains in early juveniles (Fig. 6m-r) are both observed in the cerebral ganglion (Fig. 6 m, p; Additional file 10: Fig. S14, e,  i, m, n).In the lateral somata clusters, Sce-VAChT exhibits diffuse expression (Fig. 6p -r; Additional file 11: Fig. S15e), while Sce-ChAT expression is detected at a higherintensity in the dorsal portion (Fig. 6o; Additional file 10: Fig. S14m).Notably, a pair of somata exhibiting high expression intensity of Sce-VAChT is also observed in the dorso-posterior region of the lateral somata clusters in 1 dph (Fig. 5d -f; Additional file 10: Fig. S14a, d, arrowheads, f ) which persists in early juveniles (Fig. 6p -r; Additional file 11: Fig. S15e, g).

Discussion
Our knowledge of chaetognath neurogenesis is mainly restricted to the description of the general morphology and organization of the nervous system using combinations of light microscopy, ultrastructural analyses, and generic immunohistochemical assays using antibodies directed against serotonin, FMRFamide-like peptides, RFamide-like peptides, acetylated or tyrosinated alpha tubulin [23-25, 43, 56, 60].The molecular processes underlying neurogenesis are, however, still poorly understood for Chaetognatha due to the lack of data, resulting in difficulties when comparatively investigating nervous system development and evolution in bilaterians.This is the first comprehensive study that further explores the molecular processes involved in the nervous system development of Spadella cephaloptera hatchlings.Specifically, we focused on genes involved in NS formation that have been considered to play a role during mediolateral patterning of the bilaterian nervous system (e.g.[5,9,61]) to acquire insights into evolutionarily conserved features during NS development among chaetognaths and other bilaterians.

Early patterns of neuronal development in young Spadella hatchlings
In the first 24 hours of postembryonic development, the central nervous system primarily comprises a dorsally located brain that is arranged as a circumoral loop and is linked to a VNC occupying most of the trunk (Fig. 1d,  f ) [25].Subsequently, the brain develops an anterior and a posterior domain.The anterior domain gives rise to a second circumorally arranged brain component, including vestibular and esophageal ganglia [25,43].The posterior brain domain processes the input from sensory organs and may play a role in the regulation of motor behavior in response to sensory input changes.In adults, the anterior domain functions mainly in controlling the activity of several oral components related to feeding, including the grasping spines and musculature responsible for opening and closing the mouth [43].
To provide a better picture of the nervous system in 1 dph individuals of S. cephaloptera, we describe the spatial arrangement of post-mitotic differentiating neurons by studying elav expression [62].While elav + cells are present in regions of the differentiating brain and VNC, our study highlights expression domains that have not been ascribed as neural regions before in chaetognaths [25,60].We detect strong Sce-elav-expression in a pair of cell clusters located posterior to the cerebral ganglion and laterally flanking the esophagus, which likely contribute to the nascent vestibular and esophageal ganglia.This paired structure is also Sce-foxA + and slightly Sce-VAChT + .Furthermore, a similar expression domain is observed in the early juveniles where the vestibular ganglia are more defined.While the vestibular ganglia of 1 dph S. cephaloptera only become visible by 72 h posthatching as revealed by synapsin and FMRFamide immunoreactivity [25], Sce-elav expression patterns in the head of the young hatchling suggest that the smaller and premature cephalic ganglia are already present and start developing at the same time as the cerebral ganglion.

Conserved expression of foxA in the CNS and the developing foregut
FoxA is suggested to be involved in morphogenesis and differentiation during embryonic development [63,64].It is among those genes demonstrated to possess regulatory functions in the formation and development of the different digestive tract regions in a broad range of bilaterian lineages.FoxA is expressed in the foregut of the mollusks Patella vulgata [65] and Crepidula fornicata [66], in the ectodermal foregut and hindgut of annelids [67][68][69][70] and the planarians Dugesia japonica [71] and Schmidtea polychroa [72].In addition, expression of foxA in the pharynx has also been reported for Phoronida [73], Ecdysozoa [74][75][76][77], and Chordata [78][79][80].In S. cephaloptera hatchlings, foxA-expression is also observed in various parts of the digestive tract, particularly in cells lining the foregut, corroborating its conserved regulatory functions during bilaterian digestive tract formation and regionalization [81].Moreover, Sce-foxA-expression is also detected in the developing brain and the VNC, which resembles the condition in annelids [67][68][69] and ecdysozoans [74,82,83].While some annelids probably express foxA in the ventral neuroectoderm, foxA is expressed in the floor plate of the developing neural tube of chordates [80,[84][85][86][87].This observation has been used as an argument in favor of a shared origin of the CNS among bilaterians ( [6,63], but see [16] for a different view).Although, at a later stage of neural development, it is interesting to note that foxA expression in the chaetognath hatchling is also observed in the ventral midline of the VNC.However, it remains to be investigated whether a similar expression pattern is also present during early ectoderm development of chaetognaths and whether this foxA domain is also conserved in other hitherto unstudied spiralians.
Nkx2.2 is also expressed in cells lining the gut of young Spadella hatchlings, which is an expression domain that is also present in the developing embryonic and larval gut of the annelid O. fusiformis and early juvenile of the brachiopod Terebratalia transversa [10].This indicates that nkx2.2, at least in Spiralia, might not only be involved in early neurogenesis, but also in gut differentiation.Nevertheless, more work is needed to demonstrate the functional role of nkx2.2 during gut development.

Conserved expression of pax6 in the CNS and eyes
The pax6 expression pattern in Spadella hatchlings generally resembles the expression of the developing sensory organs and nervous systems in many bilaterians.Particularly, its role during eye development has been consistently demonstrated in many spiralians, including annelids [90,91], cephalopods [92,93], and nemerteans [94], and also in several species across major ecdysozoan and deuterostome lineages [95,96].The Sce-pax6 homolog is also expressed in the developing eyes of hatchlings and weakens in later stages, suggesting its important regulatory roles during early post-embryonic eye development.Thus far, the only available data for pax6 expression in the Gnathifera clade is from the rotifer Epiphanes senta [10].The rotifer juveniles exhibit pax6 expression as two bands at the lateral regions of the brain, which is an expression domain resembling that of Spadella hatchlings.However, it is unclear whether it is also detected in their cerebral eye.Other sensory structures in different protostomes also show pax6 expression, such as the olfactory organ in squids [97,98], palps and antennae in the annelid P. dumerilii [90], and the mushroom body of the fruit fly D. melanogaster [99].Similarly, Sce-pax6 was observed in different sensory organs of Spadella hatchlings such as the corona ciliata, implying that the functional role of pax6 may have also expanded to the development of apomorphic morphological features of chaetognaths.
Moreover, various representatives of protostomes also express pax6 in the cephalic ganglia and ventral nerve cords, including mollusks [97,98,100,101], annelids [4,91,102], nemerteans [94], insects [103,104], and crustaceans [105].In the young Spadella hatchling, pax6 is also expressed in the developing brain and the VNC, particularly as paired lateral domains in the brain and in the lateral somata clusters, respectively.The specific expression domains in the CNS indicate its functions in post-embryonic neuronal patterning, a role which has also been reported in different bilaterians [91,103,106].Overall, our data on the Sce-pax6 expression pattern in Spadella support the argument for the conserved expression of pax6 in the developing eyes and CNS across bilaterian lineages.

Pax3/7 may play a role in early neuronal patterning of the chaetognath VNC
The expression of pax3/7 in the developing nervous system seems to be largely conserved in bilaterians.In several spiralians, pax3/7 is consistently expressed in the CNS: In the annelids P. dumerilii [4] and C. teleta [107], pax3/7 is expressed in two ventrolateral ectodermal bands.The brachiopod T. transversa also exhibits expression in the anterior ventral ectoderm of the larval trunk, and the cephalopod Sepia officinalis shows expression domains in ectodermal cells of different neural organs such as pedal ganglia and subesophageal region of the brain.Outside Spiralia, pax3/7 is also shown to be expressed in neural regions during development.The pax3/7 orthologs (gooseberry and gooseberry-neuro) in arthropods are also expressed in the neural ectoderm and are involved both in segmentation and the specification of neuroblast identity [108][109][110][111][112], while the chordate pax3 and pax7 are expressed in longitudinal bands in the developing neural tube of amphioxus, tunicates, and vertebrates [113][114][115][116][117]. In early-stage Spadella hatchlings, pax3/7 is detected as two thin and bilateral stripes on the dorsal hemisphere of the VNC, which suggests that pax3/7 also plays a role in the early neural patterning of the VNC, and therefore this role may be conserved in chaetognaths.
While pax3 and pax7 are generally specific markers for skeletal muscle developmental trajectories in vertebrates (e.g.[118,119]), the involvement of pax3/7 in mesoderm developmental processes is only known in few spiralians, such as the leech Helobdella robusta [120], and ecdysozoans such as Pristionchus pacificus [121].Furthermore, the mesoderm during the late elongation stage of the brachiopod N. anomala also expresses pax3/7 [10].In this study, Sce-pax3/7 is not detected in any mesodermal structures except in the presumably specialized peri-intestinal and lateral cells comprising the posterior septum [25].Further work, however, will be necessary to understand how pax3/7 functions in the formation and development of these mesodermal structures in Spadella.

Conserved expression of msx during muscle and nervous system development
Msx is known to play an important role in a variety of developmental processes in different bilaterians, more notably during neurogenesis and muscle formation.In Spiralia, msx expression is usually observed in the developing neural structures.The brachiopod T. transversa expresses msx in the ventral ectoderm, which includes the mantle and pedicle lobes [10].Also, both annelids, P. dumerilii and O. fusiformis exhibit msx + cells in the ventral neuroectoderm, while the cerebral ganglia of the annelid C. teleta also show msx expression [122].In the leech H. robusta, msx is expressed in all neurons of the anterior and posterior glial packets of the ventral nerve cord [123], and in the cephalopod S. officinalis, it is expressed in the developing branchial and stellate ganglia [92].Msx is also expressed in two longitudinal bands in the neuroectoderm of Drosophila [124,125], in the notochord and neural plate precursors of ascidians [126], and in the developing neuroectoderm during spinal cord development of vertebrates [125,127].The Sce-msx expression in Spadella hatchlings is largely observed in the VNC, but it is also detected in the brain of early juveniles.A strikingly intense level of expression is observed in some large neuronal cells in the lateral somata clusters of the young hatchling.It is, however, not clear whether these cells are identical to the aforementioned large foxA + neurons in the VNC of young hatchlings.In addition, msx is implicated during muscle differentiation and development of metazoans.The jellyfish Podocoryne carnea medusa expresses msx in the entocodon, a cell layer where the smooth and striated muscles emerge [128].Msh (ortholog of msx) in Drosophila is expressed in muscle progenitors [124,129] and in migratory limb muscle precursor cells in vertebrates [130].Also, N. anomala exhibits msx expression in the mesoderm [10].Musclespecific expression in Spadella is also observed in the longitudinal muscles along the trunk of the young hatchling.In general, our data suggest that the role of msx during muscular and nervous system development seems to be conserved in chaetognaths.

Cholinergic domains in Spadella hatchlings
ChAT and VAChT have been used as specific markers to identify acetylcholine-containing (cholinergic) neurons.ChAT is expressed in the nerve cord and brain in the xenacoelomorphs Meara stichopi and Isodiametra pulchra, in a number of cells of the apical region of the embryo and larva, in the neuropil and lateral ventral nerve cord of the juvenile annelid O. fusiformis, and in the brain, the corona ciliata, and the mastax of the rotifer E. senta [10].Structures positive for ChAT and VAChT are the brain and ventral nerve cord of the nemertean L. ruber and the anterior apical neuroectoderm of the brachiopod T. transversa [10].In the annelid P. dumerilii and vertebrates, motor neurons that express ChAT and VAChT have been shown to emerge from the pax6 + /nkx6 + progenitor domain of the ectoderm [4,131,132].While this correspondence between cholinergic motor neuronal markers (i.e.hb9, ChAT, and VAChT) and pax6 + /nkx6 + expression domains is evident in annelid and vertebrate model organisms, such expression colocalization is not observed in xenacoelomorphs and other spiralians [10].In Spadella hatchlings, ChAT and VAChT are expressed in the VNC and in the brain of later stages.We have no data on nkx6 expression, but both cholinergic neuronal markers are expressed within the pax6 + domains.This expression colocalization is also observed in the cephalopod S. officinalis, where pax6 expression is observed in the cholinergic-rich regions of the CNS [133].However, the distribution of motor neurons in the ChAT + and VAChT + domains of the VNC remains to be identified.

Expression of mediolateral patterning genes in the ventral nerve center of the Spadella hatchling
Our study demonstrates that the genes foxA, nkx2.2, pax6, pax3/7, and msx, which have been proposed to be involved in the mediolateral patterning of the bilaterian nerve cords, are also expressed in the chaetognath ventral nerve center.Nevertheless, at first glance, no similarity in the mediolateral patterning sequence of the above-mentioned genes has been observed for hatchlings (Figs. 3, 4; Fig. 7e) or early juveniles of S. cephaloptera and the latter stage, the distinct gene expression patterning observed in hatchlings is even absent (Fig. 6).The lack of staggered expression patterns of the mediolateral patterning genes may be explained by the fact that no earlier embryonic stages have been studied for S. cephaloptera or that mediolateral patterning has been secondarily lost in chaetognaths [5,12].However, it could also be that mediolateral patterning is not evolutionarily conserved among bilaterians and has evolved convergently in different organisms [10].
The lateral somata clusters enclose four trunk muscle packages and the intestine.During subsequent ontogeny, the trunk muscle packages expand outward and separate from the intestine, resulting in a large coelomic space in between the two organs (Fig. 1i).While expanding, the lateral somata clusters break away from enveloping the trunk muscles, and eventually lie on the ventral side of adults with ventral longitudinal muscles and intestine dorsally (Fig. 1i).If the ventral nerve center of hatchlings were conceptually laterally unfolded from the longitudinal muscle into a flat structure (Fig. 7f ), then the mediolateral sequence order of gene expression would be foxA, nkx2.2, pax6, msx, and pax3/7, resembling the condition observed in bilaterian models such as vertebrates [134,135], Drosophila [61,136,137], and the annelid P. dumerilii [4,138,139] (Fig. 7a -c).Interestingly, a similar observation has been studied in the egg yolk-rich embryos of the cephalopod S. officinalis where mediolateral patterning is obscured by the unique mode of gastrulation of cephalopods [92].

Conclusion
In this work, we provide insights on the molecular aspects of early post-embryonic neurogenesis in the benthic chaetognath Spadella cephaloptera.The Sceelav expression reveals previously undescribed neural domains in S. cephaloptera hatchlings, including cell clusters located posterior to the cerebral ganglion and The mediolateral extent of each neural patterning gene in the developing nerve cord is represented as a colored horizontal bar and the motor (hb9) and cholinergic (ChAT and VAChT) neuron subtype markers are labeled as yellow filled circles (modified from [5]).The vertebrate, insect, and annelid expression data are described in [61,[134][135][136][137], and [4,138,139], respectively.d A horizontal section of a 27 h post-laying embryo showing the location of the developing neuroblasts in the lateral side of the ectodermal layer that will eventually form the VNC (adapted from [42]).This is the earliest documented embryonic stage of Spadella where the VNC precursor cells (referred to as ganglion cells in [42]) were identified.e Expression patterns of mediolateral patterning genes schematized for the VNC of a S. cephaloptera hatchling (1 dph).f When the VNC is conceptually unfolded from the trunk longitudinal muscle into a flat nerve cord, the spatial extent of each gene conforms to a mediolateral system (mid and bottom images).Red arrows indicate the position and direction of the hypothetical unfolding process.Except for 7d, the dorsal and ventral side faces up and down, respectively.Question marks indicate that there is no information available.dlm: dorsal muscle; ect: ectoderm; gc: ganglion cells; in: intestine; lm: longitudinal muscle; lsc: lateral somata clusters; mes: mesodermal cells; mvs: medioventral somata clusters; no: notochord; np: neuropil; so: somites; st: stomodeum; vlm: ventral longitudinal muscle laterally flanking the esophagus, which likely contribute to the nascent vestibular and esophageal ganglia, and medioventral somata clusters of the VNC.Developmental genes and genes encoding transcription factors associated with neural differentiation during bilaterian nervous system development are also expressed in the neural domains of S. cephaloptera hatchlings, suggesting that their ancestral roles during bilaterian neurogenesis are likely conserved in Chaetognatha.Although these marker genes for neural patterning are expressed in specific neuronal cell subpopulations of the VNC, there is no evident staggered mediolateral patterning observed in the nerve cord of Spadella hatchlings, unless the latter is conceptually unfolded laterally from the longitudinal muscles into a flat structure.It would be necessary, however, to investigate earlier developmental stages directly after gastrulation to substantiate whether such mediolateral system is also present in chaetognaths.

Fig. 2
Fig. 2 Expression pattern of elav in the Spadella cephaloptera hatchling (1 dph).Gene transcripts are visualized with AP-Fast Blue (yellow) and cell nuclei with DAPI (purple).Maximum intensity projection of the (a) head, (b) trunk, and (c) tail.d Lateral projection of the head.Eyes are encircled and the dashed curve demarcates the anterior border of the lateral somata clusters.e-h Lateral projections of the trunk from the lateral region (e) to the (h) longitudinal midline.g Lateral section along the border of the gut-muscle bundle and the lateral somata cluster.i Lateral projection of a tail section.j-l Transverse sections of head showing the Sce-elav expression in (j) the cerebral ganglion, (k) the eyes (circle) and the cell clusters (with dashed borders) lateral to the esophagus and (l) slightly in the corona ciliata.Transverse sections of the (m) trunk and (n) tail.The location of the transverse section is indicated in panels a -c.Scale bars: 50 μm.The asterisk indicates the position of the future mouth opening.Orientation of specimens is indicated in the top right corner of each panel.cc, corona ciliata; cg, cerebral ganglion; cp, cephalic adhesive papillae; dlm, dorsal longitudinal muscle; es, esophagus; fn, fin; gmb, gut-muscle bundle; lsc, lateral somata clusters; mvs, medioventral somata clusters; so, ciliary sensory organ; tl, tail; vlm, ventral longitudinal muscle

Fig. 3
Fig. 3 Expression patterns of Sce-foxA and Sce-nkx2.2 in the Spadella cephaloptera hatchling (1 dph).Gene transcripts are visualized with AP-Fast Blue (yellow) and cell nuclei with DAPI (purple).a-c Sce-foxA, (d-f) Sce-nkx2.2.a, d Horizontal projection of the entire hatchling.b, e Lateral projection showing the expression pattern in the VNC.Dashed curves demarcate the posterior end of the VNC.c, f Transverse projection of the VNC.The dashed circle indicates the region of the gut (almond-shaped center)-longitudinal muscle bundle.Scale bars: 50 μm, except for panels a, d (100 μm).The asterisk indicates the position of the mouth opening.Orientation of specimens is indicated in the top right corner of each panel.See Fig. 1 for morphological description

Fig. 4
Fig. 4 Sce-pax3/7, Sce-pax6, and Sce-msx expression patterns in the hatchling of S. cephaloptera (1 dph).Gene transcripts are visualized with AP-Fast Blue (yellow) and cell nuclei with DAPI (purple).a-c Sce-pax6, (d-f) Sce-pax3/7, (g-i) Sce-msx.a, d, g Horizontal projection of the entire hatchling.(d) Sce-pax3/7 + neurons in the anterior VNC (arrowheads).b, e, h Lateral projection showing the expression pattern in the VNC.Dashed curves demarcate the posterior end of the VNC.c, f, i transverse projection of the VNC.The dashed circle indicates the region of the gut (almond-shaped center)-longitudinal muscle bundle.Scale bars: 50 μm, except panels (a, d, g;100 μm).The asterisk indicates the position of the mouth opening.Orientation of specimens is indicated in the top right corner of each panel.See Fig. 1 for morphological description

Fig. 5
Fig. 5 Expression patterns of cholinergic markers in the hatchling of Spadella cephaloptera (1 dph).Gene transcripts are visualized with AP-Fast Blue (yellow) and nuclei with DAPI (purple).a-c Sce-ChAT, (d-f) Sce-VAChT.a, d Horizontal projection of the entire hatchling.b, e Lateral projection showing the expression patterns in the VNC.Dashed curves demarcate the posterior (b) or anterior (e) end of the VNC.c, f Transverse projection of the VNC.e, f Overexpression of Sce-VAChT is observed in two large bilateral neurons in the posterior VNC (arrowheads).The dashed circle indicates the region of the gut (almond-shaped center)-longitudinal muscle bundle.Scale bars: 50 μm, except panels a, d (100 μm).The asterisk indicates the position of the mouth opening.Orientation of specimens is indicated in the top right corner of each panel.See Fig. 1 for morphological description

Fig. 6
Fig. 6 Expression of CNS patterning markers in the early Spadella cephaloptera juvenile.Gene transcripts are visualized with AP-Fast Blue (yellow) and cell nuclei with DAPI (purple).a-c Sce-foxA, (d-f) Sce-pax3/7, (g-i) Sce-pax6, (j-l) Sce-msx, (m-o) Sce-ChAT, and (p-r) Sce-VAChT.a, d, g, j, m, p Horizontal projections of entire animals (scale bar: 100 µm).b, e, h, k, n, q Lateral projections showing the expression patterns in the VNC (scale bar: 50 µm).Dashed curves demarcate the posterior (b, e, k) or anterior (h, n, q) end of the VNC.c, f, i, l, o, r show transverse projection of the VNC with lsc in yellow dashes.p, q, r Strong expression of Sce-VAChT is observed in two, large, bilateral neurons in the posterior VNC (arrowheads).The dashed circle indicates the region of the gut (almond-shaped center)-longitudinal muscle bundle.Scale bars: 50 μm, except panels a, d, g, j, m, and p (100 μm).The asterisk indicates the position of the mouth opening.Orientation of specimens is indicated in the top right corner of each panel.cc, corona ciliata; cg, cerebral ganglion; ey: eye; lsc, lateral somata clusters; mvs, medioventral somata clusters (See figure on next page.)

Fig. 7
Fig.7 Comparison of the expression profiles of mediolateral patterning in the vertebrate, insect, annelid, and chaetognath trunk nervous system.Schematic drawings of regional patterning of mediolateral genes (a) during early stages of nerve cord development: neural plate folding in the frog Xenopus and ventral ectoderm development in the fruit fly Drosophila and the annelid Platynereis; and (b) during later ontogeny, when the folding of the neural tube of vertebrates proceeds and the ventral nerve cords of insects and annelids have further developed.The vertical dashed line indicates the neural midline.c The mediolateral extent of each neural patterning gene in the developing nerve cord is represented as a colored horizontal bar and the motor (hb9) and cholinergic (ChAT and VAChT) neuron subtype markers are labeled as yellow filled circles (modified from[5]).The vertebrate, insect, and annelid expression data are described in[61,[134][135][136][137], and[4,138,139], respectively.d A horizontal section of a 27 h post-laying embryo showing the location of the developing neuroblasts in the lateral side of the ectodermal layer that will eventually form the VNC (adapted from[42]).This is the earliest documented embryonic stage of Spadella where the VNC precursor cells (referred to as ganglion cells in[42]) were identified.e Expression patterns of mediolateral patterning genes schematized for the VNC of a S. cephaloptera hatchling (1 dph).f When the VNC is conceptually unfolded from the trunk longitudinal muscle into a flat nerve cord, the spatial extent of each gene conforms to a mediolateral system (mid and bottom images).Red arrows indicate the position and direction of the hypothetical unfolding process.Except for 7d, the dorsal and ventral side faces up and down, respectively.Question marks indicate that there is no information available.dlm: dorsal muscle; ect: ectoderm; gc: ganglion cells; in: intestine; lm: longitudinal muscle; lsc: lateral somata clusters; mes: mesodermal cells; mvs: medioventral somata clusters; no: notochord; np: neuropil; so: somites; st: stomodeum; vlm: ventral longitudinal muscle